Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance

Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance | 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 Article Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance Yong Jia, Zhanghui Zeng, Yingjie Luo, Haifei Hu, Lan Lan, Baojin Guo, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6552063/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Citrus is an interesting model for plant genome evolution study due to its reticulate evolutionary history with frequent hybridization. Citrus changshan Y. B. Chang (Huyou) is a unique landrace first discovered in Zhejiang Province, China with premium fruit quality. The evolutionary origin of Huyou has puzzled local botanists. Here, we sequenced a 120-years-old “ancestral tree” of Huyou and assembled two haplotype-resolved genomes HY1 and HY2. Huyou displayed 3.07% genome heterozygosity level, the highest in published citrus genomes. K-mer-based genome tracing revealed that HY1 contained 87.8% mandarin, 7.3% pummelo, 0.2% citron origin, whereas HY2 had 85.0% pummelo, 2.9% mandarin, 0.3% citron, implying a clear hybridization origin. Phylogeny dating showed that HY1 (2.0Mya) and HY2 (2.18Mya) had diverged earlier than the split of Citrus clementina and Citrus reticulata , and the split of Citrus grandis and Citrus maxima , respectively. Further transcriptome analyses revealed a strong allelic dominance of HY2 over HY1 in root tissue and moderately in stem, leaf, flower, and fruits. We found that genes related to antioxidants biosynthesis and lipid metabolisms were most significantly affected by allelic imbalance. This first report of allelic imbalance in citrus species support Huyou as an interesting model to investigate genome evolution following distant hybridization. Biological sciences/Evolution/Molecular evolution Biological sciences/Computational biology and bioinformatics/Classification and taxonomy Citrus changshan-huyou Y. B. Chang evolutionary origin haplotype-resolved genome Hi-C highly heterozygous genome allelic imbalance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The genus Citrus belongs to the Aurantioideae subfamily of Rutaceae and contains a diverse array of species such as sweet orange ( C. sinensis ), mandarin ( C. reticulata ), pummelo ( C. grandis ), citron ( C. medica ), and lemon ( C. limon ). Their diverse flavors, nutritional benefits, and industrial applications have made them beloved fruits worldwide. Citrus ranks the third largest fruits in term of production volume ( https://www.statista.com/ ) and hold substantial agricultural, economic, and cultural values globally (Gmitter, et al. , 2012). Beyond their economic and cultural importance, citrus fruits present a fascinating subject for genomic and evolutionary studies due to their intricate origins and diversification. The genus Citrus is characterized by a reticulate evolutionary history, marked by frequent hybridization and polyploidization events (Wu, et al. , 2018). This complex evolutionary past has led to various cultivated species or landraces with different heterozygosity levels, making citrus an intriguing subject for genome evolution investigation. Citrus changshan-huyou Y. B. Chang (Huyou) is a unique Citrus species originated in the Changshan County of the Zhejiang Province in China (Li, et al. , 2019; Yin-bin, 1991). The earliest cultivation of Huyou has been recorded over 600 years ago, well before its massive planting and production over 100 years as a prominent landrace. Its fruit has a golden color and contains abundant components, such as amino acids, vitamins, naringin, limonin, citrate and other nutrients (Zhang, et al. , 2012; Guo, et al. , 2018; Sheng, et al. , 2017). These components not only invested the fruit unique aroma and taste that combines sweet, sour, and a bit of bitterness, but also health benefits, including lowering blood sugar, hepatoprotective and anti-inflammatory effects (Zhang, et al. , 2012; Guo, et al. , 2018; Jiang, et al. , 2019). These benefits have led to the widespread consumption of Huyou in Zhejiang and neighboring province. The economic importance and scientific values of Huyou are attracting increasing attention. A recent scientific dataset reported the haplotype-resolved genome assembly of a Huyou accession and transcriptome sequencing in 6 tissues (Miao, et al. , 2024). Despite the high quality Huyou genomic data, the dataset fell short on genomic origin and evolution analysis for this important natural hybrid citrus landrace. The domestication and evolutionary origin of Huyou remains to be characterized. Over the past decade, numerous citrus species have been sequenced, generating genomic data that has significantly advanced our knowledge of citrus biology and genome evolution. Key milestones include the sequencing and assembly of reference genomes for important citrus species such as sweet orange ( C. sinensis ) (Xu, et al. , 2013), clementine mandarin ( C. clementina ) (Wu, et al. , 2014), and pummelo ( C. maxima ) (Wu, et al. , 2014). These were followed by whole genome resequencing for large citrus germplasm collections that encompass diverse citrus wild, landraces, and cultivars lines (Wu, et al. , 2018; Wang, et al. , 2017). All these studies have highlighted a complex history of admixture and hybridization during citrus domestication. The sequencing projects indicate that modern citrus varieties are often the result of crosses between a few ancestral species such as mandarin, pummelo, and citron. Beyond the genomic insights into genome origin and evolution, citrus genome sequencing studies have also led to the identification of genes associated with important agronomic traits, including fruit quality, disease resistance, and stress tolerance. For example, studies have identified genes involved in flavor synthesis in lemon (Bao, et al. , 2023), disease resistance in trifoliate orange (Peng, et al. , 2020), and citric acid accumulation in oranges (Huang, et al. , 2023). Recently, citrus pangenome data for various citrus and citrus related species have been made available (Huang, et al. , 2023; Liu, et al. , 2022). These genomic resources allow researchers to delve deeper into the genetic basis of key traits, paving the way for more targeted and efficient breeding strategies (Liu, et al. , 2022; Humann, et al. , 2022). Haplotype-phased genome refers to a genome sequence where the two sets of chromosomes inherited from each parent are separately distinguished and assembled. The assembly of highly accurate haplotype-phased genomes is achieved through advanced sequencing technologies such as long-read sequencing (e.g., PacBio HiFi, Nanopore), Hi-C sequencing (chromosome conformation capture). It allows for the identification of allele-specific variations, structural variations, and gene expression patterns that would be obscured in a standard genome assembly. This is particularly crucial for genomic study on heterozygous organisms such as Citrus species, which are marked with frequent hybridization events. Recently, several haplotype-resolved genome assemblies have been generated, including C. limon (Mario, et al. , 2021), C. australis (Nakandala, et al. , 2023), C. changshanensis (Miao, et al. , 2024), Papeda (Wang, et al. , 2025), and a haploid genome assembly for pummelo (Wang, et al. , 2017), offering a more detailed view of their genetic makeup. In this study, we performed genome sequencing and assembly of a 120-years-old “ancestor tree” of Huyou currently still growing in Changshan, Zhejiang Province where Huyou was first discovered. We integrate high coverage Illumina short read and PacBio long