Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution

Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution Ningxiao Li, Jacob L. Steenwyk, Samuel O’Donnell, Emile Gluck-Thaler, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8100147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2026 Read the published version in BMC Genomics → Version 1 posted 11 You are reading this latest preprint version Abstract Background Accessory genome regions of plant pathogenic fungi, which are highly variable and consist of niche-adaptive genes, play a crucial role in shaping host-specific interactions but are notoriously difficult to assemble. Fusarium oxysporum causes some of the world’s most economically devasting diseases, however, understanding how it interacts with its host is hindered by challenges in assembly of accessory genome regions/chromosomes, even with long read sequencing technologies. F. oxysporum f. sp. lactucae (FOLac) races 1 and 4 possess highly similar core genomes but cause distinct virulence phenotypes on specific lettuce cultivars. The availability of fully assembled genomes for the two races is needed to advance our understanding of the genetic basis of pathogenicity and the evolutionary processes underlying the diversification of FOLac and other F. oxysporum pathogens. Results We developed an assembly workflow for generating gapless, telomere-to-telomere (T2T) complete genome assemblies for FOLac races 1 and 4. The T2T assemblies allowed for the identification of 16 chromosomes (5 accessory) and 20616 predicted genes for race 1 and 19 chromosomes (8 accessory) and 20292 predicted genes for race 4. Comparative genomics revealed major structural differences in their accessory genome regions, including genome rearrangement and large-scale chromosome duplication, with results suggesting transposable elements as the main drivers of those genomic changes. The analysis of Secreted in Xylem ( SIX ) effector gene profiles uncovered a similar presence/absence pattern among FOLac races 2–4, distinguishing them from race 1. Searches for genes unique to each race resulted in the identification of 689 race 1- and 536 race 4-specific genes. Assembly and genomic features comparing T2T to contig-level Illumina assemblies showed that 17–23% of genome sizes and ~ 10% of predicted genes were missing from Illumina assembly, mostly within accessory genome regions. Conclusions T2T assemblies revealed large-scale differences in accessory genome structure and content between two otherwise highly similar pathogenic races. These differences provide a framework for understanding evolutionary processes that led to the diversification of pathogens within F. oxysporum on a fine evolutionary timescale, the identification of genes that may be responsible for host-pathogen interaction, and the identification of race-specific sequences useful for diagnostics. Fusarium oxysporum f. sp. lactucae Fusarium wilt telomere-to-telomere accessory chromosomes transposable elements effectors Secreted in Xylem (SIX) comparative genomics genome evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Fusarium oxysporum is a globally distributed species that includes important agents of wilt disease, but also strains that are endophytic/presumably nonpathogenic, and saprophytes. Collectively, plant pathogenic strains of F. oxysporum have a remarkably broad host range, causing vascular wilts as well as root and crown rots on over one hundred agronomically important plant species. While host diversity is broad at a species level, individual F. oxysporum pathogens usually display a high degree of host specificity, causing disease in one or a few related plant species [ 1 , 2 ]. Pathogenic strains that infect the same plant host are grouped into the same forma specialis (f. sp.). More than 150 different formae speciales (ff. spp.) have been reported, with 106 of them having been well characterized [ 3 , 4 ]. Some ff. spp. are further divided into races based on virulence to a set of differential host genotypes and, in some cases, based on known resistance genes in these hosts [ 3 ]. Races have been described in 25 ff. spp. [ 3 ], and new races arise frequently. A well-studied example is F. oxysporum f. sp. lycopersici , which had two successive resistance-breaking races emerge within 12 years of resistance deployment, as a result of coevolution between the pathogen and resistant tomato cultivars [ 5 ]. With very few exceptions where a single f. sp. is confined in a monophyletic lineage (such as f. sp. ciceris [ 6 , 7 ]), F. oxysporum ff. spp. tend to be non-monophyletic [ 8 , 9 ], consistent with independent evolutionary origins of pathogenicity on a given host and unique evolutionary patterns of different ff. spp. within F. oxysporum . With the origin of F. oxysporum being possibly as recent as ~ 0.5 mya [ 10 ], this diversification appears to have occurred very recently. Genetic determinants of host-specificity, including effector genes, are predominantly present in transposon-rich “accessory” chromosomes (ACs), also known as lineage-specific or pathogenicity chromosomes [ 11 ]. F. oxysporum has been reported to possess one or several ACs that are highly variable and harbor unique sequences that are absent in other F. oxysporum strains, except those that share the same host. This is in sharp contrast to core chromosomes (CCs), which are essential and conserved among all strains within F. oxysporum . The most studied effector genes in F. oxysporum are the Secreted in Xylem (SIX) genes, which were first identified in F. oxysporum f. sp. lycopersici and encode small, cysteine-rich proteins that are secreted into the xylem sap of tomato plants during infection [ 12 – 14 ]. Since then, 14 SIX genes ( SIX1-14 ) and variability of them among different isolates and races within a single f. sp. have been reported in numerous F. oxysporum ff. spp. [ 15 – 19 ], with multiple studies illustrating the role of different SIX genes in conferring virulence and determining host specificity in several pathosystems [ 20 , 21 ]. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae (FOLac), has emerged as a major disease in lettuce growing areas, posing a significant threat to lettuce cultivation worldwide. The pathogen FOLac consists of four known races (races 1, 2, 3 and 4) that differ in virulence patterns on resistant or susceptible lettuce cultivars, with distinct geographic distributions. Race 1 is the most widespread and occurs in most countries where lettuce is grown, including Japan [ 22 , 23 ], the United States [ 24 ], Taiwan [ 25 ], Iran [ 26 ], Brazil [ 27 ], Argentina [ 28 ], and Europe [ 29 – 32 ]. Races 2 and 3 have very restricted distribution and are found in Japan [ 23 , 33 ] and Taiwan [ 25 ]. A new race (race 4), which is highly virulent on some race 1-resistant cultivars, was first detected in the Netherlands (Gilardi et al. 2017), where Fusarium wilt of lettuce was not previously reported. Since then, race 4 has spread rapidly to other parts of Europe, including Belgium [ 35 ], the UK [ 36 ], Italy [ 37 ], and Spain [ 38 ]. While FOLac races 2 and 3 are phylogenetically distinct from races 1 and 4 [ 25 , 34 , 39 ], all FOLac race 1 and race 4 isolates obtained from a worldwide collection were placed in a single clade based on sequences of 41 full-length, orthologous genes, with the two races being indistinguishable (Geiser, unpublished). This finding suggests recent clonal origin of FOLac race 1 and 4 pathogenicity. Using a k -mer-based approach that analyzes sequence variation directly from raw reads, FOLac races 1 and 4 can be further divided into two sub-clades, with varying levels of sequence divergence among individual isolates within each race [ 40 ]. It is speculated that intra-race genetic diversity within the race 1 and race 4 populations may contribute to the emergence of new variants or races of FOLac. This has been manifested in recent years, where novel pathogenic variants of FOLac race 1 were reported in California, with one variant (VSP-0916) exhibiting high aggressiveness on the race 1-resistant variety Costa Rica No.4, while another variant (Fol621) became less virulent on susceptible cultivars [ 41 ]. Similarly, an emerging FOLac race 4 + was detected in several farms in Belgium where FOLac race 4 intermediate resistant cultivars showed wilting and growth reduction one year after commercialization [ 42 ]. To better understand the evolutionary processes responsible for the emergence and diversification of the FOLac pathogen, a reference genome of FOLac that is fully assembled is critical to the research community. Although the disease has been reported for nearly half a century, the first relatively complete genome of FOLac was not released until recently [ 43 ]. Based on comparative genomic analyses between FOLac races 1 and 4, Bates et al. [ 43 ] showed that FOLac race 4 has a larger genome than FOLac race 1, with a lack of synteny between their accessory genome regions. Additionally, Bates et al. [ 43 ] posited that FOLac race 4 did not evolve directly from FOLac race 1, but both races inherited their accessory genomes from a common ancestor, which then underwent rearrangement/recombination events, accompanied by the acquisition of accessory regions via horizontal chromosome transfer. However, because these published FOLac assemblies are at the contig-level, with fragmented CCs and ACs, it is necessary to reconstruct a complete genome for FOLac races 1 and 4 to provide a more complete insight into the mechanisms underlying genome evolution that led to the diversification and host specialization of the pathogen. Here, we present gapless, telomere-to-telomere (T2T) complete genome assemblies for FOLac races 1 and 4. In doing so, we developed a workflow for generating T2T assemblies and highlight the importance of visual inspection of read mapping to ensure the accuracy and correctness of the assembly. Comparative genomics between the two T2T assemblies revealed major structural differences in their accessory genome regions that may underlie genetic diversification between FOLac races 1 and 4. The T2T assemblies also enabled the characterization of large-scale chromosome duplication and the recognition of a specific type of transposable elements (TEs) that may be involved. A comprehensive transcriptome dataset of FOLac, which encompassed nine RNA-seq libraries generated under different growth conditions, was used to produce a complete genome annotation for the two T2T assemblies, leading to the identification of candidate genes that may be race-specific. Moreover, this study compared assembly and genomic features between the T2T assembly and two contig-level assemblies for the same isolates, offering researchers a new perspective in assessing their sequencing and assembly strategy. Results FOLac race 4 has an expanded genome with three more chromosomes than race 1 Two previous reported F. oxysporum isolates, JCP043 (FOLac race 1, from California [ 24 , 44 ] and AT141 (FOLac race 4, from Spain [ 40 ]), were selected for long-read sequencing. The assembly workflow (Fig. 1 ) started with building a preliminary assembly using reads generated by Oxford Nanopore Technologies (ONT). Using default settings, the NECAT program assembled the ONT reads into 27 contigs for JCP043 and 25 contigs for AT141, some of which already had telomeric repeats (CCCTAA/GGGTTA) on one or both ends. The mitochondrial (mt) genome was identified from the assembly and deposited in GenBank. By mapping 50–99 kb ONT reads to the NECAT contigs, mis-assemblies were visually identified based on a sharp decrease in read depth at the misassembly junction and no reads spanning the incorrectly assembled region (example shown in Supplementary Figure S1 ) . After the assembly inspection, the NECAT contigs were broken into 44 intermediate contigs for JCP043 and 46 contigs for AT141. Iterative cycles of read mapping with 50–99 kb ONT reads followed by end extension were carried out on the intermediate contigs. In most cases, ONT reads alone were sufficient to extend the contigs to the telomeric region without taking extra steps (see detailed instructions in Methods and Supplementary File S1 ). The extended contigs with an overlap of greater than 50 kb and 95% sequence identity were joined to reconstruct full-length chromosomes. The telomeric end of the rDNA repeat region was completed through aligning 50–99 kb reads that contained rDNA sequences and flanking sequences. The rDNA copy number was estimated based on the depth of ONT read coverage relative to adjacent regions of the chromosome, which resulted in an estimated 90 copies for JCP043 and 144 copies for AT141. The final T2T assembly was evaluated for accuracy again by mapping 50–99 kb ONT reads to it, with visual inspection showing continuous and uniform read coverage throughout the chromosomes. Due to the error rates (between 4–5% at the time of data generation) of ONT data, the T2T assembly was polished using a hybrid strategy involving two runs of PacBio HiFi read mapping followed by two runs of Illumina read mapping at high stringency (to reduce problems of degenerate bases flanking repetitive regions). Although Illumina reads exhibited great read accuracy, many regions in the genome, especially those in the ACs, did not have sufficient read depth required for performing error correction ( Supplementary Table S1 and Supplementary Figure S2 ). Insufficient coverage was more pronounced in AT141 than JCP043, as the gapped genomic regions totaled 2.17 Mb in AT141 compared to 156 kb in JCP043 ( Supplementary Table S1 ) . As much as 10.5% of chromosome 16 in AT141, mainly in the 5’end of the chromosome (nonrepetitive, 280 kb), was depleted of Illumina reads. In contrast, HiFi reads had a uniformly high read coverage (> 100× coverage with reads ≥ 15 kb) across the entire genome ( Supplementary Figure S2 ). However, single-base sequence errors, including SNPs and indels, could still be detected in the HiFi-corrected assembly; therefore, additional error correction using Illumina reads was carried out under high stringency. The accuracy of the polished assembly of JCP043 was further confirmed with high-throughput chromosome conformation capture (Hi-C) analysis, and the result showed no mis-assembly ( Supplementary Figure S3 ). The number of chromosomes was also validated by centromeric interaction regions detected in the Hi-C contact map, with each contig represented as a single chromosome. The final assemblies were gapless and T2T complete, with JCP043 (race 1) and AT141 (race 4) assembled into 16 and 19 chromosomes, respectively, and the AT141 genome size being 6% or 3.86 Mb greater than that of JCP043 (Table 1 ). The benchmarking universal single-copy ortholog (BUSCO) analysis was performed to assess the genome completeness. Of the 4,494 BUSCOs searched (library hypocreales_odb10), 98.9% and 98.5% were detected as complete and single copy in JCP043 and AT141, respectively (Table 1 ), which had similar levels of completeness compared to other published FOLac genomes (98.8% for AJ520 and 98.7% for AJ516 [ 43 ]). Table 1 Comparison of assembly and genomic features between T2T, Illumina, and NECAT assemblies of FOLac race 1 isolate JCP043 and race 4 isolate AT141. JCP043 AT141 T2T Illumina NECAT T2T Illumina NECAT Assembly features Genome size (Mb) 64.64 58.59 63.73 68.50 53.58 67.10 Read depth 204× 1 146× 204× 1 176× 1 182× 176× 1 GC (%) 47.61 49.34 47.42 47.67 48.17 47.62 N50 (Mb) 4.63 0.14 4.31 4.25 0.10 4.18 L50 6 112 6 7 148 7 # of contigs 16 5,405 27 19 5,771 25 # of T2T contigs 16 0 5 19 0 12 Longest contig (Mb) 6.77 0.91 7.07 6.82 0.54 6.82 BUSCO (complete, single-copy) 98.9% 98.8% 98.9% 98.8% 98.6% 98.8% Core genome size (Mb) 48.64 46.47 2 48.09 2 49.02 43.98 2 47.44 2 Accessory genome size (Mb) 16.00 7.62 2 15.51 2 19.48 7.38 2 15.61 2 Genomic features Total genes 20,616 18,811 20,517 20,292 18,320 20,185 Total transcripts 22,319 20,120 21,983 21,918 19,601 21,572 Total proteins 22,083 - - 21,680 - - Mean gene length 1,746 bp - - 1,738 bp - - Mean exon length 630 bp - - 623 bp - - Repeat regions 17.64% 6.46% 18.29% 20.39% 4.86% 19.97% GO terms 12,293 - - 12,163 - - PFAM 13,204 - - 13,081 - - CAZYmes 700 671 700 705 659 702 Secreted CAZYmes 331 312 331 337 301 325 SM clusters 65 65 65 67 64 67 SM genes 1,051 1,036 1,051 1,028 1,004 1,026 Secreted proteins 1,433 1,316 1,422 1,451 1,303 1,414 Effector proteins 605 559 600 574 526 573 Mimp effectors 94 90 94 82 78 82 Race-specific genes 689 518 681 536 437 513 Secreted CAZYmes 4 3 4 2 2 2 Effector proteins 14 9 14 8 7 8 Mimp effectors 5 4 5 3 3 3 SM genes 3 2 3 6 5 6 1 Read coverage of T2T and NECAT assemblies was calculated by mapping 50–99 kb ONT reads to the assembly for improved mapping accuracy. 2 Core and accessory genome sizes of Illumina and NECAT assemblies were calculated based on the total length of aligned contigs to the core and accessory genome regions of their corresponding T2T assembly. - indicates comparison was not performed on the specific feature. Massive genome rearrangement in FOLac accessory chromosomes Comparison of each of the two T2T assemblies to that of Fol4287 (f. sp. lycopersici race 2), the reference genome of F. oxysporum due to its nearly complete genome featuring 11 CCs and 5 ACs, revealed that i) each of the two FOLac isolates had 11 CCs that were highly syntenic with the 11 CCs of Fol4287 (Figs. 2 A and 2 B), and exhibited common features associated with CCs, including high gene density and low abundance of TEs; ii) JCP043 had five ACs while AT141 featured eight ACs that did not show clear synteny with the Fol4287 genome, and exhibited accessory-like appearance (gene-sparse and TE-rich); iii) the accessory genome regions of JCP043 also included a 1.38-Mb terminal segment of chromosome 5 and a 0.5-Mb segment of chromosome 14, whereas a 0.9-Mb terminal segment of chromosome 8 in AT141 was part of its accessory genome. It was worth noting that many of the unplaced scaffolds of Fol4287 were aligned with several CCs of JCP043 and AT141, primarily to the sub-telomeric regions. The collinearity analysis of JCP043 and AT141 showed that the 11 CCs were highly syntenic between the two isolates, with 99%-100% sequence identity except the accessory regions located on chromosomes 5 and 14 of JCP043 and chromosome 8 of AT141, which appeared to be unique sequences to each race (Fig. 2 C). This analysis also identified one chromosomal translocation event, in which chromosome 11 of AT141 appeared to be translocated to two different chromosomes in JCP043, with nearly 80% of the chromosome landing on chromosome 14 of JCP043 and the remaining 20% fused to the terminus of chromosome 2 (Fig. 2 C). This translocation event was also observed in the synteny plot between JCP043 and Fol4287 (Fig. 2 A ) . Comparison of the ACs between JCP043 and AT141 showed an absence of large-scale synteny between the two isolates, but revealed widespread presence of smaller similar sequences, ranging from 10 to 75 kb in size and 92.99–98.92% sequence identity, between them in a non-colinear order (Fig. 2 C), which suggests massive genome rearrangements may have occurred in their ACs. One exception to this was chromosome 16 of JCP043, which appeared to have two large fragments of DNA, 480 and 580 kb in size, syntenic with chromosomes 16 and 18 of AT141, respectively. To get a better understanding of how dissimilar the unique sequences (defined by the lack of homology in any 10-kb windows) were between the two isolates, the two sets of ACs were compared at 2-kb resolution (slightly above the average gene length). The analysis, which was performed on repeat-masked ACs to avoid the detection of overwhelmingly abundant DNA repeats, showed that 32% of those unique segments were shared between the two isolates (Fig. 3 B). To determine whether the ACs of JCP043 are conserved in race 1 while variable to race 4, we conducted synteny analysis with six published contig-level FOLac genomes (three race 1 and three race 4 isolates [ 43 ]). The result showed that race 1 isolate JCP043 displayed high levels of AC synteny with previously published race 1 isolates (AJ520, AJ718 and AJ865), whereas most of the ACs in JCP043 were not syntenic with the race 4 isolates (AJ516, AJ592 and AJ705) ( Supplementary Figure S4 ). Likewise, the ACs of race 4 isolate AT141 were syntenic with the three race 4 isolates but not with race 1 ( Supplementary Figure S5 ). DNA repeats likely contribute to the expanded genome of AT141 About 18% and 20% of the JCP043 and AT141 genomes, respectively (Table 1 ), was identified as repetitive sequence, including DNA transposons ( hAT , Tc1-IS630-Pogo , MULE-MuDR , PiggyBa c and Helitron ), long terminal repeat (LTR) retrotransposons ( Gypsy/DIRS1 and Ty1/Copia ), long interspersed nuclear elements (LINEs) retrotransposons ( Tad1 and RTE ), as well as simple repeats and satellites ( Supplementary Table S2 ). Among the different classes of TEs, hAT represented the largest in length and ranked the highest in copy number, followed by Gypsy/DIRS1 and Tc1-IS630-Pogo. Based on the density plots (Fig. 3 ), ACs were enriched in different types of TEs, accounting for more than 75% of the characterized TEs in the entire genome (Fig. 4 A). While most of the repeat classes were similar in size between the two genomes, the AT141 genome contained a significantly greater amount of unknown repetitive sequence, totaling 4.34 Mb in size compared to 2.61 Mb in JCP043, accounting for 44.8% of the overall genome size difference between the two isolates. The unknown repeats were widely distributed among the ACs, especially in the accessory segment of chromosome 8 in AT141 (Fig. 3 B). Sequences of the unknown repeats are provided in Supplementary File S2 . While most of the TEs were largely less abundant in CCs, we noticed that 75% of Gypsy/DIRS elements were associated with CCs (Fig. 3 A ) . To determine whether there is any association between Gypsy/DIRS1 and the aforementioned chromosomal translocation in JCP043, we marked the breakpoint (located on chromosome 11 of AT141) and two fusion points (located on chromosomes 2 and 14 of JCP043) on the TE density plot. It was found that the breakpoint and one of the fusion points (on chromosome 2 of JCP043) overlapped with a Gypsy/DIRS1 element while the other fusion point (on chromosome 14 of JCP043) resided 15 kb upstream of three Gypsy/DIRS1 elements (Fig. 3 A). Large-scale chromosome duplication is prevalent in FOLac accessory chromosomes Besides DNA repeats, we explored the possibility of segmental chromosome duplication in the ACs contributing to the larger genome size of AT141 and the formation of three more ACs. To study this, pair-wise comparison of individual ACs of AT141 were conducted to identify similar regions greater than 20 kb (to avoid the detection of overwhelmingly abundant DNA transposons, some of which could extend up to 12 kb). The analysis resulted in the recognition of numerous intra- and inter-chromosomal duplications in all the ACs of AT141 (Fig. 5 B) that ranged in size from 20 kb to 500 kb, except for chromosome 18, which did not show any segmental duplication. Notably, we identified two nearly identical, 500-kb inverted repeats located on both ends of chromosome 17 (Fig. 5 B), flanking a 700-kb non-repetitive region. This duplication, along with a few others, might be facilitated by Gypsy/DIRS1 retrotransposons that flank the duplicated regions (Fig. 5 B). In addition, a heavy presence of hAT-Restless transposons was detected surrounding some of the other duplicated regions. While the frequency of intra-chromosomal duplication was markedly less in JCP043 compared to AT141, inter-chromosomal duplications were frequently found in the ACs of JCP043, including a 1-Mb region that was nearly identical between chromosomes 5 and 10 (Fig. 5 A). Similar to AT141, we found the association of Gypsy/DIRS1 with the boundaries of this large, duplicated region. Overall, given that the total size of duplicated regions was nearly identical between the two genomes (1.43 Mb for JCP043 vs. 1.52 Mb for AT141), it was reasonable to conclude that segmental chromosome duplication was not the contributing factor for the expanded genome of AT141, but it may play an important role in the formation of new accessory chromosomes (i.e. chromosome 17) and altering structural organization of the genome. General putative effector proteins are distributed more in the core genome regions while mimp -associated effectors are enriched in the accessory genome regions The