read sequencing with Hi-C sequencing technology for the assembly of 2 haplotype-resolved chromosome scale genomes. This is followed by in-depth genome tracing, structural variation, and comparative genomic analysis with previously established citrus ancestor species to fully characterize the unique genomic features of Huyou and to resolve its evolutionary origin. Furthermore, assisted by previously published transcriptome data and the haplotype-resolved genome assemblies in this study, we comprehensively characterized the allelic imbalance across different tissues. Gene family expansion/contraction and transcriptome profiles for key genes from important metabolic pathways related to citrus fruit quality were also analyzed. We highlight the exceptional genome heterozygosity of Huyou compared to known Citrus genomes and the unique tissue-specific allelic imbalance in the root tissue. Our study supports Huyou as a novel genomic resource for citrus breeding and also an interesting model to investigate genome evolution following distant hybridization. Materials and Methods Plant materials and nucleic acid extraction Fresh leaves, shoots and fruits were collected from a 120-year-old C. changshan-huyou Y. B. Chang tree in Chengtan Village, Qingshi Town, Changshan County (118°30′E, 28°51′N), Zhejiang Province, China, in the spring of 2022 ( Fig. 1 A ) . These tissues were quickly frozen in liquid nitrogen and then stored at − 80℃ before DNA and RNA extraction. Total genomic DNA was extracted from the leaf tissue, and its quality was assessed by the Qubit 3.0 Fluorometer (Thermo Fisher, USA), while the total RNA from leaves, shoots and fruits was extracted using TRIzol Reagent (Invitrogen). The quantity and quality of the RNA were examined using NanoDrop One spectrophotometer (Thermo Fisher, USA) and an Agilent 2100 Bioanalyser (Agilent Technologies), respectively. Genome karyotype identification Karyotype analysis was conducted with fluorescence in situ hybridization (FISH) method as previously described (Wang, et al. , 2017; Zhang, et al. , 2022), and was performed by OMIX Technologies Corporation (Chengdu, China). Briefly, excised young root tips (2-3cm) of Huyou via tissue culture (Li, et al. , 2017) were treated with nitrous oxide gas for 2h under 1 MPa, then fixed in ice-cold 90% acetic acid for 10 min. After washing in water, the root tips were diced and digested in 1% pectolyase Y23 and 2% cellulase Onozuka R-10 (Yakult Pharmaceutical, Japan) for 1 h at 37℃. After digestion, the root sections were washed in 70% ethanol three times briefly, then broken by using a needle and vortexed at maximum speed in 100% ethanol for 30 sec at room temperature to separate cells from one another. The cells were collected at the bottom of the tube by centrifugation (4000r/min) and resuspended in glacial acetic acid. The cell suspension was dropped onto glass slides in a box lined with wet paper. A telomere-specific repeat probe 5′-(TTTAGGG) 6 -3′) was used to detect the number of intact chromosomes, and 5s rDNA and 18s rDNA repeat sequence probe were used to identify multiple copies of chromosomes. The fluorescence staining of the chromosomes was performed using 4′,6-diamidino-2-phenylindole (DAPI). After DAPI staining, the dispersed metaphase chromosome cells were counted under a fluorescence microscope (Zeiss LSM880, Germany). Genome size and heterozygosity estimation For genome profiling, the DNA library was constructed with insert sizes of ~ 300 bp using Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA), and subsequently sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), which generated 150 bp pair-end reads. The software FastQC (version: 0.20.1) was used to filter the original reads and discard the low-quality reads. A total of 57.05 Gb clean data were generated by Illumina sequencing and were used to perform the genome survey for Huyou. Reference-free estimation of genome size and heterozygosity were performed using a k-mer frequency approach implemented in GenomeScope 2.0 (Ranallo-Benavidez, et al. , 2020). PacBio, Hi-C and RNA Sequencing To generate long-read sequencing reads for Huyou, DNA libraries were prepared using the SMRTbell Express Template Preparation kit 1.0 following the PacBio 20-kb protocol ( https://www.pacb.com/ ), and sequenced on the PacBio Sequel II platform (Pacific Biosciences of California, Inc.). After the data quality control, by removing sequencing adaptors and filtering low-quality short read, and correcting errors via SMRT link (v8.0), 26.17 Gb of HIFI reads were obtained (Table 1 ). The generation of a Hi-C library was performed as described previously (Strijk, et al. , 2021). Briefly, the genomic DNA was random broken into 300–700 bp fragments via DNA cross-linking, restriction enzyme digestion, cohesive end repair, DNA cyclization, and DNA purification. Then the sample was sequenced on an Illumina NovaSeq 6000 platform (PE 150). After the removal of sequencing adaptors and low-quality reads using fastp software (v 0.21.0) (Chen, et al. , 2018), 31.77 Gb Hi-C clean reads were obtained. RNA sequencing (RNA-seq) samples were obtained by mixing equal amounts of RNA extracted from each tissue (leaf, shoot, and fruit) and used for cDNA library construction. After sequencing on the Illumina NovaSeq 6000 platform, approximately 10 Gb of sequencing data was obtained. De novo genome assembly and annotation Haplotype-resolved de novo genome assembling was performed using hifiasm software (Cheng, et al. , 2021) with HIFI reads and Hi-C phasing. The default k-mer size was changed 19 to obtain correct estimation heterozygous site coverage. The resulted haplotype-resolved genome contigs were further purged for duplicates using Purge_Dups (Guan, et al. , 2020) by aligning the HIFI reads back to the assembled genomes using minimap2 tool (Li, 2018). The purged haplotype genomes were scaffolded into chromosome groups using the pipeline implemented at https://github.com/WarrenLab/hic-scaffolding-nf , which employs Chromap (Zhang, et al. , 2021) and YaHS (Zhou, et al. , 2023) for sequence alignment and Hi-C scaffolding, respectively. Genome statistics were calculated using the assembly-stats tool ( https://github.com/sanger-pathogens/assembly-stats ). Then, the juciebox software (v1.11.08) (Durand, et al. , 2016) was used to draw Hi-C heatmap based on the interaction intensity and relative position between contigs. Following manual correction, the uniquely mapped and valid interaction paired-end reads were used to build the pseudochromosome sequences. The pseudochromosomes were sorted and oriented using C. sinensis as the reference by RagTag (Alonge, et al. , 2022). The quality of the assembled genomes was evaluated by Benchmarking Universal Single-Copy Orthologs against the eukaryota_odb10 database (BUSCO v.4.1.4) (Simao, et al. , 2015). Repeat sequences of the haplotype genomes were searched using an automated pipeline implemented by EarlGrey (Baril, et al. , 2024). The soft-masked genomes were used for gene model prediction using GeMoMa v1.8 (Keilwagen, et al. , 2019) based on homology and transcriptome evidence. For homology references, the gene models from three ancestor genomes (C. sinensis, C. ichangensis, C. medica) were used. For transcriptome evidence, the RNA sequencing reads were mapped to the masked genomes using STAR (Dobin, et al. , 2013) and the resulted alignment files were used as input for GeMoMa. For noncoding RNA annotation, the transfer RNAs (tRNAs) were annotated by tRNAscan-SE (v1.23) (Chan, et al. , 2021). Other non-coding ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) and microRNAs (miRNAs) were identified using the Infernal toolkits (Nawrocki, et al. , 2009) based on information from the Rfam database. Gene function annotations were performed using Interproscan for GO annotation and kofam_scan for KEGG annotation. GO and KEGG enrichment analyses for Huyou genome specific genes were performed using TBtools (Chen, et al. , 2020). The circos v0.69 (Krzywinski, et al. , 2009) was used to visualize genomic characteristic information, including gene density, GC density, TE density, gene density, and synteny block. Variants, synteny, and genome origin analyses The genomic variations and synteny across the haplotype genomes of Huyou and their ancestor genomes C. reticulata (mandarin, NCBI accession ID: ASM325862v1) (Wang, et al. , 2018) and C. maxima (pummelo, NCBI accession ID: ASM2964120v1) (Zheng, et al. , 2023) were analyzed using SyRI software (Goel, et al. , 2019). Genome alignment was performed using minimap2 (Li, 2018). The ancestral origin of Huyou haplotype genomes were determined using the k-part tool at https://github.com/sc-zhang/kPart . Variations between HY1 and HY2 were counted per 1 Mb windown based on the output from SyRI. For ancestor tracing, genomic data for 3 ancestor genomes ( C. reticulata , C. maxima , C. medica ) were downloaded and used as references. The ancestor of Huyou genomes were assigned for each 100 kb window using kPart with -k 21 parameter ( https://github.com/sc-zhang/kPart ). The percentages of each ancestor origin were calculated. The haplotype origin for each chromosome was also phased and classified using SubPhaser program with a similar k-mer based method (Jia, et al. , 2022). Orthologs inferences, phylogeny development, and gene family analyses Protein sequences of Huyou genomes and 19 other species, including Aquilegia coerulea , Mangifera indica , Vitis vinifera , Malus domestica Golden , Solanum lycopersicum , C. sinensis , C. reticulata , C. maxima , C. medica , C. clementina , Poncirus trifoliata , Fortunella hindsii , C. ichangensis , Atalantia buxfoliata , and Litchi chinensis Sonn (downloaded from Citrus Genome Database at https://www.citrusgenomedb.org/ ) (Liu, et al. , 2022) were used as input for OrthoFinder v2.3.12 ( https://github.com/davidemms/Ortho Finder) (Emms and Kelly, 2019) for orthologs inferences. For alternative transcripts, only the sequences for the longest transcripts were used. The obtained species tree was used as input for downstream Bayesian estimation of divergence time using mcmctree from PAML v4.9 toolkits (Yang, 2007). The CDS sequences for the identified single copy genes from Orthofinder were extracted. Codon-based sequence alignments were performed using a combination of muscle and seqmagick backtrans-align functions. The concatenated CDS alignment was trimmed using Gblocks before input into mcmctree. Five divergence time points derived from https://timetree.org/ were used to calibrate the species tree. The final obtained species tree from mcmctree and orthologous gene count results from Orthofinder were input into café for gene family expansion/contraction analyses. The gene family changes along the species tree were visualized using tvBOT (Xie, et al. , 2023) online tool at https://www.chiplot.online/tvbot.html . For gene copy number analysis, the gene counts for corresponding hierarchical orthologoups from Orthofinder outputs were used. Genome divergence time analyses The genome divergence time was assessed by Ks profile analyses using WGDI program (Sun, et al. , 2022). Each citrus genome was compared to itself following the WGDI instruction. Protein sequences for the primary transcripts of each species were used for blastp using diamond program (Buchfink, et al. , 2021). Syntenic blocks were identified using the build-in function in WGDI. Codon-based sequence alignments were performed using muscle (Edgar, 2004) and pal2nal (Suyama, et al. , 2006) programs. Ks values calculation for collinear genes using yn00 from PAML v4.9 program (Yang, 2007). Ks peaks at p-value ≤ 0.05 were identified and fitted using WGDI. The whole genome duplication event in Vitis vinifera (Jaillon, et al. , 2007) was used a reference. Transcriptome analyses Transcriptome RNA sequencing data in 6 Huyou tissues (root: SRR28430799, stem: SRR28430800, leaf: SRR28430798, flower: SRR28430801, unripe fruit: SRR28430803, ripe fruit: SRR28430802) were downloaded from a previous study (Miao, et al. , 2024). Raw reads were trimmed using fastp program with --qualified_quality_phred 20 and --length_required 30 parameters. The resulted clean reads were mapped to merged haplotype genomes using STAR program, then were quantified using RSEM (Li and Dewey, 2011) based on the annotated gene models in this study. The syntenic gene pairs between HY1 and HY2 were identified using MCScanX program (Wang, et al. , 2012). The comparison of syntenic genes expression was performed by calculating the log2 value of the ratio of HY1 gene expression to HY2 gene expression. Results Genome assembly and annotation of the 120 years-old “ancestral tree” of Citrus changshan-huyou Y. B. Chang In this study, a century-old Huyou tree (120 years old as of 2025) currently growing and protected in Chentang Village, Changshan County, after which Huyou was named by Mr. Yunbing Chang in 1991, was selected for genome sequencing (Fig. 1 A). This century-old tree was believed by many local Huyou growers and botanists to be the ancestor of most Huyou trees currently cultivated across Zhejiang province and was thus termed as the “ancestral tree”. Notably, this “ancestral tree” of Huyou, despite its old age, remains highly vigorous and productive with premium fruits to date (Fig. 1 A, photographed in 2024). To determine the chromosome number and ploidy level of Huyou, high-resolution metaphase chromosome preparation assay was performed using root tip cells from tissue cultured Huyou seedlings. Results showed that the genome of Huyou is composed of 18 chromosomes (2n = 2x = 18, Fig. 1 B), which was confirmed by FISH with 5S rDNA and 18S rDNA repeat sequence probes, and consistent with those reported in other citrus species (Wang, et al. , 2017). K -mer frequency distribution analysis ( K = 21) of Illumina short reads (57.05 Gb, ∼189 × coverage) showed a distinct bimodal profile with 3.07% heterozygosity (Fig. 1 C), among the highest across all citrus genomes reported to date (Wu, et al. , 2018; Miao, et al. , 2024). Due to its exceptionally high heterozygosity level, long-read PacBio HIFI sequencing (26.17 Gb, ~ 86 ×) and the Hi-C sequencing (31.77 Gb, 105 ×) were performed to obtain high-quality chromosome-scale reference assembly for Huyou. The de novo assembling using Hifiasm with HIFI long reads and Hi-C reads as inputs produced two haplotype genomes HY1 (324 contigs, total size at 326.65 Mb with contig N50 of 21.15 Mb) and HY2 (235 contigs, total size at 322.79 Mb with contig N50 of 18.77 Mb) (Table 1 ). GC contents for HY1 (56.6%) and HY2 (55.02%) were comparable with each other. The largest contigs for HY1 and HY2 were at 46.45 Mb and 34.26 Mb, respectively. The assembled contigs were anchored into 9 pairs of chromosomes with Hi-C scaffolding. Distinct chromosomal groupings on HI-C interaction heatmap were observed in Fig. 1 D, indicating the high quality of genome anchoring. The chromosomes were further ordered and named using C. sinensis (v3.0 from the Citrus Genome Database http://citrus.hzau.edu.cn/download.php ) as reference. Particularly, 91.8% and 94.4% of the contigs for HY1 and HY2, respectively, could be assigned to chromosomes. Using the eukaryota_odb10 database as reference, the Benchmarking Universal Single-Copy Orthologs (BUSCO) revealed completeness