genomes of JCP043 and AT141 harbored 20616 and 20292 predicted genes, respectively (Table 1 ), however, only 60% of them had GO annotations. Approximately 85% of the predicted genes were associated with the core genome regions while the remaining 15% were present in the accessory genome regions (Figs. 4 A). Using the non-redundant gene sets for JCP043 and AT141, we identified 15980 pairs of putative orthologous genes. Not surprisingly, over 94% of them were associated with CCs and the co-linear order of genes between the two isolates has maintained within these chromosomes ( Supplementary Figure S7 A ). The remaining 6% were widely dispersed among different ACs ( Supplementary Figure S7 B ). The JCP043 and AT141 genomes harbored similar numbers of several specific gene classes, respectively, including 331/337 extracellularly secreted carbohydrate-active enzymes (CAZYmes), 605/574 effector proteins, and 65/67 secondary metabolite (SM) gene clusters comprising 1051/1028 genes (Table 1 ). These genes were predominantly present in CCs, representing 85–98% of the gene reservoir (Fig. 4 A and Supplementary Figure S7 ). Among the putative effector proteins identified, apoplastic effectors constituted 51–54% of the effector complement, followed by cytoplastic effectors (27–30%). The localization of the remaining effector proteins remained undetermined. According to previous studies, many effector genes (e.g. SIX genes) in F. oxysporum are located within subregions enriched for TEs, and a miniature Impala ( mimp ) element, in particular, is always present in their promoters [ 45 , 46 ]. The analysis of mimp -associated effectors identified 94 and 82 candidates in the genomes of JCP043 and AT141, respectively. Among them, 18 (for JCP043) and 11 (for AT141) mimp effectors overlapped with the effector proteins identified using the EffectorP pipeline. Nearly 95% of the candidate mimp effectors were associated with ACs (Fig. 4 A), with chromosome 10 of JCP043 (N = 29) and chromosome 5 of AT141 (N = 35) harboring the most mimp effectors ( Supplementary Figure S7 B ). Genes involved in intracellular pH homeostasis, regulation of transcription and mitochondria-nucleus signaling pathway are enriched in FOLac accessory chromosomes We conducted gene ontology (GO) enrichment analysis to identify putative biological processes and molecular functions that are enriched in the ACs of both FOLac isolates. It turned out that their ACs were significantly enriched (corrected p-value < = 0.01) for biological processes involved in intracellular monoatomic ion and cation homeostasis, regulations of intracellular pH and DNA-binding transcription factor activity, and mitochondria-nucleus signaling pathway ( Supplementary Table S3 ). Molecular functions, including various types of oxidoreductase activities and iron ion and heme-binding activities, were also enriched. In addition, the ACs of JCP043 were uniquely enriched for genes involved in lipid, peptide, and monocarboxylic acid catabolic processes, while genes involved in double-strand break repair DNA repair, regulation of nitrogen utilization, and long-chain fatty acid metabolic process were uniquely enriched in the ACs of AT141. Regarding the genes located within the duplicated regions in JCP043, they were enriched for two biological processes, including intracellular monoatomic ion and cation homeostasis and chromate transport. No GO terms were significantly enriched in the duplicated regions in AT141. SIX9.4 and SIX14 are conserved in FOLac while SIX8 and SIX9.1-9.3 are absent in race 1 Due to the lack of ability to identify all 14 SIX genes using the EffectorP and FoEC2 pipelines [ 47 ], we manually annotated the SIX gene complement in both T2T assemblies via BLAST. Moreover, we expanded the search to 53 publicly available FOLac genomes [ 40 , 41 , 43 ], including 33 race 1 isolates, 1 race 2 isolate, 1 race 3 isolate, 16 race 4 isolates, and 2 race 1 variants ( Supplementary Table S4 ), to evaluate the correlation between the SIX gene complement and race structure. Additional long-read genome assemblies representing different phylogenetic lineages and clades outside of FOLac ( Supplementary Table S4 ) were also included in this analysis to determine how similar their SIX genes are to those present in FOLac. Generally, FOLac carried three SIX genes, SIX8 , SIX9 and SIX14 , with copy number and sequence variations at the intra and inter-race levels, which are described below. None of the remaining SIX genes were identified in FOLac. SIX8 SIX8 was absent in JCP043 and 35 other FOLac race 1 isolates (including the two race 1 variants), while two identical copies of SIX8 were identified in AT141, located within a 30-kb, TE-rich, tandem repeat on chromosome 5 (Figs. 5 B and 6 A). Except for AT141 having two copies of SIX8 , a single copy of SIX8 was consistently identified in all the other FOLac race 4 isolates as well as FOLac races 2 and 3, with variation in the SIX8 sequences among isolates resulting in two SIX8 variants ( SIX8.1 and SIX8.2 ; Fig. 6 B and Supplementary Figure S8 ). SIX8 was also present in eight other unrelated F. oxysporum isolates, including ff. spp. conglutinans , lycopersici , niveum , and sesame ( Supplementary Table S4 ). The SIX8 gene phylogeny revealed that FOLac SIX8.1 and SIX8.2 were most closely related to f. sp. conglutinans (Fig. 6 B ) . SIX9 A previous study showed that FOLac had four variants of SIX9 ( SIX9.1-SIX9.4 ), with differences in copy number and sequence variation between races 1 and 4 [ 43 ]. In our study, none of the FOLac race 1 isolates had SIX9.1 , SIX9.2 , and SIX9.3 , except race 1 isolate AM163, which appeared to have one copy of SIX9.2 in its Illumina assembly. However, the read mapping analysis indicated otherwise due to the extremely low read depth (less than 8×) on SIX9.2 compared to the other SIX genes ( Supplementary Figure S9 ). Two identical copies of truncated SIX9.1 (54% in coverage), which had an unknown type of DNA repeat present upstream, were identified in JCP043 (Fig. 5 A). The truncated SIX9.1 was conserved among all the FOLac race 1 isolates. FOLac races 2, 3 and 4 possessed all four variants of SIX9 , but variation in copy numbers was observed among the FOLac race 4 isolates ( Supplementary Table S4 ). SIX9.4 appeared to be the only copy that was consistently identified from all four races, with no sequence variation among isolates (Fig. 6 F). Based on their location on the T2T assembly of AT141, the three variants of SIX9 ( SIX9.2 , SIX9.3, and SIX9.4 ) were spread sparsely on chromosome 5, flanked by DNA transposons (i.e. TcMart-Fot1 ) or unknown DNA repeats (Fig. 6 E; also observed in SIX8 as noted above). An additional copy of SIX9.4 was identified on chromosome 12 of AT141, part of a 23-kb duplicated region between chromosome 5 and 12 (Fig. 5 B). Based on the SIX9 gene phylogeny (Fig. 6 F), sequences of FOLac SIX9.1-9.4 were (nearly) identical to those in unrelated ff. spp., including apii races 3 and 4, conglutinans , lini , coriandrii, niveum , and semani, raphanin , and vasinfectum. SIX14 SIX14 was consistently present among all the FOLac isolates, with no sequence variation among isolates, the exception being race 1 isolate Fol621 and race 1 variant VSP-0916 (Fig. 6 D), which had one non-synonymous mutation. SIX14 , along with SIX8 and SIX9 , were all located on chromosome 5 of AT141 and at least 200-kb apart from one another (Fig. 5 B), indicating that chromosome 5 is a putative pathogenicity chromosome. Likewise, in the genome of JCP043, we identified chromosome 15 as a putative pathogenicity chromosome because it harbored both SIX9.4 and SIX14 (Fig. 5 A). Interestingly, the BLAST analysis revealed a second copy of SIX14 , located on chromosome 10 in JCP043, however, it appeared to be disrupted by the insertion of a Gypsy retrotransposon (Fig. 6 C), likely resulting in loss of function. This is consistent with a previous study, where a transposon has inserted into SIX14 [ 43 ]. Compared to SIX8 and SIX9, SIX14 tended to have a limited distribution among the F. oxysporum taxa we examined as it was only identified in two unrelated ff. spp., including ff. spp. niveum and lycopersici . Sequences of SIX14 identified among FOLac isolates were most closely related to those in f. sp. niveum (Fig. 6 D). Sequences of SIX8 , SIX9 , and SIX14 identified from all the F. oxysporum isolates used in this study are provided in Supplementary Files S3-S5 . Putative race-specific genes are clustered on several accessory chromosomes We identified 1010 candidate unique genes for JCP043 (Fig. 4 B), 563 of which were absent and 447 were partially present (below 90% identity or 80% coverage) in the AT141 genome. A total of 1081 candidate unique genes were identified in AT141, including 741 absent and 340 partially present. After screening the candidate genes against nine representative FOLac genomes covering all four races, 689 putative race 1-specific genes and 536 putative race 4-specific genes were identified (Table 1 and Supplementary Files S6 and S7 ). Nearly 87% of the candidate race-specific genes were associated with the accessory genome regions and often clustered together ( Supplementary Figure S7 B) . Chromosomes 7, 12 and 10 of JCP043 harbored the greatest numbers of race 1-specific genes (chr 7: 223, chr 12: 167, and chr10: 85). In AT141, chromosomes 13, 5, and 16 contained the most race 4-specific genes (chr13: 219, chr5: 76, and chr16: 60). Among the FOLac race 1-specific genes, we identified 4 secreted CAZYmes, 14 effectors, 5 mimp -associated effectors, and 3 SM genes ( Supplementary Table S5 ). As for the race 4-specific genes, 2 secreted CAZYmes, 8 effector, 3 mimp -associated effectors, and 6 SM genes were identified. Interestingly, two race-specific CAZYmes (JCP043_017764 and AT141_017966) were also predicted to be apoplastic effectors, potentially contributing to plant cell wall degradation. While most of the race-specific SM genes coded hypothetical proteins, we identified one unique SM gene cluster located on chromosome 16 in AT141 that harbored multiple race 4-specific genes, including a putative type-III polyketide synthase (AT141_019912) as the core biosynthetic enzyme, a glycoside hydrolase (GH) family protein (AT141_019910), a phosphate transporter (AT141_019913), and two hypothetical proteins (AT141_019917 and AT141_019918). The core biosynthetic enzyme had high similarity (88.5% identity in amino acid sequences) to the thiolase-like protein of F. redolens (Accession: XP_046053686.1). The T2T assembly reveals structural variants and putative genes not shown in contig-level assemblies In comparison to the T2T assemblies of JCP043 and AT141, their respective Illumina assemblies presented several limitations in characterizing assembly and genomic features as described below and summarized in Table 1 . The estimated genome sizes of JCP043 and AT141 based on their Illumina assemblies were 6.05 Mb and 14.92 Mb smaller, representing 83% and 77% of their T2T genome sizes, respectively. While the core genome regions were well represented in the Illumina assemblies of JCP043 and AT141, reflected in high BUSCO scores, only 48% and 46% of their accessory genome regions were recovered in their respective Illumina contigs, respectively. Since the Illumina assemblies were highly fragmented (5405 contigs for JCP043 and 5771 contigs for AT141), it was impossible to detect structural variants, including genome duplication and rearrangement, between the two isolates. Compared to the T2T assemblies, the Illumina assemblies of JCP043 and AT141 contained significantly fewer DNA repeats, representing only 6.46% and 4.86% of the genome sizes, respectively, in comparison to 18% and 20% in their respective T2T assemblies. Regarding the predicted genes, both Illumina assemblies turned out to miss ~ 10% of the total genes (1805 genes missing from JCP043 and 1972 missing from AT141), with 83% and 74% of them associated with ACs of JCP043 and AT141, respectively. Many of the missing genes were partially present due to the fact that they were located on the end of a contig. Illumina assemblies were able to capture the SIX genes and all but four mimp effectors, however, they fell short in identifying additional copies of SIX8 and SIX9.4 , as observed in all FOLac Illumina assemblies ( Supplementary Table S4 ). When searching for putative race 1-specific genes in the Illumina assembly of JCP043, 171 were not detected, 150 of which were associated with ACs. The Illumina assembly of AT141 had 99 of the race 4-specific genes missing, with 88 of them located on ACs. On the other hand, the NECAT assemblies of JCP043 and AT141, which were obtained by the NECAT assembler and error corrected using Illumina reads (without checking for misassembly and contig end extension), resulted in genome sizes very close to their corresponding T2T assemblies, as also observed for BUSCO scores (Table 1 ). The NECAT assemblies represented over 97% of both the core and accessory regions of each genome, with most of the NECAT contigs aligned with the T2T chromosomes ( Supplementary Figure S6 ). However, the NECAT assemblies were less contiguous and did not have telomeres on many of the contigs, and produced a much smaller number of rDNA repeats (25 copies for JCP043 and 10 copies for AT141). The large-scale chromosome duplications identified from the T2T assemblies (i.e. the 1-Mb duplicated regions in JCP043 and the 500-kb inverted repeats in AT141) were not captured in the NECAT assemblies ( Supplementary Figure S6 ). The NECAT assemblies were highly similar to the T2T assemblies regarding the amount and composition of repetitive elements, as well as predicted genes, with only ~ 100 genes absent. Not surprisingly, most of those missing genes were located within the large, duplicated regions. For instance, 92 out of 107 missing genes from the AT141 NECAT assembly were located on the 5’end of chromosome 17, which failed to be assembled in the NECAT assembly. The NECAT assemblies of both isolates possessed all the mimp effectors and rendered the same sequences and copy numbers for the SIX genes as the T2T assemblies. The NECAT assemblies possessed most of the race-specific genes, with only eight (for JCP043) and 23 genes (for AT141) missing compared to their respective T2T assemblies. Discussion This study reports a complete (gapless and T2T complete) chromosome-level assembly for two key, very closely related F. oxysporum pathogens, which due to the highly repetitive nature of their extensive accessory genome regions, have heretofore evaded complete assembly. This result permitted comparative genomic analyses of the two T2T assemblies and revealed novel structural and genetic differences between the two races, which laid the groundwork to identify putative pathogenicity genes that may be associated with the host-pathogen interaction in the FOLac-lettuce pathosystem. More broadly, this study provides a workflow to generate T2T assemblies that can significantly improve our ability to investigate genome dynamics and organism adaptation, characterize genes that may be associated with pathogenicity, and identify race-specific sequences useful for diagnostics. A novel workflow for generating gapless, T2T assemblies and things to consider In this study, we developed a novel workflow that deployed a suite of bioinformatic programs with various types of long and short sequencing technologies (ONT, Pacbio HiFi, Illumina, and Hi-C) to build F. oxysporum genome assemblies that are gapless and T2T complete. In summary, we considered the following three features as key players in achieving this goal. The first key feature relates to the abundance of long ONT reads (≥ 50 kb). These long reads are deemed crucial for T2T genome assembly because of their ability to resolve large repetitive regions in F. oxysporum accessory genome regions, some of which can reach as long as 20–30 kb in size. In our study, ONT reads that were above 50 kb provided 204× coverage for JCP043 and 176× coverage for AT141 (Table 1 ). To the best of our knowledge, such high long-read coverage has not been reported in any other F. oxysporum genome studies. The second key feature deals with misassembly, which is a major challenge in the assembly of highly repetitive accessory genome regions. Some of the misassembled regions failed to be detected by command-line programs because decent read coverage can be found at the misassembled regions but no reads entirely span them ( Supplementary Figure S1 ). Our solution to this problem was to map long reads (≥ 50 kb) to the NECAT assembly using programs like CLC Genomics Workbench (QIAGEN, Aarhus, Denmark; https://digitalinsights.qiagen.com ) and SeqMan NGen (DNASTAR, Madison, WI, USA), which enables visual scanning of the read mapping to identify misassembled regions. The third key feature involves the practice of read mapping-guided, iterative end extension on the intermediate contigs (see Methods for details). As far as we are aware of, there is no stand-alone bioinformatic software available to extend contigs using long reads beyond scaffolding and gap-filling capabilities. This is reflected in some of near complete F. oxysporum genomes (i.e. Fo47, Fo5176, and Fol4287) that have been published [ 48 – 50 ], none of which has achieved the level of genome completeness as the two FOLac assemblies presented here. An add-on value of using graphical read mapping for end extension is the ability to visually identify large-scale chromosome duplications due to read depth appearing doubled in the read mapping graphics, as observed in chromosomes 5 and 10 in JCP043 and chromosome 17 in AT141. These large, duplicated regions were not captured by the NECAT program ( Supplementary Figure S6 ) or other long-read assemblers, including Flye [ 51 ] and Canu [ 52 ] that we tested. We are currently working on automating and fine-tuning the assembly pipeline, hoping to significantly reduce the amount of time needed to generate T2T assemblies. Finally, the use of PacBio HiFi reads in conjunction with short read data for base-level error correction of the assembly is essential. The lack of Illumina read coverage in many regions of the T2T assembly ( Supplementary Table S1 ) leaves sequence errors in those gapped regions unchecked. One contributing factor may be read depletion of highly repetitive regions early in the read mapping process. In contrast to short reads, HiFi reads exhibited uniformly high read coverage (> 100× coverage with reads ≥ 15 kb) across the entire genome ( Supplementary Figures S2 ), thus allowing for gap-free error correction. Besides the heterogeneity in genome coverage, variability in Illumina read coverage was also found between different samples. A significantly higher proportion of the AT141 genome, including non-repetitive regions, was not covered by its own Illumina reads compared to that in JCP043, even though the total number of Illumina reads for AT141 was 30% greater than that for JCP043. These observations emphasize the importance of using read mapping to visually evaluate the uniformity of sequencing data used in genome polishing. T2T assemblies set a foundation for studying genome evolution in F. oxysporum The T2T assemblies reported here revealed some novel structural differences in the accessory genome regions between FOLac race 1 and race 4, providing new insight regarding genome changes that may underlie the evolution of pathogenicity. The most distinct structural difference between them was that race 4 isolate AT141 possesses a genome 3.86 Mb larger than that of race 1 isolate JCP043 and organizes differently into 19 rather than 16 chromosomes (Table 1 ). Part of the larger genome size of AT141 was related to its inflated estimated copy number of rDNA repeats, which was 144 copies vs. 90 copies in JCP043. F. oxysporum f. sp. radicis-cucumerinum isolate Forc016 was estimated to have 98 rDNA copies [ 53 ], which was similar to that of JCP043. It is widely accepted that rDNA loci are dynamic and copy number fluctuates widely between individual within a species, and even likely between cells within a single organism [ 54 ]. Further analysis using quantitative real-time PCR is needed to determine the actual copy number of rDNA for the two isolates. Although similar difference in genome size between the two FOLac races was reported previously [ 43 ], the exact chromosome number difference between the two races was not resolved from those contig-level genomes. This has been the constraint in many comparative genomic analyses for members within F. oxysporum. An expended survey of additional FOLac races 1 and 4 isolates that have T2T assemblies available will help determine whether the genome size and number of ACs are fixed within a race or not. Secondly, the T2T assemblies showed that repetitive elements made up 18–20% of the two FOLac genomes, which were substantially higher than those reported for other long-read assemblies, such as 10.54% for F. oxysporum f. sp. cepae [ 45 ], 15.4% for F. oxysporum f. sp. conglutinans and 16.42% for F. oxysporum f. sp. lycopersici [ 50 ]. Given that TEs play diverse roles in gene regulation, recombination, and adaptation to changing environment [ 55 ], the ability to identify an expanded pool of TEs from T2T assemblies serves as a valuable tool to understand how TEs contribute to pathogen diversification within F. oxysporum . Consistent with other studies [ 50 , 56 ], common TEs, including DNA transposons and retroelements, were widely distributed among the accessory genome regions, with the exception of Gypsy/DIRS1 elements, of which 75% were found in the core genome regions. The greater abundance of Gypsy/DIRS1 elements in the core genome regions of F. oxysporum has also been reported in an opportunistic human pathogen, with nearly 90% of Gypsy/DIRS1 elements found in the core genome [ 56 ]. While most of the repetitive elements were similar in copy number and length between the two isolates, a markedly larger number of unknown repeats were associated with the race 4 genome ( Supplementary Table S2 ), contributing to 44.8% (1.76 Mb in size) of the overall genome size difference between the two isolates. In particular, the accessory region on chromosome 8 in AT141 showed a very high level of unknown repeats (Fig. 3 ) that was consistently found in the three published FOLac race 4 genomes ( Supplementary Figure S5 ), suggesting that this putative proliferation may be a feature of FOLac race 4. Thirdly, the T2T assemblies offered a unique opportunity to explore how large-scale chromosome duplications and rearrangements vary between the two FOLac races with the focus to identify genome regions that are race specific. Similar to the observations reported previously [ 43 ], the syntenic analysis between race 1 isolate JCP043 and race 4 isolate AT141 demonstrated highly conserved CCs but extremely fragmented and rearranged shared sequences among ACs (Figs. 2 C and 3 B), suggesting that each race underwent very different rearrangements resulting in loss of synteny. None of the ACs appeared to be exclusively associated with either of the two isolates, including chromosome 15 of AT141, which turned out to have many small non-repetitive sequences (2–6 kb in size) shared with JCP043. Our findings pointed to a previously unreported correlation between Gypsy/DIRS1 elements and chromosomal translocation (Fig. 3 A), which appeared to be a unique event in JCP043. In addition to their potential involvement in chromosomal translocation, Gypsy/DIRS1 elements were found to be abundant near the boundaries of large, duplicated regions in JCP043 (a 1-Mb repeat duplicated between chromosomes 5 and 10) and AT141 (a 500-kb inverted repeat within chromosome 17) (Fig. 5 ), suggesting their additional role in large-scale chromosome duplications. The connection between Gypsy/DIRS1 elements and chromosome rearrangements was recently reported in two stingless bee species, Melipona quadrifasciata and M. scutellaris [ 57 ], however, it remains an open question whether there is a causative relation between Gypsy/DIRS1 elements and changes in genome architecture. A broader survey involving in-depth characterization of TEs among diverse F. oxysporum genomes representing different evolutionary lineages and ff. spp. will help better understand their role in F. oxysporum genome evolution. In addition to TEs, the enrichment of certain types of histone modifications or giant Starship elements, as described in F. graminearum [ 58 ] and Macrophomna phaseolina [ 59 ], respectively, may also