of 98.1% and 97.7% for HY1 and HY2, respectively. Using HY1 as the reference, genome alignment of HY1 and HY2 revealed overall well-conserved synteny between the 2 haplotype genomes, albeit some large structural variations were observed mainly in the central region of each chromosome, such as a large insertion in chr4 and large inversions in chr8 and chr9 (Fig. 1 E). Despite the overall synteny, the mosaic pattern and gaps in the genomic alignment plot indicated significant genetic variations between HY1 and HY2 (Fig. 1 E), consistent with the exceptional high heterozygosity in Huyou observed in this study. Repetitive sequences were identified in the two haplotype genomes HY1 (181.5 Mb or 55.56%) and HY2 (179.4 Mb or 55.58%) (Table 1 ). In both genomes, LTR represented the largest TE family, followed by DNA, LINE, Rolling Circle, sequentially ( Supplementary File S0 ). Despite a relatively smaller genome size and smaller total TE content, HY2 displayed higher number of TEs in all TE classes than that in HY1 (Table 1 ). The repeat sequences were masked before gene model prediction using both homology search ( C. sinensis , C. ichangensis , C. medica ) and RNA sequencing data (10 Gb), which led to the identification of 42091 and 42050 protein-coding genes for HY1 and HY2, respectively, with comparable mean length for transcripts, CDS, exon, and average exon number per gene (Table 1 ). HY1 displayed relatively higher number of micro-RNA (161 miRNA) and tRNA (1003) but relatively lower number of small nuclear RNA (1393 snoRNA) than that in HY2 (150 miRNA, 790 tRNA, 1463 snoRNA). However, the number of ribosomal RNA in HY2 (2686 rRNA) was much higher than that in HY1 (1590 rRNA). The distribution of TEs, genes, and non-coding RNAs in HY1 and HY2 were displayed in Fig. 2 A. Overall, we observed higher gene density in the distal regions of each chromosome compared to the central region, whereas TEs tend to be enriched in the central regions. In summary, we obtained 2 haplotype-resolved Huyou assemblies with high quality and completeness. Table 1 The statistics for genome assembly and gene annotation of Huyou. HY1 and HY2 refers the two haplotype genomes. Sequencing platform Illumina, PacBio, Hi-C Assembly HY1 HY2 Chromosome number 9 9 Total genome size 326.65 Mb 322.79 Mb Size anchored to chromosome 299.73 Mb / 91.8% 304.81 Mb / 94.4% Number of contigs 324 235 N50 contigs 21.15 Mb 18.77 Mb Largest contig 46.45 Mb 34.26 Mb Complete BUSCOs 98.1% 97.7% GC content 56.60% 55.02% Genome annotation Number of protein-coding genes 42091 42050 Mean mRNA length 2570 bp 2554 bp Mean CDS length 1245 bp 1245 bp Mean exons per gene 5.75 5.77 Mean exon length 216.40 215.87 Number of TEs DNA 14097 15545 LINE 6762 7428 SINE 248 269 LTR 50487 56301 Total size of TEs 181.5 Mb / 55.56% 179.4Mb / 55.58% Number of non-coding RNA miRNA 161 150 tRNA 1003 790 rRNA 1590 2686 snoRNA 1393 1463 Ancestral tracing of Huyou haplotype genomes HY1 and HY2 Due to the high heterozygosity level and significant structural variations between the haplotype-resolved genomes, the ancestral origin of each block (100 kb) of the HY1 and HY2 genomes were determined using a k-mer based tracing approach. The distribution of differential k-mers of homologous chromosomes indicated that HY1 and HY2 could be clearly separated into 2 distinct progenitors (Fig. 2 B), except for chr9 in HY1 which displayed a mixture origin and was clustered with HY2. Using the citrus ancestor genomes mandarin ( C. reticulata ), pummelo ( C. maxima ), and citron ( C. medica ) as references, the genomic origin of HY1 and HY2 were traced and quantified. Results showed that 87.8% of HY1 genome originated from mandarin, followed by 7.3% from pummelo, 0.2% from citron, and 4.7% unknown source (could not be explicitly assigned) (Table 2 , Supplementary File S1 ). In contrast, 85.0% of HY2 genome was derived from pummelo, followed by 2.9% from mandarin, 0.3% from citron, and 11.9% from unknown source (Table 2 , Fig. 2 A). These results strongly suggested that Huyou may have originated from a hybridization between mandarin (HY1) and pummelo (HY2). At the chromosome level, chr1-chr7 of HY1 were predominantly from mandarin, whereas chr8 (11.9% pummelo) and chr9 (54.1% pummelo) of HY1 contained significant components from pummelo, which may have resulted from chromosome recombination. Mapping the genomic origin to the chromosomes indicated clear evidence of chromosome recombination in HY1 genome, whereby one distal region of chr8 and the central chr9 were replaced by pummelo genome (Fig. 2 A). For the HY2 genome, all chromosomes were predominantly from pummelo, with minor introgression of mandarin on chr1 (3.3% mandarin), chr3 (7.6%), chr5 (4.8%), chr8 (1.2%), and chr9 (3.6%) (Table 2 ). In contrast to HY1 which retained clear segmental recombination boundary, the genome introgressions in HY2 were generally mosaic along the chromosomes (Fig. 2 A), which may provide further clue on the evolution history of Huyou following the hybridization of its pummelo and mandarin parents. Furthermore, the LTR insertion age analysis also indicated distinct profiles for HY1 and HY2 (Fig. 2 C), providing additional support for two divergent progenitors. Specifically, most of the LRTs in HY1 displayed concentrated age with a single peak of insertion time, whereas the insertion time of LRTs in HY2 was relatively dispersed and contained an additional minor peak at a much older age (Fig. 2 C). Table 2 The ancestral origin of each chromosome of haplotype-resolved Huyou. Genome tracing was implemented using a k-mer-based approach by mapping sequencing data of three reference ancestral genomes mandarin ( C. reticulata ), pummelo ( C. maxima ), and citron ( C. medica ) to HY1 and HY2 using 100kb window. See the method section and Supplementary File S1 for details. Chromosome Length (Mb) ໿Percentage (%) of sequences from ancestor species Origin tracing pummelo citron mandarin Unknown Closest Ancestor HY1 H1chr1 29.23 0.00 0.03 9.34 0.38 clementina mandarin H1chr2 32.43 0.03 0.00 10.65 0.13 clementina mandarin H1chr3 39.69 0.10 0.00 11.71 1.43 clementina mandarin H1chr4 29.54 0.00 0.01 9.68 0.17 sinensis mandarin H1chr5 50.35 0.10 0.03 16.06 0.60 reticulata mandarin H1chr6 25.60 0.00 0.00 8.44 0.10 sinensis mandarin H1chr7 29.80 0.00 0.00 9.88 0.07 sinensis mandarin H1chr8 30.36 1.20 0.03 8.37 0.52 sinensis mandarin H1chr9 32.74 5.91 0.03 3.65 1.33 sinensis pummelo Total 299.73 7.34 0.15 87.78 4.73 - - HY2 H2chr1 30.06 8.25 0.03 0.33 1.25 maxima pummelo H2chr2 34.26 9.89 0.00 0.13 1.21 grandis pummelo H2chr3 35.93 8.46 0.07 0.90 2.36 grandis pummelo H2chr4 35.11 10.63 0.00 0.23 0.66 grandis pummelo H2chr5 49.49 13.12 0.13 0.79 2.20 grandis pummelo H2chr6 24.99 8.10 0.00 0.00 0.10 grandis pummelo H2chr7 28.27 8.49 0.00 0.00 0.79 grandis pummelo H2chr8 33.75 9.55 0.03 0.13 1.36 grandis pummelo H2chr9 32.95 8.45 0.03 0.39 1.94 grandis pummelo Total 304.81 84.95 0.30 2.90 11.86 - - Phylogeny dating and gene family analyses of HY1 and HY2 To deduce the divergence time of HY1 and HY2, single copy genes (2754 in total, Supplementary File S2 ) for 13 previously published citrus genomes and 6 citrus-related genomes, together with HY1 and HY2 were identified and used for phylogeny development. Five calibration points (annotated in tree in Fig. 3 A: 3.49 ~ 7.39, 3.92 ~ 11.0, 12.8 ~ 13.0, 7.57 ~ 25.92, and 13.5 ~ 30.0 Mya) were applied to estimate the divergence time. As shown in Fig. 3 A, the 6 citrus-related species were resolved as basal branches (group F), followed by the early diverging citrus species P. trifoliata (group G). The rest citrus species could be divided into 5 major groups (A-E highlighted in Fig. 3 A). Three of those corresponded to the well-recognized citrus ancestors: mandarin (A: C. reticulata ), pumelo (C: C. maxima ), and citron (E: C. medica ). Consistent with the genome tracing results, HY1 and HY2 were grouped with mandarin and pumelo, respectively (Fig. 3 A). The divergence time of HY1 was estimated at 2.0 Mya, earlier than the split between C. clementina and C. reticulata but after the divergence of C. sinensis , while HY2 was estimated to diverge at 2.18 Mya, before the split between C. grandis and C. maxima and after the branching of C. hongheensis (Fig. 3 A). Therefore, HY1 and HY2 represented 2 novel genomes that has not been reported before. Using the earliest diverging Poncirus trifoliata as reference, ks peak fitting showed that HY1, HY2, and other Citrus species displayed a single ks peaks at similar position (Fig. 3 B), implying no additional large scale whole genome duplication events. Gene family analyses based on the developed phylogeny revealed more than 3-fold expansion (+ 345) than contraction (-102) in the branch leading to HY1. In contrast, slightly more expansion (+ 255) than contraction (-162) was observed in the branch leading to HY2 (Fig. 3 A). The percentage of single copy genes in HY1 and HY2 (6.5%) were relatively lower than that in other citrus species (8.1% ~ 12.1%), probably caused by genome annotation bias (bar plot in Fig. 3 A). Relatively higher percentage of HY2-specific genes (0.6% or 261) was identified than that in HY1 (0.4% or 158). The numbers of genes for 5 important metabolic pathways (map00900: terpenoid biosynthesis, map00906: carotenoid biosynthesis, map00940: phenylpropanoid biosynthesis, map00941: flavonoid biosynthesis, and map04626: plant-pathogen interaction) related to fruit quality were counted (heatmap in Fig. 3 A; Supplementary File S3 ). Overall, we observed a general expansion of gene members in all target pathways for citrus species, compared to the basal citrus-related species and the early diverging species P. trifoliata , implying a potential human selection impact. Particularly, we noticed that group A and C encompassing HY1 and HY2 contained the most expanded gene members compared to other citrus species, except for C. maxima Huazhouyou instead displayed relatively contracted gene families in these pathways. Further analysis is needed to investigate if this is caused by genome annotation bias or not. Noteworthy, a total of 158 and 261 species-specific genes were identified for HY1 and HY2, respectively ( Supplementary File S4 ). Enrichment analyses showed that gene ontology (GO) terms related to photosynthesis, signal transduction, and response to stimulus were significantly enriched in the HY1-specific genes (Fig. 3 C). In contrast, no significantly enriched GO term could be detected in the HY2-specific genes. Synteny and structural variations analysis of HY1 and HY2 The synteny and structural variations of HY1 and HY2 were inferred in reference to their ancestral genomes: mandarin and pummelo, respectively (Fig. 4 A). Overall, we observed much less large structural variations between mandarin and HY1, compared to that between HY1 and HY2, consistent with our ancestor tracing results that HY1 was derived from mandarin. For example, the large inversions identified between HY1 and HY2 on chromosomes chr6, chr7, and chr8 were not observed between mandarin and HY1, providing direct evidence that HY1 may have originated from a mandarin ancestor. Similar patterns could be found for other chromosomes and other structural variations as well. In contrast to the strong synteny conservation between HY1 and its ancestor genome mandarin, we observed significant large structural variations between HY2 and the pummelo genome ( C. grandis L. Osbeck.cv. Wanbaiyou) (Fig. 4 A). Noteworthy, these large structural variations such as the inversions on chromosomes chr1, chr5, and chr9 were specific to the pummelo genome used in this study but not present between HY1 and HY2 (Fig. 4 A), implying that these large structural variations between HY2 and pummelo were mainly due to genome re-arrangement in C. grandis L. Osbeck.cv. Wanbaiyou but not in the pummelo ancestor. Further analyses are needed to investigate if these large genome re-arrangements are conserved in other pummelo genomes or not. The genomic variations between HY1 and HY2 were examined specifically to investigate the allelic diversity in Huyou. We identified 1,996,631 SNPs, 150,487 insertions (1.66 Mb), and 139,519 deletions (1.57 Mb) ( Supplementary File S5 ). These variations led to the classification of 25,503 highly diverged regions, accounting for (~ 163.49 Mb or 50%) of the HY1 genome, consistent with heterozygosity. Despite these divergent regions, majority of the genome (252.08 Mb or 77.2%) were found conserved in syntenic regions, implying a generally conserved synteny between the ancestral genomes for HY1 and HY2 before their hybridization that resulted in Huyou. For structural variations, we identified a total of 73 inversions (21.04 Mb), 592 translocations (6.35 Mb), and 938 duplications (8.14 Mb), which were mainly located in the central region of each chromosome ( Supplementary File S5 ). Three large inversions between HY1 and HY2 were observed in the middle regions of chr9 and chr8, followed by a few moderate inversions on chr1, chr2, chr3, chr4 and chr6. In addition, a genomic region in the middle of chr5 in HY2 seems to be expanded through significant duplications, which may have contributed to the chromosome extension of chr5 in HY2 compared to chr5 in HY1 (Fig. 4 A). Lastly, the number of genetic variations between HY1 and HY2 were calculated per 1 Mb window along each chromosome. The obtained variation distribution profile revealed a relatively higher-level of genetic diversity in the distal regions of each chromosome compared to the central regions (Fig. 4 B). Mapping the genes from the 6 important metabolic pathways (map00020- Citrate cycle, map00195- Photosynthesis, map00900- terpenoid biosynthesis, map00906- carotenoid biosynthesis, map00940- phenylpropanoid biosynthesis, map00941- flavonoid biosynthesis) to the genome showed that many of these genes were located within the identified divergent regions, suggesting a high allelic diversity for important fruit quality characters in Huyou. Allelic imbalance of gene expression profiles in different Huyou tissues As the most heterozygous citrus genome to date, Huyou provides an invaluable opportunity to study genome evolution. To investigate the allelic imbalance post ancient hybridization, we retrieved public transcriptome data of 6 different tissues (root, stem, leaf, flower, unripe fruit, ripe fruit) in Huyou from a previous study (Miao, et al. , 2024) and compared the expression levels of collinear genes (26,092 in total) between HY1 and HY2. The percentages of genes preferentially expressed in HY1 (HY1higher (log 2 (HY1/HY2) ≥ 1) and HY2 (HY2higher log 2 (HY1/HY2) ≤ -1) were calculated across each chromosome and tissue ( Supplementary File S6 ). Overall, we observed relatively higher numbers of HY2-preferential genes than HY1-preferential genes across most tissues and chromosomes (Fig. 5 A), implying a clear transcriptional bias for HY2 genes following hybridization. Noteworthy, we found that the HY2 allelic dominance was exceptionally prominent in root tissue (HY2higher at 12.5% versus HY1higher at 7.24%) compared to that in the other tissues (leaf, stem, flower, unripe fruit, ripe fruit), which only displayed slight HY2 allelic dominance (11.67% versus 11.10% on average), implying a strong tissue-specific pattern. The exceptional HY2 