contribute to the extensive genetic and large-scale genome variation in F. oxysporum . Plasticity of SIX genes in FOLac race 4 and their putative origin With a wide collection of FOLac genomes representing all four races, the analysis of SIX genes showed the universal presence of SIX9.4 and SIX14 among all four races of FOLac. FOLac race 1 exhibits a unique SIX gene complement that distinguishes it from the three other races ( Supplementary Table S4 ). Race 1 isolates do not have SIX8 and three SIX9 variants ( SIX9.1 , SIX9.2 , and SIX9.3 ) while they are all present in races 2–4. However, in a recent study [ 42 ], PCR analysis of SIX8 , SIX9 , and SIX14 in FOLac race 4 and race4 + isolates showed the absence of SIX8 in many isolates, suggesting the instability of SIX8 in the race 4 population. Besides, the race 4 isolates also showed high levels of intra-race variation in the three SIX9 variants ( SIX9.1 , SIX9.2 , and SIX9.3 ), with AP114 (originated from Denmark) harboring three of the variants while AL127 (originated from Ireland) having only SIX9.2. In addition, we found that AT141 was the only race 4 isolate that had two copies of SIX8 , which was likely achieved through large-scale chromosome duplication (Fig. 5 B) instead of a single gene duplication mediated by TEs. In contrast, we noticed stable presence of the SIX genes among all the race 1 isolates, with no sequence variation among isolates. Given the high level of plasticity in SIX8 and SIX9 within the race 4 population, which is likely driven by TEs that are present in close proximity to the SIX genes (Fig. 6 ), one should be cautious about using only SIX genes for discerning FOLac races as it may lead to erroneous results. Interestingly, sequences of the three SIX genes present in FOLac were highly similar to those in ff. spp. that were closely related to FOLac race 3 (Geiser, unpublished, Supplementary File S8 ). Moreover, the more ancestral FOLac race 2 lineage possessed all three SIX genes, including the four variants of SIX9. Given this information, we hypothesized either that these SIX genes may have existed in F. oxysporum before lineage diversification or that they have been transferred among different lineages of F. oxysporum after lineage diversification. Future study of the SIX gene complement with more extensive sampling of F. oxysporum genomes spanning all 17 lineages as delineated by Geiser (unpublished, Supplementary File S8 ) will shed light on the evolution of the SIX gene family. Identification of putative race-specific genes provides a framework for functional characterization of candidate virulence/host-range genes The race-specific genes that were identified in this study were validated against not only FOLac race 1 and 4 genomes, but also two representative isolates from FOLac races 2 and 3, which enabled more reliable identification of race-specific genes compared to previous studies. However, due to the lack of long-read assemblies for the race 2 and 3 isolates when this study was conducted, additional validation of race-specific genes against these two isolates was achieved through read mapping analysis to ensure accuracy, similar to the approach used to confirm SIX9.2 was absent in AM163 ( Supplementary Figure S9 ). Our results identified 689 putative race 1- and 536 putative race 4-specific genes, some of which encode putative secreted CAZymes, effectors (including mimp -associated effectors), and proteins involved in SM biosynthesis (Table 1 and Supplementary Table S5 ). Not surprisingly, nearly 87% of the race-specific genes were associated with ACs ( Supplementary Figure S7 B) , with chromosomes 7 and 12 of JCP043 and chromosome 13 of AT141 each harboring over 100 race-specific genes, suggesting that they might be pathogenicity chromosomes. Chromosome 10 of JCP043 and chromosome 5 of AT141, which carried the SIX genes, also possessed many of the race-specific genes, raising the possibility of their roles in host range and virulence. Future studies are warranted for narrowing the candidates for race-specific genes, by screening them against a wide range of F. oxysporum ff. spp. and nonpathogenic isolates recovered from lettuce and other hosts. Also notable is the presence of various types of TEs within and/or in the flanking regions of the candidate race-specific genes. These observations raised the question of whether those genes are expressed in planta , due to potential disruptions in the promoter and coding sequences by transposon insertion, as observed previously in SIX14 in FOLac race 1 isolate AJ520, which was found to be unexpressed during infection due to transposon insertion [ 43 ]. Transcriptome analyses of JCP043 and AT141 during lettuce infection are needed to help identify race-specific genes that are highly expressed in planta and to guide downstream functional characterization of candidate virulence genes to investigate their function in pathogenicity and host specificity. Is T2T genome assembly necessary? While obtaining a gapless, T2T complete assembly would be ideal for genomic and genetic research, we found that NECAT assembly provides an accurate estimate of genome size, content and composition of DNA repeats, and gene content, including correct copy numbers for SIX genes (see Results for details). Moreover, generating NECAT assembly takes much less time than T2T assembly, which requires different types of bioinformatic tools and sequencing data to make it complete. Assembly using the NECAT program requires only one run of assembling ONT reads into contigs, followed by PacBio/Illumina-based error correction. Therefore, having a NECAT assembly can be an adequate solution for researchers interested in projects including pan-genome and basic repetitive sequence analyses. However, studies that aimed for identifying unique genes that may be associated with host specificity or detecting structural variants between individuals cannot be completed adequately without a T2T assembly. Short-read assembly, which has become a routine task for many research programs used in various downstream analyses, presents several additional limitations, including an incomplete set of predicted genes and inaccurate gene copy numbers, especially for those in ACs (see Results for details). Here we also cite cases where genes present in an assembly turned out to be absent based on read mapping ( Supplementary Figure S9 ), and also where sequences thought to be absent turned out to be present. The latter case can be explained by the fact that parts of those genes land on the end of different contigs. Therefore, it is recommended that a combination of BLAST and read mapping analyses be used for accurate gene prediction, if long-read assembly is inaccessible. Conclusions We present an assembly workflow that led to gapless, T2T complete genome assemblies for FOLac races 1 and 4, two devastating soilborne pathogens that have become increasingly prevalent in lettuce production areas worldwide. Comparative genomic analyses between the two isolates revealed major structural differences in the accessory genome regions and the potential involvement of Gypsy/DIRS1 elements in chromosome duplication and translocation. We identified many putative race-specific genes that were uniquely present in one race while absent in three other races and warrant further investigation through transcriptome analyses during lettuce infection to understand their role in pathogenicity and host specificity. A comprehensive genomics study of multiple isolates representing all four races, along with a broad-spectrum phenotyping study to evaluate their host range and virulence, will allow us to reconstruct the evolutionary paths that led to host-specificity of F. oxysporum towards lettuce and subsequent diversification. Ultimately, the information we gained from the genomics research will greatly advance the development of effective management strategies to control Fusarium wilts. Methods Culture growth Fresh fungal mycelia used for long-read sequencing were prepared as follows. FOLac race 1 isolate JCP043 and race 4 isolate AT141 were started on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) under dark incubation for seven days at 25°C. Six agar plugs from colony edges were transferred to two Petri plates containing 30 ml of potato dextrose broth (PDB; Difco Laboratories) and incubated unagitated for two days at 25°C. The resulting mother culture was blended with a sterile Waring laboratory blender (Conair LLC, Stamford, CT, USA) for 10 sec to make a slurry, which was then mixed with 1.2 L of PDB and distributed to 60 Petri plates for large-scale liquid culture. The plates were incubated unagitated for two days at 25°C. The resulting mycelia were harvested by centrifugation at 8,000 g for 15 min, washed twice with sterile distilled water, blotted dry, and flash frozen in liquid nitrogen before stored at -80°C. High molecular weight (HMW) DNA extraction Extraction of HMW DNA performed at the Michigan State University RTSF Genomics Core used a protocol adapted from the QIAGEN Genomic-tip protocol (QIAGEN, Germantown, MD, USA) while HMW DNA at the UC Davis DNA Technologies Core used a cetyl trimethyl ammonium bromide (CTAB)-based extraction protocol [ 60 ]. DNA quantity and purity were determined with the Qubit 4.0 fluorometer (Thermo Fisher Scientific) and the Nanodrop UV-Vis 45 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA), respectively. Integrity assessment of the genomic DNA was performed using a Femto Pulse system (Agilent Technologies, Santa Clara, CA, USA) and the Agilent 4200 TapeStation (Agilent Technologies). Short DNA fragments (≤ 25 kb) for the PacBio Revio library were eliminated using Blue Pippin (Sage Sciences, 50 Beverly, MA, USA). DNA samples with Nanodrop ratios 260/280 between 1.8-2.0, 260/230 between 2.0-2.2 and molecular weight ≥ 50 kb were selected for sequencing. Long-read whole genome sequencing Long-read genome sequencing was carried out using HMW DNA on ONT PromethION and Pacific Biosciences HiFi platforms. The ONT sequencing was performed separately by two sequencing facilities, Michigan State University (for JCP043) and UC Davis (for AT141). Library for ONT sequencing was made using the Oxford Nanopore SQK-LSK114 Ligation Sequencing Kit V14 and run on a PromethION R10.4.1 flow cell. Sequencing was performed following manufacturer's recommendations. MinKNOW software v.22.10.7 was used for data acquisition and base calling was achieved using Guppy v.6.3.9. Total yields for JCP043 and AT141 were 122Gb (7 million reads; read length N50 of 32 kb) and 149Gb (11 million reads; read length N50 of 21 kb), respectively. Raw reads were filtered with a Q score ≥ 9.0 and a minimum length of 10k, resulting in a total of 5.45 million reads for JCP043 and 9.45 million reads for AT141. HiFi reads were generated by the UC Davis DNA Technologies Core, with sequencing of JCP043 performed on the Sequel II system while AT141 on the Revio system. Total yields for JCP043 and AT141 were 12.5 Gb (1.1 M reads; read length N50 of 12 kb) and 62.7 Gb (5.9 M reads; read length N50 of 13 kb), respectively. T2T assembly As summarized in Fig. 1 , we developed a bioinformatic workflow that implemented a series of command-line and graphic user interface programs to assemble the two FOLac genomes, which is described in greater details in Supplementary File S1 . Briefly, the Nanopore data assembler, NECAT version 0.0.1_update20200803 [ 61 ] was used to assemble ONT reads into a preliminary genome assembly, which was then visually inspected for mis-assembly by mapping 50–99 kb ONT reads to the contigs using CLC Genomics Workbench 22.0 (QIAGEN, Aarhus, Denmark; https://digitalinsights.qiagen.com/ ) and SeqMan NGen 16.0 (DNASTAR, Madison, WI, USA). Each program uses a different approach for read mapping that complements each other. CLC Genomics uses a percent identity and fraction overlap approach while SeqMan NGen uses a k- mer approach. The mis-assembled contigs were split into sub-contigs at the breakpoints before subject to a second run of read mapping, followed by end extension using the “Extend contig ends” function of the Genome Finishing Module on CLC Genomics. Other contigs, which did not have misassembled regions but had no telomeric repeats on either or both ends, were also included in the end extension analysis. After this procedure was repeated multiple times, the extended contigs and sub-contigs were merged when there was a minimum of 50-kb overlap using the nucmer module of the MUMmer package version 3.23 [ 62 ]. Once the T2T assembly was obtained, the continuity and correctness of the assembly was checked by re-mapping 50–99 kb ONT reads to the assembly using CLC Genomics, followed by repeating the read mapping using SeqMan NGen with a stringency setting of 95% and k -mer size of 30. The final T2T assembly was polished with two runs of HiFi reads mapping, followed by two runs of Illumina HiSeq reads mapping using SeqMan NGen with a stringency of 99% and k -mer size of 30. The NECAT contigs that contained the mt sequences were removed from the assembly, BLASTed against a reference mt genome of Fo47 (GenBank: LT906306.1) to identify a complete single-copy genome, and error corrected using Illumina data. Hi-C analysis . To verify that the T2T assembly of JCP043 is correct with no mis-assembled contigs, a proximity ligation-based method called, Hi-C analysis [ 63 ], was performed via Dovetail Omni-C library construction and sequencing on an Illumina HiSeqX platform by Cantata Bio. BWA-MEM version 0.7.17 [ 64 ] was used to align the Omni-C reads to the T2T assembly of JCP043, followed by Hi-C analysis using the SALSA2 pipeline [ 65 ]. The resulting alignment file was converted to create a .hic file and then visualized with JuiceBox [ 66 ]. Genome assessment QUAST version 5.3.0 [ 67 ] was used to assess the quality of the T2T assembly and to evaluate the genome completeness of the Illumina and NECAT assemblies with flags --eukaryote --fungus to specify a fungal organism, and --features set to gene to analyze gene content. Gene content of the assembly was determined based on the presence of BUSCOs using BUSCO version 5.4.5 [ 68 ], with the hypocreales_odb10 database. Analysis of the repeat content of the genome We identified and classified repetitive and low complexity regions in the genome using RepeatModeler version 2.0.4 ( http://www.repeatmasker.org ). Then, the repeat library was used to analyze repetitive regions, as well as soft-masking the genome using RepeatMasker version 4.1.5 ( https://www.repeatmasker.org/ ). Whole-genome comparison to identify core and accessory chromosomes Core and accessory genome regions and chromosomes of JCP043 and AT141 were identified based on whole-genome alignment with the Fol4287 reference genome (GCA_000149955.2) using the nucmer module of the MUMmer package with the parameter –max-match, -L 10000. The synteny plot was generated using TBtools-II v. 2.311 [ 69 ]. Gene prediction and functional annotation After repeat masking, we annotated the genome using Funannotate version 1.8.16 [ 70 ]. The masked genome file along with the RNA-seq data generated from mycelia of JCP043 grown under nine different environmental conditions (see below) were used as inputs to Funannotate to train the gene prediction models, followed by funannotate predict and funannotate update commands to annotate untranslated regions (UTRs) and refine gene model predictions. InterProScan5 version 5.64-96.0 [ 71 ] was used to assign functional annotation to predicted genes. Diamond blastp [ 72 ] was used to search UniProt DB v. 2023_04 [ 73 ] and MERPOP v. 12.0 [ 74 ] databases to aid in functional annotation and eggNOG terms were identified using eggNOG-mapper v. 2.1.12 [ 75 ]. Pfam domains were identified using PFAM v. 36.0 [ 76 ], and CAZymes were annotated using dbCAN v.12.0 [ 77 ]. Putative secreted proteins (secretomes) were identified through prediction of signal peptides using SignalP v.4.1 [ 78 ] and removing those predicted to contain transmembrane domains using the DeepTMHMM v.1.0 web server [ 79 ]. The resulting secretomes were used to predict effector proteins using EffectorP 3.0 [ 80 ]. Secondary metabolite gene clusters were identified using the antiSMASH fungal v. 8.0 web server [ 81 ] with the detection strictness set to relaxed. Prior to the antiSMASH analysis, the genome annotation file was filtered using AGAT v.1.4.3 [ 82 ] to retain only the longest isoform per gene. Genomic features, including gene density, distribution of TEs, and genome duplication events were visualized using TBtools-II v. 2.311. To conduct functional enrichment analysis, we used GOATOOLS v1.5.1 [ 83 ]. GO data v.1.2 (release date: 2025-07-22) was obtained from the Gene Ontology consortia [ 84 , 85 ].To avoid false positives, p-values were multi-test corrected using the Bonferroni method; the resulting adjusted p-values were subject to a significance threshold of 0.01. Identification of putative race-specific genes To identify putative race-specific genes for FOLac races 1 and 4, we identified unique genes in JCP043 and AT141 first. CDS transcripts of the predicted genes were clustered to create a non-redundant gene set using the CD-HIT program version 4.8.1 [ 86 ]. Based on the criteria that were previously applied in similar analyses [ 45 ], the analysis was carried out with minimum 90% identity and minimum 80% coverage as the thresholds for clustering genes (cd-hit -c 0.9 -s 0.8). Pairs of homologous genes between the two genomes were then identified from the non-redundant gene sets using the RBBH module of the MMseq2 software version 18.8cc5c [ 87 ] with the parameters mmseqs easy-rbh --search-type 3 --min-seq-id 0.90 -c 0.8 --cov-mode 0. The synteny of the homologous genes in the core and accessory chromosomes was visualized using TBtools-II v. 2.311. The resulting non-homologous genes in JCP043 were extracted and BLASTed against the AT141 assembly using Geneious Prime version 24.0.7 (໿ http://www.geneious.com/ ). The genes that were either absent in AT141 or partially present (below 90% identity or 80% coverage) were considered unique genes in JCP043. To validate their uniqueness to JCP043, raw Illumina reads of AT141 (SRR28734917) were aligned to the unique genes using SeqMan NGen with a stringency of 90% sequence identity and k -mer size of 21. Read coverage of the alignments was visually inspected with SeqMan Pro. Similarly, the unique genes in AT141 were identified following the same procedure and validated with read mapping analysis using raw Illumina reads of JCP043 (SRR28734937). To further narrow down the unique genes that may be race-specific, the presence/absence analysis of the unique genes, including mimp -associated effectors (see below), for FOLac race 1 was carried out on four representative FOLac race 1 isolates (AJ520, AJ718, AJ865, and AT142), one isolate each for race 2 (F9501) and race 3 (FLK1001), and three FOLac race 4 isolates (AJ516, AJ592, AJ705), most of which had long-read assemblies. Details of the isolates can be found in Supplementary Table S4 . BLAST search was conducted by querying the unique genes to each genome assembly via Megablast (Max E-value = 1e-20, Match Mismatch = 1, -2, Gap Cost = linear) using Geneious Prime. A hit was considered present if the sequence identity and coverage were above 95%. The unique genes were also evaluated by read mapping analysis using SeqMan NGen for additional validation. Identification of mimp -associated effector proteins To identify putative effectors associated with mimps for each FOLac genome, we employed the FoEC2 pipeline [ 47 ], which was designed specifically for identification of mimp effectors, with the parameters -g and -a . Sequences of the resulting candidate effector protein set between 30 aa and 300 aa were then extracted from each genome and clustered using CD-HIT version 4.8.1 to create a non-redundant candidate effector set. To determine the presence/absence of JCP043 candidate effectors in AT141, initially, TBLASTN search using protein sequences of the candidate mimp effectors against the genome was performed, however the results seemed inaccurate due to introns that resulted in fragmented or incomplete hits. Instead, genomic DNA sequences of the non-redundant candidate mimp effectors of JCP043 were used to query against the genome of AT141 using Megablast on Geneious Prime, with an e-value cut-off of 1e − 20 and a percentage identity and coverage threshold of 90% and 80%, respectively. To identify candidate mimp effectors that are race 1 specific, we included nine additional FOLac isolates (the same set used in race-specific gene analysis as described above) in this analysis. Similarly, the presence/absence of AT141 candidate effectors in JCP043 and putative race 4-specific mimp effectors were identified following the same procedure. In silico assessment of SIX genes To identify the SIX genes present in the F. oxysporum isolates used in this study ( Supplementary Table S4 ), BLAST search was conducted by querying the SIX genes obtained from NCBI ( SIX1 : MK906592.1; SIX2 : MK906595.1; SIX3 : MK906598.1; SIX4 :GQ268951.1; SIX5 : MK906607.1; SIX6 : MK906615.1; SIX7 :GQ268954.1; SIX8 : FJ755837.1; SIX9 : KC701447.1; SIX10 : MK906667.1; SIX11 : MK906677.1; SIX12 : MW160867.1; SIX13 : MK906693.1; SIX14 : KC701452.1) to each genome assembly via Discontiguous Megablast (Max E-value = 0.05, Match Mismatch = 2, -3, Gap Cost = 5, 2) using Geneious Prime. As for SIX9 , sequences corresponding to the four variants of SIX9 ( SIX9.1- SIX9.4 ), which were reported in FOLac race 4 [ 43 ], were used as additional references to account for sequence diversity. A hit is valid if the query identity and coverage meet the 65% threshold. If the hit was similar to the reference (≥ 65% identity) but was low in coverage, Illumina reads of the target isolate were mapped to the corresponding SIX reference to retrieve full-length sequence using SeqMan NGen with stringency cutoffs of 90% sequence identity and k -mer size of 30. Sequences of the SIX genes were aligned using MUSCLE 5.1 [ 88 ] and then used as input for IQ-TREE v2.2.2.6 [ 89 ] with parameters ' -m MFP -B 1000 ' to create a phylogeny. The resulting maximum likelihood tree was visualized in FigTree, version 1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). RNA extraction and sequencing For F. oxysporum genome annotation, RNA-seq data obtained from PDB-cultured mycelia has been commonly used as the standard to train the gene prediction models. Considering that environmental factors (i.e. pH, nutrient composition, temperature and light) have significant impact on fungal gene expression [ 90 , 91 ], we generated nine RNA-seq libraries from race 1 isolate JCP043 that was grown under different growth conditions (see below) to expand the expression of a wide variety of genes. Six agar plugs of five-day old JCP043 culture were inoculated in 30 ml PDB at 25°C for two days. The mycelia were washed twice with sterile water before blended with water. Ten milliliters of the mycelial slurry were transferred to each bottle containing 100 ml of a specific type of media listed below, mixed well, and then distributed to four petri plates to grow for 48 hours at 25°C in the darkness. The media included PDB (pH of 6), PDB with 4% NaCl, PDB adjusted to a pH of 9 with NaOH, PDB amended with 0.5% and 1% peptone, respectively, carboxymethyl cellulose (CMC), and filter-sterilized macerated lettuce crown extract, prepared according to the method described previously [ 92 ]. Additional growth conditions include mycelia grown in PDB for 20 h at 25°C before switching to 37°C for 4 h, and mycelia grown in PDB under 24-h light. Mycelia were collected from the nine growth conditions by removing liquid from the tissue using filtration, washing the tissue with sterile water twice, and blotting the tissue dry. The tissue mat was then partitioned into 2 ml Eppendorf tubes, each containing approximately 100 mg of wet tissue, and immediately flash frozen in liquid nitrogen. The frozen tissue was stored at -80C until the RNA was extracted. RNA was extracted using TRIzol reagent (Life Technology, Karlsruhe, Germany) and purified with a Zymo RNA clean and Concentrate kit (Zymo, Irvine, CA, USA), according to the manufacturer’s instructions. The quality and quantity of purified RNA were determined using the NanoDrop spectrophotometer and Agilent 4200 TapeStation. All the RNA samples, which yielded RIN scores above 9, were used for sequencing. Libraries were prepared at the Michigan State University RTSF Genomics Core using the Watchmaker Genomics mRNA Library Preparation kit (Watchmaker Genomics, Boulder, CO) with IDT xGEN 10nt Unique Dual-Index primers (Integrated DNA Technologies, Coralville, IA) following manufacturer’s recommendations. Completed libraries were assessed for quality and quantified using a combination of Biotium AccuGreen High Sensitivity dsDNA (Biotium, Frement, CA, USA) and Agilent 4200 TapeStation. All libraries were barcoded, normalized, and pooled equimolarly for sequencing in an AVITI Cloudbreak Freestyle High Output flow cell in a 2 × 150 bp paired end format. Base calling was done by AVITIOS v3.2.0 and the output was demultiplexed and converted to FastQ format using Element Biosciences bases2fastq v2.1.0. Approximately 55–70 million high-quality (% ≥ Q30 reads was ∼90%) paired-end reads were obtained for each library with a total yield of 183.5 Gb of sequencing data. Abbreviations CC Core chromosome AC Accessory chromosome FOLac F. oxysporum f. sp. lactucae T2T Telomere-to-telomere SIX Secreted in Xylem ff. spp. formae speciales f. sp. forma specialis TE Transposable element ONT Oxford Nanopore Technologies mt mitochondrial rDNA Ribosomal deoxyribonucleic acid SNP Single nucleotide polymorphism BUSCO Benchmarking universal single-copy ortholog BLAST Basic local alignment search tool LTR Long terminal repeat LINE Long interspersed nuclear element CAZYme Carbohydrate-active enzyme SM Secondary metabolite GO Gene ontology mimp Miniature impala GH Glycoside hydrolase PDA Potato dextrose agar PDB Potato dextrose broth HMW High molecular weight CTAB Cetyl trimethyl ammonium bromide Hi-C High-throughput chromosome conformation capture UTR Untranslated region CDS Coding sequence RNA Ribonucleic acid CMC carboxymethyl cellulose aa Amino acid Declarations Acknowledgements We gratefully acknowledge Mathieu Pel for supplying FOLac race 4 isolate AT141 and other international isolates of races 1and 4. We also thank the DNA Technologies Core at the UC Davis Genome Center and the Michigan State University RTSF Genomics Core for their assistance in long-read and Illumina sequencing. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. The USDA is an equal opportunity provider and employer. Authors' contributions NL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. JLS: Formal analysis, Investigation, Methodology, Software, Writing – review & editing. SOD: Formal analysis, Methodology, Writing – review & editing. EGT: Investigation, Writing – review & editing. SMK: Data curation, Formal analysis. DMG: Conceptualization, Funding acquisition, Project administration, Supervision, Writing review & editing. FNM: Conceptualization, Funding acquisition, Project administration, Supervision, Investigation, Formal analysis, Writing – review & editing. Funding This research was funded by USDA ARS Project 2038-22000-016-00D, California Department of Food and Agriculture, Specialty Crop Block Grant Program (18-0001-059-SC, 21-0001-050-SF), National Science Foundation (DEB-1655980), and Pennsylvania State Agricultural Experiment Station Project (Project number: 4655). JLS is a Howard Hughes Medical Institute Awardee of the Life Sciences Research Foundation. EGT and SO are supported by the Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison with funding from the Wisconsin Alumni Research Foundation and the Department of Plant Pathology at the University of Wisconsin-Madison. Availability of data and materials The datasets generated for this study can be found in the article and Supplementary Material. All raw sequencing data have been submitted to the NCBI under the BioProject ID RJNA1098703 with the accession numbers of SRR35856514- SRR35856527. The final assembled genomes are deposited under the same BioProject at NCBI. The assembled genomes and genome annotation files have been submitted to the online open access repository Figshare for peer-review only (https://figshare.com/s/60447a577ab459285236). Further inquiries can be directed to the corresponding author. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests JLS is an advisor to ForensisGroup Inc. JLS is a scientific consultant to FutureHouse Inc. References Gordon TR, Martyn RD. The evolutionary biology of Fusarium oxysporum . Annu Rev Phytopathol. 1997;35:111–28. https://doi.org/10.1146/annurev.phyto.35.1.111 . Armstrong GM, Armstrong JK. Formae speciales and races of Fusarium oxysporum causing wilt diseases. In: Nelson PE, Toussoun TA, Cook RJ, editors. 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10:27:48","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":302814,"visible":true,"origin":"","legend":"","description":"","filename":"f5e91a22cb8141a99f619f4df9a53ed21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/6b74d52300bfafe7108b704e.xml"},{"id":96918339,"identity":"8b04834a-11af-483b-9c51-a89f4e871f43","added_by":"auto","created_at":"2025-11-27 14:11:46","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":332471,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/dc78f21b5692f19632a69ce9.html"},{"id":96812766,"identity":"670b116b-6c78-4677-a096-ebd709cff275","added_by":"auto","created_at":"2025-11-26 10:27:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1891555,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart depicting the assembly workflow to generate gapless, T2T complete genome assemblies for FOLac\u003cem\u003e \u003c/em\u003eisolates JCP043 and AT141. Details of the workflow are delineated in Methods and Supplementary File S1. * Hi-C analysis was performed on JCP043 only.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/fc49909c9e83dc36bc201689.png"},{"id":96812775,"identity":"a812847e-1a0f-4cd3-9d10-edb226d8b1f3","added_by":"auto","created_at":"2025-11-26 10:27:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15154903,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny of chromosomes between FOLac race 1 JCP043, race 4 AT14 and \u003cem\u003eF. oxysporum \u003c/em\u003ef. sp. \u003cem\u003elycopersici \u003c/em\u003eFol4287. \u003cstrong\u003eA\u003c/strong\u003eCircos plot of JCP043 vs. Fol4287. \u003cstrong\u003eB\u003c/strong\u003e Circos plot of AT141 vs. Fol4287. \u003cstrong\u003eC\u003c/strong\u003eCircos plot of JCP043 vs. AT141. Order of tracks from outward to inward (a-d): a: Cyan and purple boxes represent core and accessory chromosomes/genome regions, respectively; b: The abundance of annotated genes in each 100 kb window. Black tic markers occur every 200 kb on the outside of the ideogram; c: The proportion of each 100 kb window covered by transposable elements; d: Lines that connect the chromosomes between the two genomes represent similar regions greater than 10 kb in size.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/9f25b63fffd6ab6fe1e2260c.png"},{"id":96812786,"identity":"3b4491a1-a154-43bf-92f8-c9ffc8fe5ca1","added_by":"auto","created_at":"2025-11-26 10:27:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4931335,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of different classes of transposable elements in the core and accessory chromosomes of JCP043 and AT141. \u003cstrong\u003eA\u003c/strong\u003e Core chromosomes of JCP043 and AT141. The breakpoint on chromosome 11 of AT141 and the two fusion points on chromosomes 2 and 14 of JCP043, which are highlighted in black arrows, overlap with \u003cem\u003eGypsy/DIRS1\u003c/em\u003eelements (highlight in blue). \u003cstrong\u003eB\u003c/strong\u003e Accessory chromosomes of JCP043 and AT141. Lines in the center represent shared regions (≥ 2 kb in size and ≥ 85% of sequence identity) within the large unique regions that are unshared between the two FOLac isolates. Repeat-masked accessory chromosomes are used in this analysis. Order of tracks from outward to inward (a-i): a: \u003cem\u003eGypsy/DIRS1\u003c/em\u003e, b: unknown repetitive sequence, c: \u003cem\u003ehAT-Restless\u003c/em\u003e, d: \u003cem\u003eTc1/Mar\u003c/em\u003e-\u003cem\u003eFot1\u003c/em\u003e, e: \u003cem\u003eMULE-MuDR\u003c/em\u003e, f: \u003cem\u003ePiggyBa\u003c/em\u003ec, g: \u003cem\u003eHelitron,\u003c/em\u003e h: \u003cem\u003eTy1/Copia\u003c/em\u003e, and i: \u003cem\u003eTad1.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/b180c6aa59348d5c0850e92b.png"},{"id":96916621,"identity":"2c9d4701-037b-4abc-bb48-c02dc5d9f333","added_by":"auto","created_at":"2025-11-27 14:08:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1004836,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic features of JCP043 and AT141. \u003cstrong\u003eA\u003c/strong\u003e Distribution of genomic features in the core vs. accessory genome regions of JCP043 and AT141, including genes that encode secreted CAZYmes (carbohydrate-activate enzymes), effectors, SM (secondary metabolite) genes, transposable elements, and \u003cem\u003emimp-\u003c/em\u003eassociated effectors. \u003cstrong\u003eB\u003c/strong\u003eVenn diagrams depicting shared and unique genes between JCP043 and AT141 genomes. The numbers marked with asterisks are based on the gene set of AT141. As for JCP043, the total shared genes for all, core and accessory genome regions are 19,606, 17,309, and 2,297, respectively, due to the fact that some of the shared genes are in multiple copies in JCP043.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/2bc2dcb79e0ff0fab39c875e.png"},{"id":96916544,"identity":"f69754a6-8f53-4f7b-918e-2c1a333aa2ef","added_by":"auto","created_at":"2025-11-27 14:08:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2627975,"visible":true,"origin":"","legend":"\u003cp\u003eLarge-scale chromosome duplication in JCP043 and AT141 and the distribution of \u003cem\u003eSIX \u003c/em\u003egenes among accessory chromosomes/genome regions. \u003cstrong\u003eA\u003c/strong\u003e Accessory chromosomes/genome regions of JCP043. \u003cstrong\u003eB\u003c/strong\u003e Accessory chromosomes/genome regions of AT141. The accessory regions of each genome were aligned against themselves using the nucmer package (nucmer –maxmatch; -L 20000) to identify highly similar regions. Lines in the center represent homologous regions (≥ 2 kb in size and ≥ 85% of sequence identity). The distribution of \u003cem\u003ehAT-Restless\u003c/em\u003e and \u003cem\u003eGypsy/DIRS1 \u003c/em\u003eis shown on track a and b, respectively. \u003cem\u003eSIX \u003c/em\u003egenes located within the duplicated regions are marked in pink while single-copy \u003cem\u003eSIX \u003c/em\u003egenes are noted in grey.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/608e5e13292a877bd6bf7816.png"},{"id":96917120,"identity":"e01e7e25-d265-4605-bf99-66001ed593de","added_by":"auto","created_at":"2025-11-27 14:09:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4869045,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003eSIX8, SIX9, \u003c/em\u003eand\u003cem\u003e SIX14 \u003c/em\u003eusing 87 \u003cem\u003eF. oxysporum\u003c/em\u003e genomes. \u003cstrong\u003eA, C and E\u003c/strong\u003e Schematic representations of the genomic regions harboring the two identical copies of \u003cem\u003eSIX8\u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e), the truncated version of \u003cem\u003eSIX14 \u003c/em\u003e(\u003cstrong\u003eC\u003c/strong\u003e), and the three variants of \u003cem\u003eSIX9 \u003c/em\u003e(\u003cstrong\u003eE\u003c/strong\u003e) in the context of transposable elements. \u003cstrong\u003eB, D and F\u003c/strong\u003e Maximum likelihood trees of \u003cem\u003eSIX8 \u003c/em\u003e(\u003cstrong\u003eB\u003c/strong\u003e), \u003cem\u003eSIX14 \u003c/em\u003e(\u003cstrong\u003eD\u003c/strong\u003e), and \u003cem\u003eSIX9 \u003c/em\u003e(\u003cstrong\u003eF\u003c/strong\u003e), inferred using IQ-TREE 2 [89]. Each tree is rooted through the \u003cem\u003eF. oxysporum \u003c/em\u003ef. sp. \u003cem\u003elycopersici SIX \u003c/em\u003egene references. The numbers above the branches indicate bootstrap values. Taxon names of FOLac isolates are highlighted in blue.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/b2fd9c3198511abd67518850.png"},{"id":106808759,"identity":"3d0f93cb-e2e1-41ad-8a7e-1182020d7105","added_by":"auto","created_at":"2026-04-13 16:00:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32170148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/34df312f-8d3a-4cc0-b1ec-80e7e951ea74.pdf"},{"id":96812797,"identity":"9335c2e6-1b74-4a11-a0ad-c86a977e3cae","added_by":"auto","created_at":"2025-11-26 10:27:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7692181,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/3b46b594365dbec3729fcd34.docx"},{"id":96918012,"identity":"2c7f5f98-118a-46cd-ae64-829a9fba9ed3","added_by":"auto","created_at":"2025-11-27 14:10:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20117,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/045db684140b8a931cafed7e.docx"},{"id":96918102,"identity":"b672c68c-7b60-45c1-8571-5ed33e7c9d9e","added_by":"auto","created_at":"2025-11-27 14:11:09","extension":"fasta","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7802093,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS2.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/abaf34817f784b28bc72b843.fasta"},{"id":96812767,"identity":"2af1ec0d-bada-4c64-ab9a-6c81fadfd677","added_by":"auto","created_at":"2025-11-26 10:27:47","extension":"fasta","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25604,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS3.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/028dfa06ded60efcda542a11.fasta"},{"id":96917080,"identity":"59004bba-7907-4d9b-934d-2de926485e8b","added_by":"auto","created_at":"2025-11-27 14:09:14","extension":"fasta","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":60957,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS4.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/28bebf599f837976f1cb6349.fasta"},{"id":96917798,"identity":"472ed079-2890-48e5-8117-552186519105","added_by":"auto","created_at":"2025-11-27 14:10:35","extension":"fasta","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":22023,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS5.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/2e82200fd0a4b7cadc69bd24.fasta"},{"id":96812780,"identity":"999de0bb-14fc-46da-a119-9ca2f163248e","added_by":"auto","created_at":"2025-11-26 10:27:48","extension":"fasta","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1216524,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS6.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/802888338a280cc82ac51acf.fasta"},{"id":96917111,"identity":"feca5d8b-04f2-418f-b250-2b4ba4839674","added_by":"auto","created_at":"2025-11-27 14:09:16","extension":"fasta","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":627037,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS7.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8100147/v1/afaeee15df3ef7578fb4f28c.fasta"}],"financialInterests":"Competing interest reported. JLS is an advisor to ForensisGroup Inc. JLS is a scientific consultant to FutureHouse Inc.","formattedTitle":"Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e is a globally distributed species that includes important agents of wilt disease, but also strains that are endophytic/presumably nonpathogenic, and saprophytes. Collectively, plant pathogenic strains of \u003cem\u003eF. oxysporum\u003c/em\u003e have a remarkably broad host range, causing vascular wilts as well as root and crown rots on over one hundred agronomically important plant species. While host diversity is broad at a species level, individual \u003cem\u003eF. oxysporum\u003c/em\u003e pathogens usually display a high degree of host specificity, causing disease in one or a few related plant species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pathogenic strains that infect the same plant host are grouped into the same \u003cem\u003eforma specialis\u003c/em\u003e (f. sp.). More than 150 different \u003cem\u003eformae speciales\u003c/em\u003e (ff. spp.) have been reported, with 106 of them having been well characterized [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Some ff. spp. are further divided into races based on virulence to a set of differential host genotypes and, in some cases, based on known resistance genes in these hosts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Races have been described in 25 ff. spp. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and new races arise frequently. A well-studied example is \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, which had two successive resistance-breaking races emerge within 12 years of resistance deployment, as a result of coevolution between the pathogen and resistant tomato cultivars [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith very few exceptions where a single f. sp. is confined in a monophyletic lineage (such as f. sp. \u003cem\u003eciceris\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]), \u003cem\u003eF. oxysporum\u003c/em\u003e ff. spp. tend to be non-monophyletic [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], consistent with independent evolutionary origins of pathogenicity on a given host and unique evolutionary patterns of different ff. spp. within \u003cem\u003eF. oxysporum\u003c/em\u003e. With the origin of \u003cem\u003eF. oxysporum\u003c/em\u003e being possibly as recent as ~\u0026thinsp;0.5 mya [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], this diversification appears to have occurred very recently. Genetic determinants of host-specificity, including effector genes, are predominantly present in transposon-rich \u0026ldquo;accessory\u0026rdquo; chromosomes (ACs), also known as lineage-specific or pathogenicity chromosomes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eF. oxysporum\u003c/em\u003e has been reported to possess one or several ACs that are highly variable and harbor unique sequences that are absent in other \u003cem\u003eF. oxysporum\u003c/em\u003e strains, except those that share the same host. This is in sharp contrast to core chromosomes (CCs), which are essential and conserved among all strains within \u003cem\u003eF. oxysporum\u003c/em\u003e. The most studied effector genes in \u003cem\u003eF. oxysporum\u003c/em\u003e are the \u003cem\u003eSecreted in Xylem (SIX)\u003c/em\u003e genes, which were first identified in \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e and encode small, cysteine-rich proteins that are secreted into the xylem sap of tomato plants during infection [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Since then, 14 \u003cem\u003eSIX\u003c/em\u003e genes (\u003cem\u003eSIX1-14\u003c/em\u003e) and variability of them among different isolates and races within a single f. sp. have been reported in numerous \u003cem\u003eF. oxysporum\u003c/em\u003e ff. spp. [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with multiple studies illustrating the role of different \u003cem\u003eSIX\u003c/em\u003e genes in conferring virulence and determining host specificity in several pathosystems [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFusarium wilt of lettuce, caused by \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elactucae\u003c/em\u003e (FOLac), has emerged as a major disease in lettuce growing areas, posing a significant threat to lettuce cultivation worldwide. The pathogen FOLac consists of four known races (races 1, 2, 3 and 4) that differ in virulence patterns on resistant or susceptible lettuce cultivars, with distinct geographic distributions. Race 1 is the most widespread and occurs in most countries where lettuce is grown, including Japan [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the United States [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Taiwan [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], Iran [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Brazil [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], Argentina [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Europe [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Races 2 and 3 have very restricted distribution and are found in Japan [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Taiwan [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A new race (race 4), which is highly virulent on some race 1-resistant cultivars, was first detected in the Netherlands (Gilardi et al. 2017), where Fusarium wilt of lettuce was not previously reported. Since then, race 4 has spread rapidly to other parts of Europe, including Belgium [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], the UK [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], Italy [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and Spain [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile FOLac races 2 and 3 are phylogenetically distinct from races 1 and 4 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], all FOLac race 1 and race 4 isolates obtained from a worldwide collection were placed in a single clade based on sequences of 41 full-length, orthologous genes, with the two races being indistinguishable (Geiser, unpublished). This finding suggests recent clonal origin of FOLac race 1 and 4 pathogenicity. Using a \u003cem\u003ek\u003c/em\u003e-mer-based approach that analyzes sequence variation directly from raw reads, FOLac races 1 and 4 can be further divided into two sub-clades, with varying levels of sequence divergence among individual isolates within each race [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is speculated that intra-race genetic diversity within the race 1 and race 4 populations may contribute to the emergence of new variants or races of FOLac. This has been manifested in recent years, where novel pathogenic variants of FOLac race 1 were reported in California, with one variant (VSP-0916) exhibiting high aggressiveness on the race 1-resistant variety Costa Rica No.4, while another variant (Fol621) became less virulent on susceptible cultivars [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Similarly, an emerging FOLac race 4\u0026thinsp;+\u0026thinsp;was detected in several farms in Belgium where FOLac race 4 intermediate resistant cultivars showed wilting and growth reduction one year after commercialization [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo better understand the evolutionary processes responsible for the emergence and diversification of the FOLac pathogen, a reference genome of FOLac that is fully assembled is critical to the research community. Although the disease has been reported for nearly half a century, the first relatively complete genome of FOLac was not released until recently [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Based on comparative genomic analyses between FOLac races 1 and 4, Bates et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] showed that FOLac race 4 has a larger genome than FOLac race 1, with a lack of synteny between their accessory genome regions. Additionally, Bates et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] posited that FOLac race 4 did not evolve directly from FOLac race 1, but both races inherited their accessory genomes from a common ancestor, which then underwent rearrangement/recombination events, accompanied by the acquisition of accessory regions via horizontal chromosome transfer. However, because these published FOLac assemblies are at the contig-level, with fragmented CCs and ACs, it is necessary to reconstruct a complete genome for FOLac races 1 and 4 to provide a more complete insight into the mechanisms underlying genome evolution that led to the diversification and host specialization of the pathogen.\u003c/p\u003e\u003cp\u003eHere, we present gapless, telomere-to-telomere (T2T) complete genome assemblies for FOLac races 1 and 4. In doing so, we developed a workflow for generating T2T assemblies and highlight the importance of visual inspection of read mapping to ensure the accuracy and correctness of the assembly. Comparative genomics between the two T2T assemblies revealed major structural differences in their accessory genome regions that may underlie genetic diversification between FOLac races 1 and 4. The T2T assemblies also enabled the characterization of large-scale chromosome duplication and the recognition of a specific type of transposable elements (TEs) that may be involved. A comprehensive transcriptome dataset of FOLac, which encompassed nine RNA-seq libraries generated under different growth conditions, was used to produce a complete genome annotation for the two T2T assemblies, leading to the identification of candidate genes that may be race-specific. Moreover, this study compared assembly and genomic features between the T2T assembly and two contig-level assemblies for the same isolates, offering researchers a new perspective in assessing their sequencing and assembly strategy.