allelic dominance in root tissue across the 9 chromosomes could be seen in Fig. 5 B, relatively more evident on chromosomes chr1-7 and relatively mild on chr8 and chr9. This might be caused by the large segmental replacements observed on chr8 and chr9. Indeed, at the chromosome level, chr9 displayed the lowest level of total allelic divergence (HY1higher + HY2higher = 16.77%) and followed by chr8 (21.29%) and other chromosomes (22.30-24.27%). Due to the exceptional HY2 allelic dominance in the root tissue, the collinear genes with transcriptional preference from HY1 (1354 HY1higher genes) and HY2 (2432 HY2higher genes) were used for KEGG pathway enrichment analysis (corrected p-value ≤ 1-e5). Interestingly, these two sets of genes shared some commonly enriched pathways that are mainly related to antioxidants biosynthesis, including flavonoid, phenylpropanoid, isoflavonoid, carotenoid, benzoxazinoid, flavone, flavonol, alkaloid, ascorbate, and steroid biosynthesis (Fig. 5 C). It is well-known that antoxidant biosynthesis plays critical role in plants’ adaptation to various biotic and abiotic stresses. The allelic imbalance of genes within these pathways in the root tissue may have important implication in Huyou’s environmental adaptation following its ancient genome hybridization. In addition to these common pathways, HY1 preferential genes were uniquely enriched with amino acid and linoleic acid metabolism and the biosynthesis of cutin, wax, diterpenoid, triterpenoid, sesquiterpenoid, stilbenoid, diarylheptanoid, and gingerol which are closely linked to environmental adaptation (Fig. 5 C). In contrast, HY2 preferential genes were uniquely enriched with protein families related to signaling and cellular processes, including transporter genes, sphingolipid metabolism, and genes responsible for fructose, mannose, pentose, glucuronate interconversions, glycerolphospholipid, peptidoglycan metabolism. In addition, the biosynthesis of anthocyanin, folate, and various secondary metabolites were also enriched in HY2 preferential genes but not in HY1 preferential genes. In addition to the root tissue, the allelic imbalance genes from HY1 (2053HY1higher genes) and HY2 (2084 HY2higher genes) in ripe fruit tissue were also investigated. Overall, we observed a strikingly similar enrichment profiles with that in the root tissue (Fig. 5 D), suggesting that the allelic imbalance at the gene functional level may be conserved across tissues despite the variation in gene numbers. In details, like that in the root tissue, the commonly enriched pathways from HY1 and HY2 were also prominently related to antioxidants biosynthesis such as flavonoid, phenylpropanoid, isoflavonoid, flavone, flavonol, isoquinoline alkaloid, carotenoid, benzoxazinoid, and glucosinolate. Other common pathway terms included lipid, cofactors and vitamin, amino acid, porphyrin and chlorophyll metabolism which were also detected in the root tissue (Fig. 5 D). The unique pathway terms in HY1 (linolenic acid metabolism and the biosynthesis of cutin, suberine, wax, stilbenoid, diarylheptanoid, gingerol, sesquiterpenoid, triterpenoid) and HY2 (tryptophan, ascorbate, aldarate, sphingolipid, pentose, glucuronate, terpenoids metabolisms and the biosynthesis of secondary metabolites, anthocyanin, folate, peptidoglycan) in ripe fruit tissue were also similar to that in the root tissue (Fig. 5 D). Tissue-specific allelic imbalance in key pathways Carotenoid biosynthesis pathway (map00906) plays a critical role in citrus fruit quality, controlling fruit color and flavor. To investigate the genomic characteristics of key Huyou candidate genes in this pathway, we first investigated the allelic imbalance by mapping the ratio of HY1 and HY2 collinear gene transcription to this pathway. Whilst the major components of the pathway displayed no allelic preference, we observed a clear tissue-specific allelic imbalance for some key genes responsible for the astaxanthin biosynthesis specifically (Fig. 6 ). For instance, we found one enzyme (EC 1.14.15.24) encoding beta-carotene 3-hydroxylase displayed a consistent HY2 dominance in the root tissue but a HY1 dominance in the stem, flower, unripe fruit, and ripe fruit tissue and no imbalance in leaf. Another enzyme (EC 1.23.5.1) encoding violaxanthin de-epoxidase displayed HY1 dominance in root, leaf, ripe fruit but no imbalance in the other tissue. Conversely, the enzyme (EC 1.2.3.14) encoding abscisic-aldehyde oxidase display HY2 dominance in stem, leaf, and unripe fruit but not in other tissue. These observations provided direct support of various allelic imbalance present in Huyou genome and shed valuable insights into genome evolution following ancient hybridization. To characterize the complete profile of the carotenoid biosynthesis pathway in Huyou, we further extracted the gene copy number information of key genes (represented by 39 KEGG orthologs) in HY1, HY2, and another 8 citrus species and explored their transcriptional profiles in 6 Huyou tissues. Overall, we observed conserved gene presence and copy number pattern across different citrus species, with cytochrome P450 (CYP701, CYP97C1, CYP707A, CYP97A3, CYP82G1) as the most expanded protein families within the carotenoid pathway. Copy number variations (CNVs) were observed in all KO orthologs except for 2 single copy genes K15744 (zeta-carotene isomerase) and K17911 (beta-carotene isomerase) (Fig. 7 A). Similarly, HY1 and HY2 displayed varied copy numbers in 33 out of the 39 KO orthologs. Noteworthy, Huyou (HY1 or HY2) displayed the highest copy numbers in 21 out of the 39 KO orthologs (Fig. 7 A). At the gene expression level, we observed varied transcriptional profiles across the 6 tissues investigated (Fig. 7 B). Interestingly, we found that over half of the KO orthologous genes in Huyou displayed the highest expression in the flower tissue, followed by the root tissue. Instead, out of the 39 KO orthologs, we found 17 KOs displayed above median transcription in fruit tissues (either unripe or ripe), of which 6 KOs (FDFT1, LCYB, LCYE, CCS1, AOG, DWARF27) displayed the highest expression in fruit tissues (highlighted in red rectangles in Fig. 7 C), which require more attention for their potential critical roles in fruit development, despite that some of these genes may not be expanded in Huyou (Fig. 7 C). At the pathway map level, we found most of the genes leading to the terpenoid biosynthesis (C30, C15, C5, C10, C20) were found expanded compared to other species, whilst this pattern was not prominent for the carotenoid biosynthesis (C40) (Fig. 7 C). Taken together, CNV and transcriptome analyses revealed unique genomic characteristics for the carotenoid biosynthesis pathway in Huyou which may have contributed to its distinct fruit flavor against other citrus species. The ratios of HY1 and HY2 collinear genes (log2 (HY1/HY2) ) were mapped to KEGG pathway map00906 using TBtools. Transcriptome data in 6 Huyou tissues (root, stem, leaf, flower, unripe fruit and ripe fruit) from a previous study was downloaded and analyzed. Yellow, red, and green highlights indicated neutral, HY1 dominant, and HY2 dominant, respectively. Only the Astaxanthin biosynthesis module of map00906 were displayed. Discussion As a unique natural hybrid citrus landrace first discovered and widely grown in Zhejiang province in China, Huyou holds important agricultural, economic, and medicinal values due to its premium fruit quality characteristics such as a golden skin, abundant bioactive components, extended storage life. In this study, we leverage PacBio long-read and Hi-C sequencing technologies and presented 2 high-quality haplotype-resolved genome assemblies HY1 and HY2 of 120 years old Huyou “ancestral tree”. Our in-depth ancestor tracing, phylogeny dating, genome structure, and transcriptome analyses offered an unprecedent view of Huyou’s genetic makeup and evolutionary origin. The explicit determination of Huyou’s genome origin from a mandarin and pummelo hybridization event solved a long biological mystery that has puzzled local Huyou growers and botanists (Li, et al. , 2019; Chen, et al. , 2002; Chen, et al. , 2006; Xu, et al. , 2006). Based on whole genome survey of 58 worldwide diverse citrus germplasms, hybrid citrus species derived from 2 or more ancestral species have been found to have generally high genome heterozygosity at 1.5–2.4% (Wu, et al. , 2018). Here, we highlight the exceptional genome heterozygosity of Huyou at 3.07%, among the highest across known citrus genomes, which will make Huyou an interesting subject for genome evolution studies by broad citrus researchers in the future. The haplotype genomes HY1 and HY2 obtained in this study essentially represent 2 novel ancestral citrus genomes not reported before, thus would significantly contribute to the currently limited citrus genetic pool. This study will enable citrus breeders to harness the genetic potential of Huyou more effectively, potentially accelerating the development of improved citrus varieties with unique traits. The explicit resolution of Huyou’s genome origin in this study highlights the power of haplotype-resolved genomes in dissecting the complex evolutionary origin of citrus species. In the citrus genus, there are a total of 30 species, most of which have been derived from the hybridization across 3 ancestral species: mandarin ( C. reticulata ), pummelo ( C. maxima ), citron ( C. medica ). The ancient hybrids were subjected to further reticulate hybridization during citrus domestication, leading to a complex history of admixture in current citrus cultivars and landraces characterized by high heterozygosity (Wu, et al. , 2018). Tracing the evolution and domestication history of citrus species has been extremely challenging in traditional citrus genome studies based on short read sequencing, which often produced a mosaic genome assembly and failed to account for allelic variants, leading to incomplete or inaccurate representations of the genome. By leveraging a k-mer-based tracing approach on the haplotype-resolved genomes, we are able to resolve and quantify the ancestral components of Huyou to specific genomic regions. In addition, the haplotype genome tracing also allows us to dissect the segmental insertions, deletions, inversions, recombinations and other structural variations between HY1 and HY2, particularly the recombination events on chr8 and chr9 in HY2. These unprecedented genomic insights are critical for the accurate interpretation of the genomic and phenotypic features of Huyou. Our study was inspired by a recent study in which the haplotype-resolved lemon genome was traced to be an admixture of 3 ancestor genomes of citron, mandarin, and pummelo (Bao, et al. , 2023). Notably, numerous studies highlight the increasing trend of generating chromosome-level and haplotype-resolved genome assemblies across the plant kingdom, such as potato, lychee, tea, and strawberry. The advantages of haplotype-resolved genome for citrus genome are being increasingly appreciated in several recent citrus genome research including C. limon (Mario, et al. , 2021), C. australis (Nakandala, et al. , 2023), C. changshanensis (Miao, et al. , 2024), Papeda (Wang, et al. , 2025). However, we noticed that only the collapsed genome sequence was reported and analyzed in several haplotype-resolved genome studies, which in our opinion should be avoided, particularly for those organisms with high heterozygosity such as Citrus species. We expect an increasingly more haplotype-resolved citrus genomes to be generated and published, which is positioned to revolutionize the citrus genome evolution and domestication research. Allelic imbalance (AI) refers to the unequal expression of alleles from a heterozygous locus, leading to differential gene expression. This is another critical insight gained from haplotype-resolved genome assembly. In this study, based on gene expression analysis of collinear genes between HY1 and HY2, we observed clear evidence of AI (HY2 dominant over HY1) across all 6 tissues investigated, suggesting AI may be a prevalent phenomenon in genome evolution in the plant kingdom. One of the most notable findings in this study is that we observed AI tends to be imbalanced across tissues, i.e the HY2 allelic dominance was much more prominent in some tissues such as root over the others. Metabolic pathway enrichment analyses of AI genes in Huyou root tissue revealed a strong over-representation of genes related to various antioxidant production, such as ໿flavonoid, phenylpropanoid, isoflavonoid, carotenoid, benzoxazinoid, flavone, flavonol, alkaloid, ascorbate, and steroid biosynthesis. These antioxidants have well-known biological functions in plants’ adaptation to various biotic and abiotic stresses. The allelic imbalance of genes within these pathways in the root tissue may have important implication in Huyou’s environmental adaptation following its ancient genome hybridization. It is interesting to observe that these antioxidant biosynthesis terms were over-represented in both HY1-higher and HY2-higher genes, suggesting an overall evolutionary preference of AI genes related to environmental adaptation. At the plant tissue level, we observed overall conservation of AI functions across tissues such as root and ripe fruit, despite a significantly higher degree of AI in root. This novel insight needs to be verified in future analysis in other species. In plants, especially those with high heterozygosity, AI plays a significant role in genome evolution, influencing traits such as disease resistance, growth patterns, and adaptation to environmental changes (Jang, et al. , 2024; Djari, et al. , 2024; Li, et al. , 2023). The impact of AI on plants’ genome evolution and environmental adaption has been well recognized in several recent haplotype-resolved genome studies. For example, a study in Populus tomentosa identified heterozygous alleles associated with adaptive traits, highlighting the role of AI in evolutionary processes (Li, et al. , 2023). Similarly, haplotype-resolved assemblies grapevine identified structural variations between haplotypes associated with allelic expression linked to disease resistance and fruit quality (Djari, et al. , 2024). Another recent study in Pinus densiflora revealed the functional contributions of AI to flowering and abiotic stress-related traits (Jang, et al. , 2024). In addition to the overlapping AI functions, haplotype-specific AI functions were also observed. The biological implications of our observed AI patterns remain to be characterized, particularly for citrus genome research. As far as the authors are concerns, our study is the first to demonstrate the presence of strong AI in citrus hybrid genome. Based on our observation of AI in Huyou, we reason that AI should be prevalent across the various citrus genomes due to the presence of frequent hybridization events. Given the still limited haplotype resolved genome assemblies for citrus species, the importance of AI in citrus genome evolution and its interplay with phenotypic diversity in citrus deserves much more attention in future research. Conclusions We sequenced a 120-year-old "ancestral tree" of C. changshan Y. B. Chang (Huyou), a unique natural hybrid citrus landrace first discovered in Changshan, Zhejiang province, China. Huyou