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFOLac race 4 has an expanded genome with three more chromosomes than race 1\u003c/h2\u003e\u003cp\u003eTwo previous reported \u003cem\u003eF. oxysporum\u003c/em\u003e isolates, JCP043 (FOLac race 1, from California [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and AT141 (FOLac race 4, from Spain [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]), were selected for long-read sequencing. The assembly workflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) started with building a preliminary assembly using reads generated by Oxford Nanopore Technologies (ONT). Using default settings, the NECAT program assembled the ONT reads into 27 contigs for JCP043 and 25 contigs for AT141, some of which already had telomeric repeats (CCCTAA/GGGTTA) on one or both ends. The mitochondrial (mt) genome was identified from the assembly and deposited in GenBank. By mapping 50\u0026ndash;99 kb ONT reads to the NECAT contigs, mis-assemblies were visually identified based on a sharp decrease in read depth at the misassembly junction and no reads spanning the incorrectly assembled region (example shown in \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. After the assembly inspection, the NECAT contigs were broken into 44 intermediate contigs for JCP043 and 46 contigs for AT141.\u003c/p\u003e\u003cp\u003eIterative cycles of read mapping with 50\u0026ndash;99 kb ONT reads followed by end extension were carried out on the intermediate contigs. In most cases, ONT reads alone were sufficient to extend the contigs to the telomeric region without taking extra steps (see detailed instructions in Methods and \u003cb\u003eSupplementary File S1\u003c/b\u003e). The extended contigs with an overlap of greater than 50 kb and 95% sequence identity were joined to reconstruct full-length chromosomes. The telomeric end of the rDNA repeat region was completed through aligning 50\u0026ndash;99 kb reads that contained rDNA sequences and flanking sequences. The rDNA copy number was estimated based on the depth of ONT read coverage relative to adjacent regions of the chromosome, which resulted in an estimated 90 copies for JCP043 and 144 copies for AT141. The final T2T assembly was evaluated for accuracy again by mapping 50\u0026ndash;99 kb ONT reads to it, with visual inspection showing continuous and uniform read coverage throughout the chromosomes.\u003c/p\u003e\u003cp\u003eDue to the error rates (between 4\u0026ndash;5% at the time of data generation) of ONT data, the T2T assembly was polished using a hybrid strategy involving two runs of PacBio HiFi read mapping followed by two runs of Illumina read mapping at high stringency (to reduce problems of degenerate bases flanking repetitive regions). Although Illumina reads exhibited great read accuracy, many regions in the genome, especially those in the ACs, did not have sufficient read depth required for performing error correction (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Insufficient coverage was more pronounced in AT141 than JCP043, as the gapped genomic regions totaled 2.17 Mb in AT141 compared to 156 kb in JCP043 (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. As much as 10.5% of chromosome 16 in AT141, mainly in the 5\u0026rsquo;end of the chromosome (nonrepetitive, 280 kb), was depleted of Illumina reads. In contrast, HiFi reads had a uniformly high read coverage (\u0026gt;\u0026thinsp;100\u0026times; coverage with reads\u0026thinsp;\u0026ge;\u0026thinsp;15 kb) across the entire genome (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). However, single-base sequence errors, including SNPs and indels, could still be detected in the HiFi-corrected assembly; therefore, additional error correction using Illumina reads was carried out under high stringency. The accuracy of the polished assembly of JCP043 was further confirmed with high-throughput chromosome conformation capture (Hi-C) analysis, and the result showed no mis-assembly (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). The number of chromosomes was also validated by centromeric interaction regions detected in the Hi-C contact map, with each contig represented as a single chromosome.\u003c/p\u003e\u003cp\u003eThe final assemblies were gapless and T2T complete, with JCP043 (race 1) and AT141 (race 4) assembled into 16 and 19 chromosomes, respectively, and the AT141 genome size being 6% or 3.86 Mb greater than that of JCP043 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The benchmarking universal single-copy ortholog (BUSCO) analysis was performed to assess the genome completeness. Of the 4,494 BUSCOs searched (library hypocreales_odb10), 98.9% and 98.5% were detected as complete and single copy in JCP043 and AT141, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which had similar levels of completeness compared to other published FOLac genomes (98.8% for AJ520 and 98.7% for AJ516 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of assembly and genomic features between T2T, Illumina, and NECAT assemblies of FOLac race 1 isolate JCP043 and race 4 isolate AT141.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eJCP043\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eAT141\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT2T\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIllumina\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNECAT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eT2T\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIllumina\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNECAT\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eAssembly features\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenome size (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e64.64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e68.50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e53.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e67.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRead depth\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e204\u0026times;\u003c/b\u003e\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e146\u0026times;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e204\u0026times;\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e176\u0026times;\u003c/b\u003e\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e182\u0026times;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e176\u0026times;\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGC (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e47.61\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e47.67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e48.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eN50 (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e4.63\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e4.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eL50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e# of contigs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5,405\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5,771\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e# of T2T contigs\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLongest contig (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e6.77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e6.82\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBUSCO (complete, single-copy)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e98.9%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.8%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e98.8%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e98.8%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCore genome size (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e48.64\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.47\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48.09\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e49.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.98\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.44\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAccessory genome size (Mb)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16.00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.62\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.51\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e19.48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.38\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.61\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenomic features\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e20,616\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18,811\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20,517\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e20,292\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18,320\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20,185\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal transcripts\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e22,319\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20,120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21,983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e21,918\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19,601\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e21,572\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal proteins\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e22,083\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e21,680\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean gene length\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1,746 bp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,738 bp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMean exon length\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e630 bp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e623 bp\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRepeat regions\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e17.64%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.46%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.29%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e20.39%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.86%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19.97%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGO terms\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e12,293\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e12,163\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePFAM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e13,204\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e13,081\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCAZYmes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e700\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e671\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e705\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e659\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e702\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSecreted CAZYmes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e331\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e331\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e337\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e301\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e325\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSM clusters\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSM genes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1,051\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,028\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1,004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1,026\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSecreted proteins\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1,433\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,316\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,422\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,451\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1,303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1,414\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEffector proteins\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e605\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e574\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e526\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e573\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMimp effectors\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e94\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e82\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRace-specific genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e689\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e681\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e536\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e513\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSecreted CAZYmes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEffector proteins\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMimp effectors\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSM genes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003eRead coverage of T2T and NECAT assemblies was calculated by mapping 50\u0026ndash;99 kb ONT reads to the assembly for improved mapping accuracy.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e2\u003c/sup\u003eCore and accessory genome sizes of Illumina and NECAT assemblies were calculated based on the total length of aligned contigs to the core and accessory genome regions of their corresponding T2T assembly.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e- indicates comparison was not performed on the specific feature.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMassive genome rearrangement in FOLac accessory chromosomes\u003c/h3\u003e\n\u003cp\u003eComparison of each of the two T2T assemblies to that of Fol4287 (f. sp. \u003cem\u003elycopersici\u003c/em\u003e race 2), the reference genome of \u003cem\u003eF. oxysporum\u003c/em\u003e due to its nearly complete genome featuring 11 CCs and 5 ACs, revealed that i) each of the two FOLac isolates had 11 CCs that were highly syntenic with the 11 CCs of Fol4287 (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and exhibited common features associated with CCs, including high gene density and low abundance of TEs; ii) JCP043 had five ACs while AT141 featured eight ACs that did not show clear synteny with the Fol4287 genome, and exhibited accessory-like appearance (gene-sparse and TE-rich); iii) the accessory genome regions of JCP043 also included a 1.38-Mb terminal segment of chromosome 5 and a 0.5-Mb segment of chromosome 14, whereas a 0.9-Mb terminal segment of chromosome 8 in AT141 was part of its accessory genome. It was worth noting that many of the unplaced scaffolds of Fol4287 were aligned with several CCs of JCP043 and AT141, primarily to the sub-telomeric regions.\u003c/p\u003e\u003cp\u003eThe collinearity analysis of JCP043 and AT141 showed that the 11 CCs were highly syntenic between the two isolates, with 99%-100% sequence identity except the accessory regions located on chromosomes 5 and 14 of JCP043 and chromosome 8 of AT141, which appeared to be unique sequences to each race (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This analysis also identified one chromosomal translocation event, in which chromosome 11 of AT141 appeared to be translocated to two different chromosomes in JCP043, with nearly 80% of the chromosome landing on chromosome 14 of JCP043 and the remaining 20% fused to the terminus of chromosome 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This translocation event was also observed in the synteny plot between JCP043 and Fol4287 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eComparison of the ACs between JCP043 and AT141 showed an absence of large-scale synteny between the two isolates, but revealed widespread presence of smaller similar sequences, ranging from 10 to 75 kb in size and 92.99\u0026ndash;98.92% sequence identity, between them in a non-colinear order (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), which suggests massive genome rearrangements may have occurred in their ACs. One exception to this was chromosome 16 of JCP043, which appeared to have two large fragments of DNA, 480 and 580 kb in size, syntenic with chromosomes 16 and 18 of AT141, respectively. To get a better understanding of how dissimilar the unique sequences (defined by the lack of homology in any 10-kb windows) were between the two isolates, the two sets of ACs were compared at 2-kb resolution (slightly above the average gene length). The analysis, which was performed on repeat-masked ACs to avoid the detection of overwhelmingly abundant DNA repeats, showed that 32% of those unique segments were shared between the two isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo determine whether the ACs of JCP043 are conserved in race 1 while variable to race 4, we conducted synteny analysis with six published contig-level FOLac genomes (three race 1 and three race 4 isolates [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]). The result showed that race 1 isolate JCP043 displayed high levels of AC synteny with previously published race 1 isolates (AJ520, AJ718 and AJ865), whereas most of the ACs in JCP043 were not syntenic with the race 4 isolates (AJ516, AJ592 and AJ705) (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). Likewise, the ACs of race 4 isolate AT141 were syntenic with the three race 4 isolates but not with race 1 (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eDNA repeats likely contribute to the expanded genome of AT141\u003c/h3\u003e\n\u003cp\u003eAbout 18% and 20% of the JCP043 and AT141 genomes, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), was identified as repetitive sequence, including DNA transposons (\u003cem\u003ehAT\u003c/em\u003e, \u003cem\u003eTc1-IS630-Pogo\u003c/em\u003e, \u003cem\u003eMULE-MuDR\u003c/em\u003e, \u003cem\u003ePiggyBa\u003c/em\u003ec and \u003cem\u003eHelitron\u003c/em\u003e), long terminal repeat (LTR) retrotransposons (\u003cem\u003eGypsy/DIRS1\u003c/em\u003e and \u003cem\u003eTy1/Copia\u003c/em\u003e), long interspersed nuclear elements (LINEs) retrotransposons (\u003cem\u003eTad1\u003c/em\u003e and \u003cem\u003eRTE\u003c/em\u003e), as well as simple repeats and satellites (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Among the different classes of TEs, \u003cem\u003ehAT\u003c/em\u003e represented the largest in length and ranked the highest in copy number, followed by \u003cem\u003eGypsy/DIRS1\u003c/em\u003e and \u003cem\u003eTc1-IS630-Pogo.\u003c/em\u003e Based on the density plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), ACs were enriched in different types of TEs, accounting for more than 75% of the characterized TEs in the entire genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). While most of the repeat classes were similar in size between the two genomes, the AT141 genome contained a significantly greater amount of unknown repetitive sequence, totaling 4.34 Mb in size compared to 2.61 Mb in JCP043, accounting for 44.8% of the overall genome size difference between the two isolates. The unknown repeats were widely distributed among the ACs, especially in the accessory segment of chromosome 8 in AT141 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Sequences of the unknown repeats are provided in \u003cb\u003eSupplementary File S2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWhile most of the TEs were largely less abundant in CCs, we noticed that 75% of \u003cem\u003eGypsy/DIRS\u003c/em\u003e elements were associated with CCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To determine whether there is any association between \u003cem\u003eGypsy/DIRS1\u003c/em\u003e and the aforementioned chromosomal translocation in JCP043, we marked the breakpoint (located on chromosome 11 of AT141) and two fusion points (located on chromosomes 2 and 14 of JCP043) on the TE density plot. It was found that the breakpoint and one of the fusion points (on chromosome 2 of JCP043) overlapped with a \u003cem\u003eGypsy/DIRS1\u003c/em\u003e element while the other fusion point (on chromosome 14 of JCP043) resided 15 kb upstream of three \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\n\u003ch3\u003eLarge-scale chromosome duplication is prevalent in FOLac accessory chromosomes\u003c/h3\u003e\n\u003cp\u003eBesides DNA repeats, we explored the possibility of segmental chromosome duplication in the ACs contributing to the larger genome size of AT141 and the formation of three more ACs. To study this, pair-wise comparison of individual ACs of AT141 were conducted to identify similar regions greater than 20 kb (to avoid the detection of overwhelmingly abundant DNA transposons, some of which could extend up to 12 kb). The analysis resulted in the recognition of numerous intra- and inter-chromosomal duplications in all the ACs of AT141 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) that ranged in size from 20 kb to 500 kb, except for chromosome 18, which did not show any segmental duplication. Notably, we identified two nearly identical, 500-kb inverted repeats located on both ends of chromosome 17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), flanking a 700-kb non-repetitive region. This duplication, along with a few others, might be facilitated by \u003cem\u003eGypsy/DIRS1\u003c/em\u003e retrotransposons that flank the duplicated regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, a heavy presence of \u003cem\u003ehAT-Restless\u003c/em\u003e transposons was detected surrounding some of the other duplicated regions.\u003c/p\u003e\u003cp\u003eWhile the frequency of intra-chromosomal duplication was markedly less in JCP043 compared to AT141, inter-chromosomal duplications were frequently found in the ACs of JCP043, including a 1-Mb region that was nearly identical between chromosomes 5 and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar to AT141, we found the association of \u003cem\u003eGypsy/DIRS1\u003c/em\u003e with the boundaries of this large, duplicated region.\u003c/p\u003e\u003cp\u003eOverall, given that the total size of duplicated regions was nearly identical between the two genomes (1.43 Mb for JCP043 vs. 1.52 Mb for AT141), it was reasonable to conclude that segmental chromosome duplication was not the contributing factor for the expanded genome of AT141, but it may play an important role in the formation of new accessory chromosomes (i.e. chromosome 17) and altering structural organization of the genome.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eGeneral putative effector proteins are distributed more in the core genome regions while\u003c/b\u003e \u003cb\u003emimp\u003c/b\u003e\u003cb\u003e-associated effectors are enriched in the accessory genome regions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genomes of JCP043 and AT141 harbored 20616 and 20292 predicted genes, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), however, only 60% of them had GO annotations. Approximately 85% of the predicted genes were associated with the core genome regions while the remaining 15% were present in the accessory genome regions (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Using the non-redundant gene sets for JCP043 and AT141, we identified 15980 pairs of putative orthologous genes. Not surprisingly, over 94% of them were associated with CCs and the co-linear order of genes between the two isolates has maintained within these chromosomes (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA\u003c/b\u003e). The remaining 6% were widely dispersed among different ACs (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThe JCP043 and AT141 genomes harbored similar numbers of several specific gene classes, respectively, including 331/337 extracellularly secreted carbohydrate-active enzymes (CAZYmes), 605/574 effector proteins, and 65/67 secondary metabolite (SM) gene clusters comprising 1051/1028 genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These genes were predominantly present in CCs, representing 85\u0026ndash;98% of the gene reservoir (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e\u003c/b\u003e). Among the putative effector proteins identified, apoplastic effectors constituted 51\u0026ndash;54% of the effector complement, followed by cytoplastic effectors (27\u0026ndash;30%). The localization of the remaining effector proteins remained undetermined.\u003c/p\u003e\u003cp\u003eAccording to previous studies, many effector genes (e.g. \u003cem\u003eSIX\u003c/em\u003e genes) in \u003cem\u003eF. oxysporum\u003c/em\u003e are located within subregions enriched for TEs, and a miniature Impala (\u003cem\u003emimp\u003c/em\u003e) element, in particular, is always present in their promoters [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The analysis of \u003cem\u003emimp\u003c/em\u003e-associated effectors identified 94 and 82 candidates in the genomes of JCP043 and AT141, respectively. Among them, 18 (for JCP043) and 11 (for AT141) \u003cem\u003emimp\u003c/em\u003e effectors overlapped with the effector proteins identified using the EffectorP pipeline. Nearly 95% of the candidate \u003cem\u003emimp\u003c/em\u003e effectors were associated with ACs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), with chromosome 10 of JCP043 (N\u0026thinsp;=\u0026thinsp;29) and chromosome 5 of AT141 (N\u0026thinsp;=\u0026thinsp;35) harboring the most \u003cem\u003emimp\u003c/em\u003e effectors (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenes involved in intracellular pH homeostasis, regulation of transcription and mitochondria-nucleus signaling pathway are enriched in FOLac accessory chromosomes\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe conducted gene ontology (GO) enrichment analysis to identify putative biological processes and molecular functions that are enriched in the ACs of both FOLac isolates. It turned out that their ACs were significantly enriched (corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.01) for biological processes involved in intracellular monoatomic ion and cation homeostasis, regulations of intracellular pH and DNA-binding transcription factor activity, and mitochondria-nucleus signaling pathway (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). Molecular functions, including various types of oxidoreductase activities and iron ion and heme-binding activities, were also enriched. In addition, the ACs of JCP043 were uniquely enriched for genes involved in lipid, peptide, and monocarboxylic acid catabolic processes, while genes involved in double-strand break repair DNA repair, regulation of nitrogen utilization, and long-chain fatty acid metabolic process were uniquely enriched in the ACs of AT141. Regarding the genes located within the duplicated regions in JCP043, they were enriched for two biological processes, including intracellular monoatomic ion and cation homeostasis and chromate transport. No GO terms were significantly enriched in the duplicated regions in AT141.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eSIX9.4\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSIX14\u003c/b\u003e \u003cb\u003eare conserved in FOLac while\u003c/b\u003e \u003cb\u003eSIX8\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSIX9.1-9.3\u003c/b\u003e \u003cb\u003eare absent in race 1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDue to the lack of ability to identify all 14 \u003cem\u003eSIX\u003c/em\u003e genes using the EffectorP and FoEC2 pipelines [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], we manually annotated the \u003cem\u003eSIX\u003c/em\u003e gene complement in both T2T assemblies via BLAST. Moreover, we expanded the search to 53 publicly available FOLac genomes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], including 33 race 1 isolates, 1 race 2 isolate, 1 race 3 isolate, 16 race 4 isolates, and 2 race 1 variants (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e), to evaluate the correlation between the \u003cem\u003eSIX\u003c/em\u003e gene complement and race structure. Additional long-read genome assemblies representing different phylogenetic lineages and clades outside of FOLac (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e) were also included in this analysis to determine how similar their \u003cem\u003eSIX\u003c/em\u003e genes are to those present in FOLac. Generally, FOLac carried three \u003cem\u003eSIX\u003c/em\u003e genes, \u003cem\u003eSIX8\u003c/em\u003e, \u003cem\u003eSIX9\u003c/em\u003e and \u003cem\u003eSIX14\u003c/em\u003e, with copy number and sequence variations at the intra and inter-race levels, which are described below. None of the remaining \u003cem\u003eSIX\u003c/em\u003e genes were identified in FOLac.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSIX8\u003c/strong\u003e\u003cp\u003e\u003cem\u003eSIX8\u003c/em\u003e was absent in JCP043 and 35 other FOLac race 1 isolates (including the two race 1 variants), while two identical copies of \u003cem\u003eSIX8\u003c/em\u003e were identified in AT141, located within a 30-kb, TE-rich, tandem repeat on chromosome 5 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Except for AT141 having two copies of \u003cem\u003eSIX8\u003c/em\u003e, a single copy of \u003cem\u003eSIX8\u003c/em\u003e was consistently identified in all the other FOLac race 4 isolates as well as FOLac races 2 and 3, with variation in the \u003cem\u003eSIX8\u003c/em\u003e sequences among isolates resulting in two \u003cem\u003eSIX8\u003c/em\u003e variants (\u003cem\u003eSIX8.1\u003c/em\u003e and \u003cem\u003eSIX8.2\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e\u003c/b\u003e). \u003cem\u003eSIX8\u003c/em\u003e was also present in eight other unrelated \u003cem\u003eF. oxysporum\u003c/em\u003e isolates, including \u003cem\u003eff. spp. conglutinans\u003c/em\u003e, \u003cem\u003elycopersici\u003c/em\u003e, \u003cem\u003eniveum\u003c/em\u003e, and \u003cem\u003esesame\u003c/em\u003e (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). The \u003cem\u003eSIX8\u003c/em\u003e gene phylogeny revealed that FOLac \u003cem\u003eSIX8.1\u003c/em\u003e and \u003cem\u003eSIX8.2\u003c/em\u003e were most closely related to f. sp. \u003cem\u003econglutinans\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSIX9\u003c/strong\u003e\u003cp\u003eA previous study showed that FOLac had four variants of \u003cem\u003eSIX9\u003c/em\u003e (\u003cem\u003eSIX9.1-SIX9.4\u003c/em\u003e), with differences in copy number and sequence variation between races 1 and 4 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, none of the FOLac race 1 isolates had \u003cem\u003eSIX9.1\u003c/em\u003e, \u003cem\u003eSIX9.2\u003c/em\u003e, and \u003cem\u003eSIX9.3\u003c/em\u003e, except race 1 isolate AM163, which appeared to have one copy of \u003cem\u003eSIX9.2\u003c/em\u003e in its Illumina assembly. However, the read mapping analysis indicated otherwise due to the extremely low read depth (less than 8\u0026times;) on \u003cem\u003eSIX9.2\u003c/em\u003e compared to the other \u003cem\u003eSIX\u003c/em\u003e genes (\u003cb\u003eSupplementary Figure S9\u003c/b\u003e). Two identical copies of truncated \u003cem\u003eSIX9.1\u003c/em\u003e (54% in coverage), which had an unknown type of DNA repeat present upstream, were identified in JCP043 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The truncated \u003cem\u003eSIX9.1\u003c/em\u003e was conserved among all the FOLac race 1 isolates. FOLac races 2, 3 and 4 possessed all four variants of \u003cem\u003eSIX9\u003c/em\u003e, but variation in copy numbers was observed among the FOLac race 4 isolates (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). \u003cem\u003eSIX9.4\u003c/em\u003e appeared to be the only copy that was consistently identified from all four races, with no sequence variation among isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Based on their location on the T2T assembly of AT141, the three variants of \u003cem\u003eSIX9\u003c/em\u003e (\u003cem\u003eSIX9.2\u003c/em\u003e, \u003cem\u003eSIX9.3, and SIX9.4\u003c/em\u003e) were spread sparsely on chromosome 5, flanked by DNA transposons (i.e. \u003cem\u003eTcMart-Fot1\u003c/em\u003e) or unknown DNA repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE; also observed in \u003cem\u003eSIX8\u003c/em\u003e as noted above). An additional copy of \u003cem\u003eSIX9.4\u003c/em\u003e was identified on chromosome 12 of AT141, part of a 23-kb duplicated region between chromosome 5 and 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Based on the \u003cem\u003eSIX9\u003c/em\u003e gene phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), sequences of FOLac \u003cem\u003eSIX9.1-9.4\u003c/em\u003e were (nearly) identical to those in unrelated ff. spp., including \u003cem\u003eapii\u003c/em\u003e races 3 and 4, \u003cem\u003econglutinans\u003c/em\u003e, \u003cem\u003elini\u003c/em\u003e, \u003cem\u003ecoriandrii, niveum\u003c/em\u003e, and \u003cem\u003esemani, raphanin\u003c/em\u003e, and \u003cem\u003evasinfectum.\u003c/em\u003e\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSIX14\u003c/strong\u003e\u003cp\u003e\u003cem\u003eSIX14\u003c/em\u003e was consistently present among all the FOLac isolates, with no sequence variation among isolates, the exception being race 1 isolate Fol621 and race 1 variant VSP-0916 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which had one non-synonymous mutation. \u003cem\u003eSIX14\u003c/em\u003e, along with \u003cem\u003eSIX8\u003c/em\u003e and \u003cem\u003eSIX9\u003c/em\u003e, were all located on chromosome 5 of AT141 and at least 200-kb apart from one another (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that chromosome 5 is a putative pathogenicity chromosome. Likewise, in the genome of JCP043, we identified chromosome 15 as a putative pathogenicity chromosome because it harbored both \u003cem\u003eSIX9.4\u003c/em\u003e and \u003cem\u003eSIX14\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Interestingly, the BLAST analysis revealed a second copy of \u003cem\u003eSIX14\u003c/em\u003e, located on chromosome 10 in JCP043, however, it appeared to be disrupted by the insertion of a \u003cem\u003eGypsy\u003c/em\u003e retrotransposon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), likely resulting in loss of function. This is consistent with a previous study, where a transposon has inserted into \u003cem\u003eSIX14\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Compared to \u003cem\u003eSIX8\u003c/em\u003e and \u003cem\u003eSIX9, SIX14\u003c/em\u003e tended to have a limited distribution among the \u003cem\u003eF. oxysporum\u003c/em\u003e taxa we examined as it was only identified in two unrelated ff. spp., including ff. spp. \u003cem\u003eniveum\u003c/em\u003e and \u003cem\u003elycopersici\u003c/em\u003e. Sequences of \u003cem\u003eSIX14\u003c/em\u003e identified among FOLac isolates were most closely related to those in f. sp. \u003cem\u003eniveum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Sequences of \u003cem\u003eSIX8\u003c/em\u003e, \u003cem\u003eSIX9\u003c/em\u003e, and \u003cem\u003eSIX14\u003c/em\u003e identified from all the \u003cem\u003eF. oxysporum\u003c/em\u003e isolates used in this study are provided in \u003cb\u003eSupplementary Files S3-S5\u003c/b\u003e.\u003c/p\u003e\u003c/p\u003e\n\u003ch3\u003ePutative race-specific genes are clustered on several accessory chromosomes\u003c/h3\u003e\n\u003cp\u003eWe identified 1010 candidate unique genes for JCP043 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), 563 of which were absent and 447 were partially present (below 90% identity or 80% coverage) in the AT141 genome. A total of 1081 candidate unique genes were identified in AT141, including 741 absent and 340 partially present. After screening the candidate genes against nine representative FOLac genomes covering all four races, 689 putative race 1-specific genes and 536 putative race 4-specific genes were identified (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cb\u003eSupplementary Files S6 and S7\u003c/b\u003e). Nearly 87% of the candidate race-specific genes were associated with the accessory genome regions and often clustered together (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB)\u003c/b\u003e. Chromosomes 7, 12 and 10 of JCP043 harbored the greatest numbers of race 1-specific genes (chr 7: 223, chr 12: 167, and chr10: 85). In AT141, chromosomes 13, 5, and 16 contained the most race 4-specific genes (chr13: 219, chr5: 76, and chr16: 60).\u003c/p\u003e\u003cp\u003eAmong the FOLac race 1-specific genes, we identified 4 secreted CAZYmes, 14 effectors, 5 \u003cem\u003emimp\u003c/em\u003e-associated effectors, and 3 SM genes (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). As for the race 4-specific genes, 2 secreted CAZYmes, 8 effector, 3 \u003cem\u003emimp\u003c/em\u003e-associated effectors, and 6 SM genes were identified. Interestingly, two race-specific CAZYmes (JCP043_017764 and AT141_017966) were also predicted to be apoplastic effectors, potentially contributing to plant cell wall degradation. While most of the race-specific SM genes coded hypothetical proteins, we identified one unique SM gene cluster located on chromosome 16 in AT141 that harbored multiple race 4-specific genes, including a putative type-III polyketide synthase (AT141_019912) as the core biosynthetic enzyme, a glycoside hydrolase (GH) family protein (AT141_019910), a phosphate transporter (AT141_019913), and two hypothetical proteins (AT141_019917 and AT141_019918). The core biosynthetic enzyme had high similarity (88.5% identity in amino acid sequences) to the thiolase-like protein of \u003cem\u003eF. redolens\u003c/em\u003e (Accession: XP_046053686.1).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eThe T2T assembly reveals structural variants and putative genes not shown in contig-level assemblies\u003c/h2\u003e\u003cp\u003eIn comparison to the T2T assemblies of JCP043 and AT141, their respective Illumina assemblies presented several limitations in characterizing assembly and genomic features as described below and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The estimated genome sizes of JCP043 and AT141 based on their Illumina assemblies were 6.05 Mb and 14.92 Mb smaller, representing 83% and 77% of their T2T genome sizes, respectively. While the core genome regions were well represented in the Illumina assemblies of JCP043 and AT141, reflected in high BUSCO scores, only 48% and 46% of their accessory genome regions were recovered in their respective Illumina contigs, respectively. Since the Illumina assemblies were highly fragmented (5405 contigs for JCP043 and 5771 contigs for AT141), it was impossible to detect structural variants, including genome duplication and rearrangement, between the two isolates. Compared to the T2T assemblies, the Illumina assemblies of JCP043 and AT141 contained significantly fewer DNA repeats, representing only 6.46% and 4.86% of the genome sizes, respectively, in comparison to 18% and 20% in their respective T2T assemblies. Regarding the predicted genes, both Illumina assemblies turned out to miss\u0026thinsp;~\u0026thinsp;10% of the total genes (1805 genes missing from JCP043 and 1972 missing from AT141), with 83% and 74% of them associated with ACs of JCP043 and AT141, respectively. Many of the missing genes were partially present due to the fact that they were located on the end of a contig. Illumina assemblies were able to capture the \u003cem\u003eSIX\u003c/em\u003e genes and all but four \u003cem\u003emimp\u003c/em\u003e effectors, however, they fell short in identifying additional copies of \u003cem\u003eSIX8\u003c/em\u003e and \u003cem\u003eSIX9.4\u003c/em\u003e, as observed in all FOLac Illumina assemblies (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). When searching for putative race 1-specific genes in the Illumina assembly of JCP043, 171 were not detected, 150 of which were associated with ACs. The Illumina assembly of AT141 had 99 of the race 4-specific genes missing, with 88 of them located on ACs.\u003c/p\u003e\u003cp\u003eOn the other hand, the NECAT assemblies of JCP043 and AT141, which were obtained by the NECAT assembler and error corrected using Illumina reads (without checking for misassembly and contig end extension), resulted in genome sizes very close to their corresponding T2T assemblies, as also observed for BUSCO scores (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The NECAT assemblies represented over 97% of both the core and accessory regions of each genome, with most of the NECAT contigs aligned with the T2T chromosomes (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/b\u003e). However, the NECAT assemblies were less contiguous and did not have telomeres on many of the contigs, and produced a much smaller number of rDNA repeats (25 copies for JCP043 and 10 copies for AT141). The large-scale chromosome duplications identified from the T2T assemblies (i.e. the 1-Mb duplicated regions in JCP043 and the 500-kb inverted repeats in AT141) were not captured in the NECAT assemblies (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/b\u003e). The NECAT assemblies were highly similar to the T2T assemblies regarding the amount and composition of repetitive elements, as well as predicted genes, with only\u0026thinsp;~\u0026thinsp;100 genes absent. Not surprisingly, most of those missing genes were located within the large, duplicated regions. For instance, 92 out of 107 missing genes from the AT141 NECAT assembly were located on the 5\u0026rsquo;end of chromosome 17, which failed to be assembled in the NECAT assembly. The NECAT assemblies of both isolates possessed all the \u003cem\u003emimp\u003c/em\u003e effectors and rendered the same sequences and copy numbers for the \u003cem\u003eSIX\u003c/em\u003e genes as the T2T assemblies. The NECAT assemblies possessed most of the race-specific genes, with only eight (for JCP043) and 23 genes (for AT141) missing compared to their respective T2T assemblies.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reports a complete (gapless and T2T complete) chromosome-level assembly for two key, very closely related \u003cem\u003eF. oxysporum\u003c/em\u003e pathogens, which due to the highly repetitive nature of their extensive accessory genome regions, have heretofore evaded complete assembly. This result permitted comparative genomic analyses of the two T2T assemblies and revealed novel structural and genetic differences between the two races, which laid the groundwork to identify putative pathogenicity genes that may be associated with the host-pathogen interaction in the FOLac-lettuce pathosystem. More broadly, this study provides a workflow to generate T2T assemblies that can significantly improve our ability to investigate genome dynamics and organism adaptation, characterize genes that may be associated with pathogenicity, and identify race-specific sequences useful for diagnostics.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eA novel workflow for generating gapless, T2T assemblies and things to consider\u003c/b\u003e\u003c/div\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn this study, we developed a novel workflow that deployed a suite of bioinformatic programs with various types of long and short sequencing technologies (ONT, Pacbio HiFi, Illumina, and Hi-C) to build \u003cem\u003eF. oxysporum\u003c/em\u003e genome assemblies that are gapless and T2T complete. In summary, we considered the following three features as key players in achieving this goal. The first key feature relates to the abundance of long ONT reads (\u0026ge;\u0026thinsp;50 kb). These long reads are deemed crucial for T2T genome assembly because of their ability to resolve large repetitive regions in \u003cem\u003eF. oxysporum\u003c/em\u003e accessory genome regions, some of which can reach as long as 20\u0026ndash;30 kb in size. In our study, ONT reads that were above 50 kb provided 204\u0026times; coverage for JCP043 and 176\u0026times; coverage for AT141 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To the best of our knowledge, such high long-read coverage has not been reported in any other \u003cem\u003eF. oxysporum\u003c/em\u003e genome studies. The second key feature deals with misassembly, which is a major challenge in the assembly of highly repetitive accessory genome regions. Some of the misassembled regions failed to be detected by command-line programs because decent read coverage can be found at the misassembled regions but no reads entirely span them (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Our solution to this problem was to map long reads (\u0026ge;\u0026thinsp;50 kb) to the NECAT assembly using programs like CLC Genomics Workbench (QIAGEN, Aarhus, Denmark; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://digitalinsights.qiagen.com\u003c/span\u003e\u003cspan address=\"https://digitalinsights.qiagen.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SeqMan NGen (DNASTAR, Madison, WI, USA), which enables visual scanning of the read mapping to identify misassembled regions. The third key feature involves the practice of read mapping-guided, iterative end extension on the intermediate contigs (see Methods for details). As far as we are aware of, there is no stand-alone bioinformatic software available to extend contigs using long reads beyond scaffolding and gap-filling capabilities. This is reflected in some of near complete \u003cem\u003eF. oxysporum\u003c/em\u003e genomes (i.e. Fo47, Fo5176, and Fol4287) that have been published [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], none of which has achieved the level of genome completeness as the two FOLac assemblies presented here. An add-on value of using graphical read mapping for end extension is the ability to visually identify large-scale chromosome duplications due to read depth appearing doubled in the read mapping graphics, as observed in chromosomes 5 and 10 in JCP043 and chromosome 17 in AT141. These large, duplicated regions were not captured by the NECAT program (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e\u003c/b\u003e) or other long-read assemblers, including Flye [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and Canu [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] that we tested. We are currently working on automating and fine-tuning the assembly pipeline, hoping to significantly reduce the amount of time needed to generate T2T assemblies.\u003c/p\u003e\u003cp\u003eFinally, the use of PacBio HiFi reads in conjunction with short read data for base-level error correction of the assembly is essential. The lack of Illumina read coverage in many regions of the T2T assembly (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) leaves sequence errors in those gapped regions unchecked. One contributing factor may be read depletion of highly repetitive regions early in the read mapping process. In contrast to short reads, HiFi reads exhibited uniformly high read coverage (\u0026gt;\u0026thinsp;100\u0026times; coverage with reads\u0026thinsp;\u0026ge;\u0026thinsp;15 kb) across the entire genome (\u003cb\u003eSupplementary Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), thus allowing for gap-free error correction. Besides the heterogeneity in genome coverage, variability in Illumina read coverage was also found between different samples. A significantly higher proportion of the AT141 genome, including non-repetitive regions, was not covered by its own Illumina reads compared to that in JCP043, even though the total number of Illumina reads for AT141 was 30% greater than that for JCP043. These observations emphasize the importance of using read mapping to visually evaluate the uniformity of sequencing data used in genome polishing.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eT2T assemblies set a foundation for studying genome evolution in\u003c/b\u003e \u003cb\u003eF. oxysporum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe T2T assemblies reported here revealed some novel structural differences in the accessory genome regions between FOLac race 1 and race 4, providing new insight regarding genome changes that may underlie the evolution of pathogenicity. The most distinct structural difference between them was that race 4 isolate AT141 possesses a genome 3.86 Mb larger than that of race 1 isolate JCP043 and organizes differently into 19 rather than 16 chromosomes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Part of the larger genome size of AT141 was related to its inflated estimated copy number of rDNA repeats, which was 144 copies vs. 90 copies in JCP043. \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003eradicis-cucumerinum\u003c/em\u003e isolate Forc016 was estimated to have 98 rDNA copies [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], which was similar to that of JCP043. It is widely accepted that rDNA loci are dynamic and copy number fluctuates widely between individual within a species, and even likely between cells within a single organism [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Further analysis using quantitative real-time PCR is needed to determine the actual copy number of rDNA for the two isolates. Although similar difference in genome size between the two FOLac races was reported previously [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], the exact chromosome number difference between the two races was not resolved from those contig-level genomes. This has been the constraint in many comparative genomic analyses for members within \u003cem\u003eF. oxysporum.\u003c/em\u003e An expended survey of additional FOLac races 1 and 4 isolates that have T2T assemblies available will help determine whether the genome size and number of ACs are fixed within a race or not.\u003c/p\u003e\u003cp\u003eSecondly, the T2T assemblies showed that repetitive elements made up 18\u0026ndash;20% of the two FOLac genomes, which were substantially higher than those reported for other long-read assemblies, such as 10.54% for \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecepae\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], 15.4% for \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003econglutinans\u003c/em\u003e and 16.42% for \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Given that TEs play diverse roles in gene regulation, recombination, and adaptation to changing environment [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], the ability to identify an expanded pool of TEs from T2T assemblies serves as a valuable tool to understand how TEs contribute to pathogen diversification within \u003cem\u003eF. oxysporum\u003c/em\u003e. Consistent with other studies [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], common TEs, including DNA transposons and retroelements, were widely distributed among the accessory genome regions, with the exception of \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements, of which 75% were found in the core genome regions. The greater abundance of \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements in the core genome regions of \u003cem\u003eF. oxysporum\u003c/em\u003e has also been reported in an opportunistic human pathogen, with nearly 90% of \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements found in the core genome [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. While most of the repetitive elements were similar in copy number and length between the two isolates, a markedly larger number of unknown repeats were associated with the race 4 genome (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), contributing to 44.8% (1.76 Mb in size) of the overall genome size difference between the two isolates. In particular, the accessory region on chromosome 8 in AT141 showed a very high level of unknown repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) that was consistently found in the three published FOLac race 4 genomes (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e), suggesting that this putative proliferation may be a feature of FOLac race 4.\u003c/p\u003e\u003cp\u003eThirdly, the T2T assemblies offered a unique opportunity to explore how large-scale chromosome duplications and rearrangements vary between the two FOLac races with the focus to identify genome regions that are race specific. Similar to the observations reported previously [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], the syntenic analysis between race 1 isolate JCP043 and race 4 isolate AT141 demonstrated highly conserved CCs but extremely fragmented and rearranged shared sequences among ACs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that each race underwent very different rearrangements resulting in loss of synteny. None of the ACs appeared to be exclusively associated with either of the two isolates, including chromosome 15 of AT141, which turned out to have many small non-repetitive sequences (2\u0026ndash;6 kb in size) shared with JCP043. Our findings pointed to a previously unreported correlation between \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements and chromosomal translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which appeared to be a unique event in JCP043. In addition to their potential involvement in chromosomal translocation, \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements were found to be abundant near the boundaries of large, duplicated regions in JCP043 (a 1-Mb repeat duplicated between chromosomes 5 and 10) and AT141 (a 500-kb inverted repeat within chromosome 17) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting their additional role in large-scale chromosome duplications. The connection between \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements and chromosome rearrangements was recently reported in two stingless bee species, \u003cem\u003eMelipona quadrifasciata\u003c/em\u003e and \u003cem\u003eM. scutellaris\u003c/em\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], however, it remains an open question whether there is a causative relation between \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements and changes in genome architecture. A broader survey involving in-depth characterization of TEs among diverse \u003cem\u003eF. oxysporum\u003c/em\u003e genomes representing different evolutionary lineages and ff. spp. will help better understand their role in \u003cem\u003eF. oxysporum\u003c/em\u003e genome evolution. In addition to TEs, the enrichment of certain types of histone modifications or giant \u003cem\u003eStarship\u003c/em\u003e elements, as described in \u003cem\u003eF. graminearum\u003c/em\u003e [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and \u003cem\u003eMacrophomna phaseolina\u003c/em\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], respectively, may also contribute to the extensive genetic and large-scale genome variation in \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasticity of\u003c/b\u003e \u003cb\u003eSIX\u003c/b\u003e \u003cb\u003egenes in FOLac race 4 and their putative origin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWith a wide collection of FOLac genomes representing all four races, the analysis of \u003cem\u003eSIX\u003c/em\u003e genes showed the universal presence of \u003cem\u003eSIX9.4\u003c/em\u003e and \u003cem\u003eSIX14\u003c/em\u003e among all four races of FOLac. FOLac race 1 exhibits a unique \u003cem\u003eSIX\u003c/em\u003e gene complement that distinguishes it from the three other races (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). Race 1 isolates do not have \u003cem\u003eSIX8\u003c/em\u003e and three \u003cem\u003eSIX9\u003c/em\u003e variants (\u003cem\u003eSIX9.1\u003c/em\u003e, \u003cem\u003eSIX9.2\u003c/em\u003e, and \u003cem\u003eSIX9.3\u003c/em\u003e) while they are all present in races 2\u0026ndash;4. However, in a recent study [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], PCR analysis of \u003cem\u003eSIX8\u003c/em\u003e, \u003cem\u003eSIX9\u003c/em\u003e, and \u003cem\u003eSIX14\u003c/em\u003e in FOLac race 4 and race4\u0026thinsp;+\u0026thinsp;isolates showed the absence of \u003cem\u003eSIX8\u003c/em\u003e in many isolates, suggesting the instability of \u003cem\u003eSIX8\u003c/em\u003e in the race 4 population. Besides, the race 4 isolates also showed high levels of intra-race variation in the three \u003cem\u003eSIX9\u003c/em\u003e variants (\u003cem\u003eSIX9.1\u003c/em\u003e, \u003cem\u003eSIX9.2\u003c/em\u003e, and \u003cem\u003eSIX9.3\u003c/em\u003e), with AP114 (originated from Denmark) harboring three of the variants while AL127 (originated from Ireland) having only \u003cem\u003eSIX9.2.\u003c/em\u003e In addition, we found that AT141 was the only race 4 isolate that had two copies of \u003cem\u003eSIX8\u003c/em\u003e, which was likely achieved through large-scale chromosome duplication (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) instead of a single gene duplication mediated by TEs. In contrast, we noticed stable presence of the \u003cem\u003eSIX\u003c/em\u003e genes among all the race 1 isolates, with no sequence variation among isolates. Given the high level of plasticity in \u003cem\u003eSIX8\u003c/em\u003e and \u003cem\u003eSIX9\u003c/em\u003e within the race 4 population, which is likely driven by TEs that are present in close proximity to the \u003cem\u003eSIX\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), one should be cautious about using only \u003cem\u003eSIX\u003c/em\u003e genes for discerning FOLac races as it may lead to erroneous results.\u003c/p\u003e\u003cp\u003eInterestingly, sequences of the three \u003cem\u003eSIX\u003c/em\u003e genes present in FOLac were highly similar to those in ff. spp. that were closely related to FOLac race 3 (Geiser, unpublished, \u003cb\u003eSupplementary File S8\u003c/b\u003e). Moreover, the more ancestral FOLac race 2 lineage possessed all three \u003cem\u003eSIX\u003c/em\u003e genes, including the four variants of \u003cem\u003eSIX9.\u003c/em\u003e Given this information, we hypothesized either that these \u003cem\u003eSIX\u003c/em\u003e genes may have existed in \u003cem\u003eF. oxysporum\u003c/em\u003e before lineage diversification or that they have been transferred among different lineages of \u003cem\u003eF. oxysporum\u003c/em\u003e after lineage diversification. Future study of the \u003cem\u003eSIX\u003c/em\u003e gene complement with more extensive sampling of \u003cem\u003eF. oxysporum\u003c/em\u003e genomes spanning all 17 lineages as delineated by Geiser (unpublished, \u003cb\u003eSupplementary File S8\u003c/b\u003e) will shed light on the evolution of the \u003cem\u003eSIX\u003c/em\u003e gene family.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of putative race-specific genes provides a framework for functional characterization of candidate virulence/host-range genes\u003c/h2\u003e\u003cp\u003eThe race-specific genes that were identified in this study were validated against not only FOLac race 1 and 4 genomes, but also two representative isolates from FOLac races 2 and 3, which enabled more reliable identification of race-specific genes compared to previous studies. However, due to the lack of long-read assemblies for the race 2 and 3 isolates when this study was conducted, additional validation of race-specific genes against these two isolates was achieved through read mapping analysis to ensure accuracy, similar to the approach used to confirm \u003cem\u003eSIX9.2\u003c/em\u003e was absent in AM163 (\u003cb\u003eSupplementary Figure S9\u003c/b\u003e). Our results identified 689 putative race 1- and 536 putative race 4-specific genes, some of which encode putative secreted CAZymes, effectors (including \u003cem\u003emimp\u003c/em\u003e-associated effectors), and proteins involved in SM biosynthesis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e). Not surprisingly, nearly 87% of the race-specific genes were associated with ACs (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB)\u003c/b\u003e, with chromosomes 7 and 12 of JCP043 and chromosome 13 of AT141 each harboring over 100 race-specific genes, suggesting that they might be pathogenicity chromosomes. Chromosome 10 of JCP043 and chromosome 5 of AT141, which carried the \u003cem\u003eSIX\u003c/em\u003e genes, also possessed many of the race-specific genes, raising the possibility of their roles in host range and virulence. Future studies are warranted for narrowing the candidates for race-specific genes, by screening them against a wide range of \u003cem\u003eF. oxysporum\u003c/em\u003e ff. spp. and nonpathogenic isolates recovered from lettuce and other hosts. Also notable is the presence of various types of TEs within and/or in the flanking regions of the candidate race-specific genes. These observations raised the question of whether those genes are expressed \u003cem\u003ein planta\u003c/em\u003e, due to potential disruptions in the promoter and coding sequences by transposon insertion, as observed previously in \u003cem\u003eSIX14\u003c/em\u003e in FOLac race 1 isolate AJ520, which was found to be unexpressed during infection due to transposon insertion [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Transcriptome analyses of JCP043 and AT141 during lettuce infection are needed to help identify race-specific genes that are highly expressed \u003cem\u003ein planta\u003c/em\u003e and to guide downstream functional characterization of candidate virulence genes to investigate their function in pathogenicity and host specificity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIs T2T genome assembly necessary?\u003c/h2\u003e\u003cp\u003eWhile obtaining a gapless, T2T complete assembly would be ideal for genomic and genetic research, we found that NECAT assembly provides an accurate estimate of genome size, content and composition of DNA repeats, and gene content, including correct copy numbers for \u003cem\u003eSIX\u003c/em\u003e genes (see Results for details). Moreover, generating NECAT assembly takes much less time than T2T assembly, which requires different types of bioinformatic tools and sequencing data to make it complete. Assembly using the NECAT program requires only one run of assembling ONT reads into contigs, followed by PacBio/Illumina-based error correction. Therefore, having a NECAT assembly can be an adequate solution for researchers interested in projects including pan-genome and basic repetitive sequence analyses. However, studies that aimed for identifying unique genes that may be associated with host specificity or detecting structural variants between individuals cannot be completed adequately without a T2T assembly. Short-read assembly, which has become a routine task for many research programs used in various downstream analyses, presents several additional limitations, including an incomplete set of predicted genes and inaccurate gene copy numbers, especially for those in ACs (see Results for details). Here we also cite cases where genes present in an assembly turned out to be absent based on read mapping (\u003cb\u003eSupplementary Figure S9\u003c/b\u003e), and also where sequences thought to be absent turned out to be present. The latter case can be explained by the fact that parts of those genes land on the end of different contigs. Therefore, it is recommended that a combination of BLAST and read mapping analyses be used for accurate gene prediction, if long-read assembly is inaccessible.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe present an assembly workflow that led to gapless, T2T complete genome assemblies for FOLac races 1 and 4, two devastating soilborne pathogens that have become increasingly prevalent in lettuce production areas worldwide. Comparative genomic analyses between the two isolates revealed major structural differences in the accessory genome regions and the potential involvement of \u003cem\u003eGypsy/DIRS1\u003c/em\u003e elements in chromosome duplication and translocation. We identified many putative race-specific genes that were uniquely present in one race while absent in three other races and warrant further investigation through transcriptome analyses during lettuce infection to understand their role in pathogenicity and host specificity. A comprehensive genomics study of multiple isolates representing all four races, along with a broad-spectrum phenotyping study to evaluate their host range and virulence, will allow us to reconstruct the evolutionary paths that led to host-specificity of \u003cem\u003eF. oxysporum\u003c/em\u003e towards lettuce and subsequent diversification. Ultimately, the information we gained from the genomics research will greatly advance the development of effective management strategies to control Fusarium wilts.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e\u003c/h2\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eCulture growth\u003c/h2\u003e\u003cp\u003eFresh fungal mycelia used for long-read sequencing were prepared as follows. FOLac race 1 isolate JCP043 and race 4 isolate AT141 were started on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) under dark incubation for seven days at 25°C. Six agar plugs from colony edges were transferred to two Petri plates containing 30 ml of potato dextrose broth (PDB; Difco Laboratories) and incubated unagitated for two days at 25°C. The resulting mother culture was blended with a sterile Waring laboratory blender (Conair LLC, Stamford, CT, USA) for 10 sec to make a slurry, which was then mixed with 1.2 L of PDB and distributed to 60 Petri plates for large-scale liquid culture. The plates were incubated unagitated for two days at 25°C. The resulting mycelia were harvested by centrifugation at 8,000 g for 15 min, washed twice with sterile distilled water, blotted dry, and flash frozen in liquid nitrogen before stored at -80°C.\u003c/p\u003e\u003cb\u003eHigh molecular weight (HMW) DNA extraction\u003c/b\u003e\u003cp\u003eExtraction of HMW DNA performed at the Michigan State University RTSF Genomics Core used a protocol adapted from the QIAGEN Genomic-tip protocol (QIAGEN, Germantown, MD, USA) while HMW DNA at the UC Davis DNA Technologies Core used a cetyl trimethyl ammonium bromide (CTAB)-based extraction protocol [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. DNA quantity and purity were determined with the Qubit 4.0 fluorometer (Thermo Fisher Scientific) and the Nanodrop UV-Vis 45 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA), respectively. Integrity assessment of the genomic DNA was performed using a Femto Pulse system (Agilent Technologies, Santa Clara, CA, USA) and the Agilent 4200 TapeStation (Agilent Technologies). Short DNA fragments (≤ 25 kb) for the PacBio Revio library were eliminated using Blue Pippin (Sage Sciences, 50 Beverly, MA, USA). DNA samples with Nanodrop ratios 260/280 between 1.8-2.0, 260/230 between 2.0-2.2 and molecular weight ≥ 50 kb were selected for sequencing.\u003c/p\u003e\u003ch2\u003eLong-read whole genome sequencing\u003c/h2\u003e\u003cp\u003eLong-read genome sequencing was carried out using HMW DNA on ONT PromethION and Pacific Biosciences HiFi platforms. The ONT sequencing was performed separately by two sequencing facilities, Michigan State University (for JCP043) and UC Davis (for AT141). Library for ONT sequencing was made using the Oxford Nanopore SQK-LSK114 Ligation Sequencing Kit V14 and run on a PromethION R10.4.1 flow cell. Sequencing was performed following manufacturer's recommendations. MinKNOW software v.22.10.7 was used for data acquisition and base calling was achieved using Guppy v.6.3.9. Total yields for JCP043 and AT141 were 122Gb (7\u0026nbsp;million reads; read length N50 of 32 kb) and 149Gb (11\u0026nbsp;million reads; read length N50 of 21 kb), respectively. Raw reads were filtered with a Q score ≥ 9.0 and a minimum length of 10k, resulting in a total of 5.45\u0026nbsp;million reads for JCP043 and 9.45\u0026nbsp;million reads for AT141. HiFi reads were generated by the UC Davis DNA Technologies Core, with sequencing of JCP043 performed on the Sequel II system while AT141 on the Revio system. Total yields for JCP043 and AT141 were 12.5 Gb (1.1 M reads; read length N50 of 12 kb) and 62.7 Gb (5.9 M reads; read length N50 of 13 kb), respectively.\u003c/p\u003e\u003ch2\u003eT2T assembly\u003c/h2\u003e\u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we developed a bioinformatic workflow that implemented a series of command-line and graphic user interface programs to assemble the two FOLac genomes, which is described in greater details in \u003cb\u003eSupplementary File S1\u003c/b\u003e. Briefly, the Nanopore data assembler, NECAT version 0.0.1_update20200803 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] was used to assemble ONT reads into a preliminary genome assembly, which was then visually inspected for mis-assembly by mapping 50–99 kb ONT reads to the contigs using CLC Genomics Workbench 22.0 (QIAGEN, Aarhus, Denmark; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://digitalinsights.qiagen.com/\u003c/span\u003e\u003cspan address=\"https://digitalinsights.qiagen.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SeqMan NGen 16.0 (DNASTAR, Madison, WI, USA). Each program uses a different approach for read mapping that complements each other. CLC Genomics uses a percent identity and fraction overlap approach while SeqMan NGen uses a \u003cem\u003ek-\u003c/em\u003emer approach. The mis-assembled contigs were split into sub-contigs at the breakpoints before subject to a second run of read mapping, followed by end extension using the “Extend contig ends” function of the Genome Finishing Module on CLC Genomics. Other contigs, which did not have misassembled regions but had no telomeric repeats on either or both ends, were also included in the end extension analysis. After this procedure was repeated multiple times, the extended contigs and sub-contigs were merged when there was a minimum of 50-kb overlap using the nucmer module of the MUMmer package version 3.23 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Once the T2T assembly was obtained, the continuity and correctness of the assembly was checked by re-mapping 50–99 kb ONT reads to the assembly using CLC Genomics, followed by repeating the read mapping using SeqMan NGen with a stringency setting of 95% and \u003cem\u003ek\u003c/em\u003e-mer size of 30. The final T2T assembly was polished with two runs of HiFi reads mapping, followed by two runs of Illumina HiSeq reads mapping using SeqMan NGen with a stringency of 99% and \u003cem\u003ek\u003c/em\u003e-mer size of 30. The NECAT contigs that contained the mt sequences were removed from the assembly, BLASTed against a reference mt genome of Fo47 (GenBank: LT906306.1) to identify a complete single-copy genome, and error corrected using Illumina data.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHi-C analysis\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo verify that the T2T assembly of JCP043 is correct with no mis-assembled contigs, a proximity ligation-based method called, Hi-C analysis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], was performed via Dovetail Omni-C library construction and sequencing on an Illumina HiSeqX platform by Cantata Bio. BWA-MEM version 0.7.17 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] was used to align the Omni-C reads to the T2T assembly of JCP043, followed by Hi-C analysis using the SALSA2 pipeline [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The resulting alignment file was converted to create a .hic file and then visualized with JuiceBox [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eGenome assessment\u003c/h2\u003e\u003cp\u003eQUAST version 5.3.0 [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] was used to assess the quality of the T2T assembly and to evaluate the genome completeness of the Illumina and NECAT assemblies with flags --eukaryote --fungus to specify a fungal organism, and --features set to gene to analyze gene content. Gene content of the assembly was determined based on the presence of BUSCOs using BUSCO version 5.4.5 [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], with the hypocreales_odb10 database.\u003c/p\u003e\u003ch2\u003eAnalysis of the repeat content of the genome\u003c/h2\u003e\u003cp\u003eWe identified and classified repetitive and low complexity regions in the genome using RepeatModeler version 2.0.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.repeatmasker.org\u003c/span\u003e\u003cspan address=\"http://www.repeatmasker.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, the repeat library was used to analyze repetitive regions, as well as soft-masking the genome using RepeatMasker version 4.1.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.repeatmasker.org/\u003c/span\u003e\u003cspan address=\"https://www.repeatmasker.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eWhole-genome comparison to identify core and accessory chromosomes\u003c/h2\u003e\u003cp\u003eCore and accessory genome regions and chromosomes of JCP043 and AT141 were identified based on whole-genome alignment with the Fol4287 reference genome (GCA_000149955.2) using the nucmer module of the MUMmer package with the parameter –max-match, -L 10000. The synteny plot was generated using TBtools-II v. 2.311 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eGene prediction and functional annotation\u003c/h2\u003e\u003cp\u003eAfter repeat masking, we annotated the genome using Funannotate version 1.8.16 [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The masked genome file along with the RNA-seq data generated from mycelia of JCP043 grown under nine different environmental conditions (see below) were used as inputs to Funannotate to train the gene prediction models, followed by funannotate predict and funannotate update commands to annotate untranslated regions (UTRs) and refine gene model predictions. InterProScan5 version 5.64-96.0 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] was used to assign functional annotation to predicted genes. Diamond blastp [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] was used to search UniProt DB v. 2023_04 [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] and MERPOP v. 12.0 [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] databases to aid in functional annotation and eggNOG terms were identified using eggNOG-mapper v. 2.1.12 [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Pfam domains were identified using PFAM v. 36.0 [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], and CAZymes were annotated using dbCAN v.12.0 [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Putative secreted proteins (secretomes) were identified through prediction of signal peptides using SignalP v.4.1 [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and removing those predicted to contain transmembrane domains using the DeepTMHMM v.1.0 web server [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The resulting secretomes were used to predict effector proteins using EffectorP 3.0 [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Secondary metabolite gene clusters were identified using the antiSMASH fungal v. 8.0 web server [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e] with the detection strictness set to relaxed. Prior to the antiSMASH analysis, the genome annotation file was filtered using AGAT v.1.4.3 [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e] to retain only the longest isoform per gene. Genomic features, including gene density, distribution of TEs, and genome duplication events were visualized using TBtools-II v. 2.311. To conduct functional enrichment analysis, we used GOATOOLS v1.5.1 [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. GO data v.1.2 (release date: 2025-07-22) was obtained from the Gene Ontology consortia [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].To avoid false positives, p-values were multi-test corrected using the Bonferroni method; the resulting adjusted p-values were subject to a significance threshold of 0.01.\u003c/p\u003e\u003ch2\u003eIdentification of putative race-specific genes\u003c/h2\u003e\u003cp\u003eTo identify putative race-specific genes for FOLac races 1 and 4, we identified unique genes in JCP043 and AT141 first. CDS transcripts of the predicted genes were clustered to create a non-redundant gene set using the CD-HIT program version 4.8.1 [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Based on the criteria that were previously applied in similar analyses [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], the analysis was carried out with minimum 90% identity and minimum 80% coverage as the thresholds for clustering genes (cd-hit -c 0.9 -s 0.8). Pairs of homologous genes between the two genomes were then identified from the non-redundant gene sets using the RBBH module of the MMseq2 software version 18.8cc5c [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e] with the parameters mmseqs easy-rbh --search-type 3 --min-seq-id 0.90 -c 0.8 --cov-mode 0. The synteny of the homologous genes in the core and accessory chromosomes was visualized using TBtools-II v. 2.311. The resulting non-homologous genes in JCP043 were extracted and BLASTed against the AT141 assembly using Geneious Prime version 24.0.7 (໿\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneious.com/\u003c/span\u003e\u003cspan address=\"http://www.geneious.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genes that were either absent in AT141 or partially present (below 90% identity or 80% coverage) were considered unique genes in JCP043. To validate their uniqueness to JCP043, raw Illumina reads of AT141 (SRR28734917) were aligned to the unique genes using SeqMan NGen with a stringency of 90% sequence identity and \u003cem\u003ek\u003c/em\u003e-mer size of 21. Read coverage of the alignments was visually inspected with SeqMan Pro. Similarly, the unique genes in AT141 were identified following the same procedure and validated with read mapping analysis using raw Illumina reads of JCP043 (SRR28734937).\u003c/p\u003e\u003cp\u003eTo further narrow down the unique genes that may be race-specific, the presence/absence analysis of the unique genes, including \u003cem\u003emimp\u003c/em\u003e-associated effectors (see below), for FOLac race 1 was carried out on four representative FOLac race 1 isolates (AJ520, AJ718, AJ865, and AT142), one isolate each for race 2 (F9501) and race 3 (FLK1001), and three FOLac race 4 isolates (AJ516, AJ592, AJ705), most of which had long-read assemblies. Details of the isolates can be found in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e. BLAST search was conducted by querying the unique genes to each genome assembly via Megablast (Max E-value = 1e-20, Match Mismatch = 1, -2, Gap Cost = linear) using Geneious Prime. A hit was considered present if the sequence identity and coverage were above 95%. The unique genes were also evaluated by read mapping analysis using SeqMan NGen for additional validation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003emimp\u003c/b\u003e\u003cb\u003e-associated effector proteins\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify putative effectors associated with \u003cem\u003emimps\u003c/em\u003e for each FOLac genome, we employed the FoEC2 pipeline [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which was designed specifically for identification of \u003cem\u003emimp\u003c/em\u003e effectors, with the parameters -g \u0026lt; genome_folder \u0026gt; and -a \u0026lt; annotation_folder\u0026gt;. Sequences of the resulting candidate effector protein set between 30 aa and 300 aa were then extracted from each genome and clustered using CD-HIT version 4.8.1 to create a non-redundant candidate effector set. To determine the presence/absence of JCP043 candidate effectors in AT141, initially, TBLASTN search using protein sequences of the candidate \u003cem\u003emimp\u003c/em\u003e effectors against the genome was performed, however the results seemed inaccurate due to introns that resulted in fragmented or incomplete hits. Instead, genomic DNA sequences of the non-redundant candidate \u003cem\u003emimp\u003c/em\u003e effectors of JCP043 were used to query against the genome of AT141 using Megablast on Geneious Prime, with an e-value cut-off of 1e\u003csup\u003e− 20\u003c/sup\u003e and a percentage identity and coverage threshold of 90% and 80%, respectively. To identify candidate \u003cem\u003emimp\u003c/em\u003e effectors that are race 1 specific, we included nine additional FOLac isolates (the same set used in race-specific gene analysis as described above) in this analysis. Similarly, the presence/absence of AT141 candidate effectors in JCP043 and putative race 4-specific \u003cem\u003emimp\u003c/em\u003e effectors were identified following the same procedure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eassessment of\u003c/b\u003e \u003cb\u003eSIX\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the \u003cem\u003eSIX\u003c/em\u003e genes present in the \u003cem\u003eF. oxysporum\u003c/em\u003e isolates used in this study (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e), BLAST search was conducted by querying the \u003cem\u003eSIX\u003c/em\u003e genes obtained from NCBI (\u003cem\u003eSIX1\u003c/em\u003e: MK906592.1; \u003cem\u003eSIX2\u003c/em\u003e: MK906595.1; \u003cem\u003eSIX3\u003c/em\u003e: MK906598.1; \u003cem\u003eSIX4\u003c/em\u003e:GQ268951.1; \u003cem\u003eSIX5\u003c/em\u003e: MK906607.1; \u003cem\u003eSIX6\u003c/em\u003e: MK906615.1; \u003cem\u003eSIX7\u003c/em\u003e:GQ268954.1; \u003cem\u003eSIX8\u003c/em\u003e: FJ755837.1; \u003cem\u003eSIX9\u003c/em\u003e: KC701447.1; \u003cem\u003eSIX10\u003c/em\u003e: MK906667.1; \u003cem\u003eSIX11\u003c/em\u003e: MK906677.1; \u003cem\u003eSIX12\u003c/em\u003e: MW160867.1; \u003cem\u003eSIX13\u003c/em\u003e: MK906693.1; \u003cem\u003eSIX14\u003c/em\u003e: KC701452.1) to each genome assembly via Discontiguous Megablast (Max E-value = 0.05, Match Mismatch = 2, -3, Gap Cost = 5, 2) using Geneious Prime. As for \u003cem\u003eSIX9\u003c/em\u003e, sequences corresponding to the four variants of \u003cem\u003eSIX9\u003c/em\u003e (\u003cem\u003eSIX9.1- SIX9.4\u003c/em\u003e), which were reported in FOLac race 4 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], were used as additional references to account for sequence diversity. A hit is valid if the query identity and coverage meet the 65% threshold. If the hit was similar to the reference (≥ 65% identity) but was low in coverage, Illumina reads of the target isolate were mapped to the corresponding \u003cem\u003eSIX\u003c/em\u003e reference to retrieve full-length sequence using SeqMan NGen with stringency cutoffs of 90% sequence identity and \u003cem\u003ek\u003c/em\u003e-mer size of 30. Sequences of the \u003cem\u003eSIX\u003c/em\u003e genes were aligned using MUSCLE 5.1 [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and then used as input for IQ-TREE v2.2.2.6 [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] with parameters ' -m MFP -B 1000 ' to create a phylogeny. The resulting maximum likelihood tree was visualized in FigTree, version 1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eRNA extraction and sequencing\u003c/h2\u003e\u003cp\u003eFor \u003cem\u003eF. oxysporum\u003c/em\u003e genome annotation, RNA-seq data obtained from PDB-cultured mycelia has been commonly used as the standard to train the gene prediction models. Considering that environmental factors (i.e. pH, nutrient composition, temperature and light) have significant impact on fungal gene expression [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e], we generated nine RNA-seq libraries from race 1 isolate JCP043 that was grown under different growth conditions (see below) to expand the expression of a wide variety of genes. Six agar plugs of five-day old JCP043 culture were inoculated in 30 ml PDB at 25°C for two days. The mycelia were washed twice with sterile water before blended with water. Ten milliliters of the mycelial slurry were transferred to each bottle containing 100 ml of a specific type of media listed below, mixed well, and then distributed to four petri plates to grow for 48 hours at 25°C in the darkness. The media included PDB (pH of 6), PDB with 4% NaCl, PDB adjusted to a pH of 9 with NaOH, PDB amended with 0.5% and 1% peptone, respectively, carboxymethyl cellulose (CMC), and filter-sterilized macerated lettuce crown extract, prepared according to the method described previously [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Additional growth conditions include mycelia grown in PDB for 20 h at 25°C before switching to 37°C for 4 h, and mycelia grown in PDB under 24-h light. Mycelia were collected from the nine growth conditions by removing liquid from the tissue using filtration, washing the tissue with sterile water twice, and blotting the tissue dry. The tissue mat was then partitioned into 2 ml Eppendorf tubes, each containing approximately 100 mg of wet tissue, and immediately flash frozen in liquid nitrogen. The frozen tissue was stored at -80C until the RNA was extracted.\u003c/p\u003e\u003cp\u003eRNA was extracted using TRIzol reagent (Life Technology, Karlsruhe, Germany) and purified with a Zymo RNA clean and Concentrate kit (Zymo, Irvine, CA, USA), according to the manufacturer’s instructions. The quality and quantity of purified RNA were determined using the NanoDrop spectrophotometer and Agilent 4200 TapeStation. All the RNA samples, which yielded RIN scores above 9, were used for sequencing. Libraries were prepared at the Michigan State University RTSF Genomics Core using the Watchmaker Genomics mRNA Library Preparation kit (Watchmaker Genomics, Boulder, CO) with IDT xGEN 10nt Unique Dual-Index primers (Integrated DNA Technologies, Coralville, IA) following manufacturer’s recommendations. Completed libraries were assessed for quality and quantified using a combination of Biotium AccuGreen High Sensitivity dsDNA (Biotium, Frement, CA, USA) and Agilent 4200 TapeStation. All libraries were barcoded, normalized, and pooled equimolarly for sequencing in an AVITI Cloudbreak Freestyle High Output flow cell in a 2 × 150 bp paired end format. Base calling was done by AVITIOS v3.2.0 and the output was demultiplexed and converted to FastQ format using Element Biosciences bases2fastq v2.1.0. Approximately 55–70\u0026nbsp;million high-quality (% ≥ Q30 reads was ∼90%) paired-end reads were obtained for each library with a total yield of 183.5 Gb of sequencing data.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCore chromosome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAccessory chromosome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFOLac\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elactucae\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eT2T\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTelomere-to-telomere\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eSIX\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eSecreted in Xylem\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eff. spp.\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eformae speciales\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ef. sp.\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eforma specialis\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransposable element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eONT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOxford Nanopore Technologies\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emt\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emitochondrial\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003erDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRibosomal deoxyribonucleic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSingle nucleotide polymorphism\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBUSCO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBenchmarking universal single-copy ortholog\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBasic local alignment search tool\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLTR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLong terminal repeat\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLINE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLong interspersed nuclear element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAZYme\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbohydrate-active enzyme\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSecondary metabolite\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003emimp\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMiniature impala\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlycoside hydrolase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePotato dextrose agar\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePotato dextrose broth\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMW\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh molecular weight\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTAB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCetyl trimethyl ammonium bromide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHi-C\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh-throughput chromosome conformation capture\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUTR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUntranslated region\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoding sequence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRibonucleic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCMC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecarboxymethyl cellulose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eaa\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAmino acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Mathieu Pel for supplying FOLac race 4 isolate AT141 and other international isolates of races 1and 4. We also thank the DNA Technologies Core at the UC Davis Genome Center and the Michigan State University RTSF Genomics Core for their assistance in long-read and Illumina sequencing. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. The USDA is an equal opportunity provider and employer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. JLS: Formal analysis, Investigation, Methodology, Software, Writing \u0026ndash; review \u0026amp; editing. SOD: Formal analysis, Methodology, Writing \u0026ndash; review \u0026amp; editing. EGT: Investigation, Writing \u0026ndash; review \u0026amp; editing. SMK: Data curation, Formal analysis. DMG: Conceptualization, Funding acquisition, Project administration, Supervision, Writing review \u0026amp; editing. FNM: Conceptualization, Funding acquisition, Project administration, Supervision, Investigation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by USDA ARS Project 2038-22000-016-00D, California Department of Food and Agriculture, Specialty Crop Block Grant Program (18-0001-059-SC, 21-0001-050-SF), National Science Foundation (DEB-1655980), and Pennsylvania State Agricultural Experiment Station Project (Project number: 4655). JLS is a Howard Hughes Medical Institute Awardee of the Life Sciences Research Foundation. EGT and SO are supported by the Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison with funding from the Wisconsin Alumni Research Foundation and the Department of Plant Pathology at the University of Wisconsin-Madison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated for this study can be found in the article and Supplementary Material. All raw sequencing data have been submitted to the NCBI under the BioProject ID RJNA1098703 with the accession numbers of SRR35856514- SRR35856527. The final assembled genomes are deposited under the same BioProject at NCBI. The assembled genomes and genome annotation files have been submitted to the online open access repository Figshare for peer-review only (https://figshare.com/s/60447a577ab459285236). Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJLS is an advisor to ForensisGroup Inc. JLS is a scientific consultant to FutureHouse Inc.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGordon TR, Martyn RD. The evolutionary biology of \u003cem\u003eFusarium oxysporum\u003c/em\u003e. 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Plant Dis. 2019;103:504\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PDIS-05-18-0814-RE\u003c/span\u003e\u003cspan address=\"10.1094/PDIS-05-18-0814-RE\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fusarium oxysporum f. sp. lactucae, Fusarium wilt, telomere-to-telomere, accessory chromosomes, transposable elements, effectors, Secreted in Xylem (SIX), comparative genomics, genome evolution","lastPublishedDoi":"10.21203/rs.3.rs-8100147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8100147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAccessory genome regions of plant pathogenic fungi, which are highly variable and consist of niche-adaptive genes, play a crucial role in shaping host-specific interactions but are notoriously difficult to assemble. \u003cem\u003eFusarium oxysporum\u003c/em\u003e causes some of the world\u0026rsquo;s most economically devasting diseases, however, understanding how it interacts with its host is hindered by challenges in assembly of accessory genome regions/chromosomes, even with long read sequencing technologies. \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elactucae\u003c/em\u003e (FOLac) races 1 and 4 possess highly similar core genomes but cause distinct virulence phenotypes on specific lettuce cultivars. The availability of fully assembled genomes for the two races is needed to advance our understanding of the genetic basis of pathogenicity and the evolutionary processes underlying the diversification of FOLac and other \u003cem\u003eF. oxysporum\u003c/em\u003e pathogens.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe developed an assembly workflow for generating gapless, telomere-to-telomere (T2T) complete genome assemblies for FOLac races 1 and 4. The T2T assemblies allowed for the identification of 16 chromosomes (5 accessory) and 20616 predicted genes for race 1 and 19 chromosomes (8 accessory) and 20292 predicted genes for race 4. Comparative genomics revealed major structural differences in their accessory genome regions, including genome rearrangement and large-scale chromosome duplication, with results suggesting transposable elements as the main drivers of those genomic changes. The analysis of \u003cem\u003eSecreted in Xylem\u003c/em\u003e (\u003cem\u003eSIX\u003c/em\u003e) effector gene profiles uncovered a similar presence/absence pattern among FOLac races 2\u0026ndash;4, distinguishing them from race 1. Searches for genes unique to each race resulted in the identification of 689 race 1- and 536 race 4-specific genes. Assembly and genomic features comparing T2T to contig-level Illumina assemblies showed that 17\u0026ndash;23% of genome sizes and ~\u0026thinsp;10% of predicted genes were missing from Illumina assembly, mostly within accessory genome regions.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eT2T assemblies revealed large-scale differences in accessory genome structure and content between two otherwise highly similar pathogenic races. These differences provide a framework for understanding evolutionary processes that led to the diversification of pathogens within \u003cem\u003eF. oxysporum\u003c/em\u003e on a fine evolutionary timescale, the identification of genes that may be responsible for host-pathogen interaction, and the identification of race-specific sequences useful for diagnostics.\u003c/p\u003e","manuscriptTitle":"Assembling telomere-to-telomere genomes of Fusarium oxysporum f. sp. lactucae provides a roadmap for studying genome and phenotype evolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 10:27:42","doi":"10.21203/rs.3.rs-8100147/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-24T06:26:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T14:30:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327600012431322642987532900425793828089","date":"2025-12-14T05:41:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T01:10:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302263421905209966127202848922185650982","date":"2025-11-21T13:29:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289646825184352877615603159325184990082","date":"2025-11-18T04:45:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-18T03:36:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-14T09:23:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-13T12:54:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-13T12:50:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-11-13T00:20:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43f7fb31-6880-4f20-b437-89ec36570389","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T15:59:55+00:00","versionOfRecord":{"articleIdentity":"rs-8100147","link":"https://doi.org/10.1186/s12864-026-12744-5","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-04-07 15:57:09","publishedOnDateReadable":"April 7th, 2026"},"versionCreatedAt":"2025-11-26 10:27:42","video":"","vorDoi":"10.1186/s12864-026-12744-5","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12744-5","workflowStages":[]},"version":"v1","identity":"rs-8100147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8100147","identity":"rs-8100147","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}