displayed genome heterozygosity at 3.07%, among the highest across known citrus genomes. We presented two high-quality haplotype-resolved genomes HY1 and HY2 and determined that Huyou had originated from a mandarin and pummelo hybridisation event, resolving a mystery that has puzzled local Huyou growers and botanists. HY1 and HY2 had diverged at 2.0 Mya and 2.18 Mya, earlier than the split of C. clementina and C. reticulata , and the split of C. grandis and C. maxima , respectively. Haplotype-based transcriptome analyses also demonstrated the presence of strong tissue-specific allelic imbalance in Huyou’s genome. The haplotype genomes HY1 and HY2 represent two novel ancestral citrus genomes not previously reported, contributing to the limited citrus genetic pool and enabling citrus breeders to harness Huyou's genetic potential. The explicit resolution of Huyou’s genome origin highlights the power of haplotype-resolved genomes in dissecting the complex evolutionary origin of citrus species. Declarations Competing interests The authors declare no competing interest. Acknowledgements We acknowledge the relevant citrus research communication for making the citrus genomic data available to the public, particularly the recently published Huyou transcriptome data (Miao , et al. , 2024). Author contributions YJ, CL, ZZ designed and supervised the study. ZZ, YJ, TX, YL, HH, LL, BG, PZ, CT, TQ, ZY, ZC performed data analysis. YJ, ZZ, XH, YL wrote the manuscript. TX, CL, LW edited the manuscript. All authors have read and approved the manuscript. Data availability The raw sequencing data has been deposited at NCBI with project ID PRJNA1090545 (release upon acceptance). The genome assemblies and gene annotations are available at https://figshare.com/s/ba2b2081a2512770c31a Funding This work was supported by the National Natural Science Foundation of China (32301872), the Natural Science Foundation of Zhejiang Province (LQ23C130003), and Interdisciplinary Research Project of Hangzhou Normal University (2025JCXK01). 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Plant Breeding , 125 : 519–522. Nakandala, U, Masouleh, AK, Smith, MW, Furtado, A, Mason, P, Constantin, L, Henry, RJ . 2023 . Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes. Horticulture Research , 10 : uhad058. Jang, MJ, Cho, HJ, Park, YS, Lee, HY, Bae, EK, Jung, S, Jin, HS, Woo, J, Park, E, Kim, SJ et al. 2024 . Haplotype-resolved genome assembly and resequencing analysis provide insights into genome evolution and allelic imbalance in. Nature Genetics , 56 : 2551–2561. Djari, A, Madignier, G, Di Valentin, O, Gillet, T, Frasse, P, Djouhri, A, Hu, GJ, Julliard, S, Liu, MC, Zhang, Y et al. 2024 . Haplotype- resolved genome assembly and implementation of VitExpress, an open interactive transcriptomic platform for grapevine. Proceedings of the National Academy of Sciences of the United States of America , 121 : e2403750121. Li, P, Xiao, L, Du, QZ, Quan, MY, Song, YP, He, YL, Huang, WX, Xie, JB, Lv, CF, Wang, D et al. 2023 . Genomic insights into selection for heterozygous alleles and woody traits in. Plant Biotechnology Journal , 21 : 2002–2018. Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarydata.xlsx Supplementary File S0. TE annotation for HY1 and HY2. Supplementary File S1. k-mer tracing results of HY1 and HY2. Supplementary File S2. Orthofinder gene matrix for target species in this study. Supplementary File S3. List of Orthogroup IDs identified for key KEGG pathways. Supplementary File S4. List of species-specific gene in HY1 and HY2. Supplementary File S5. SyRI summary for HY1 and HY2 synteny analysis. Supplementary File S6. RNAseq results (quantified TPM and HY1/HY2 ratio) HY1 and HY2 collinear genes in six tissues. Supplementary File S7. Percentage of allelic imbalance genes in each chromosome across 6 tissues. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6552063","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455752939,"identity":"4dc590c3-dd3d-4b36-aaec-63b83eb9db56","order_by":0,"name":"Yong 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Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhehao","middleName":"","lastName":"Chen","suffix":""},{"id":455752950,"identity":"584dadb7-3bad-46e9-b461-278b552fe505","order_by":11,"name":"Zhiming Yu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Yu","suffix":""},{"id":455752951,"identity":"20d83b98-7e57-49c8-8d05-0f32763237dd","order_by":12,"name":"Lilin Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lilin","middleName":"","lastName":"Wang","suffix":""},{"id":455752952,"identity":"0c1767b4-daf6-42c6-ae7a-853d76d315c0","order_by":13,"name":"Taihe Xiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Taihe","middleName":"","lastName":"Xiang","suffix":""},{"id":455752953,"identity":"c188b47d-cd57-4421-890b-c736430b4cc8","order_by":14,"name":"Chengdao Li","email":"","orcid":"https://orcid.org/0000-0002-9653-2700","institution":"Murdoch University","correspondingAuthor":false,"prefix":"","firstName":"Chengdao","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-04-29 03:35:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6552063/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6552063/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82806364,"identity":"90378335-5783-428a-84d3-c2d646ff4b3f","added_by":"auto","created_at":"2025-05-15 12:26:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":761508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuyou tree characteristics, karyotype\u003c/strong\u003e \u003cstrong\u003eanalysis, and Genome survey.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. The 120 years old “ancestral” Huyou tree sequenced in this study (whole tree, stem, leaves, flowers, and fruits). \u003cstrong\u003eB\u003c/strong\u003e. Karyotype analysison chromosomes of Huyou root tips. Karyotype pictures of Huyou using DAPI staining (top left), FISH analysis with a telomere specific repeat probe (top right), and 5S rDNA (red) and 18S rDNA (green) repeat sequence probes (bottom). Bar = 5 μm. \u003cstrong\u003eC\u003c/strong\u003e. Genome size and heterozygosity estimation based on K-mer frequency analysis (K = 21). The fist peak reveals heterozygous k-mers while the second peak indicates the homozygous k-mers. \u003cstrong\u003eD\u003c/strong\u003e. Hi-C interaction heatmap for the assembled HY1 and HY2 genomes. Blue lines indicated chromosomal boundary. The 18 chromosomes were sorted based on length in decreasing order. The bottom-right cluster represented unanchored contigs. \u003cstrong\u003eE\u003c/strong\u003e. Dot plots displaying the synteny between HY1 and HY2 for each chromosome.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/fe10017287afd13ac6434efc.jpeg"},{"id":82805171,"identity":"6e327622-b9a8-4a66-a75e-9ea90dbfac7d","added_by":"auto","created_at":"2025-05-15 12:18:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAncestral tracing and comparative genome analysis of Huyou.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Circles from outside to inside represent chromosomes of two phased haplotypes (mandarin, orange; pummelo, purple; citron, green; undetermined, white), gene density (blue), transposable element density (red), tRNA density (green), small nucleolar RNA density (orange) and microRNA density (plum). The curved lines in the center indicate patterns of gene collinearity. All distributions are drawn in a window size of 100kb.\u003cstrong\u003e B. \u003c/strong\u003eThe histogram of differential k-mers among homoeologous chromosomes of HY1 and HY2.\u003cstrong\u003e C\u003c/strong\u003e. Insertion time of subgenome-specific LTR-RTs (output from SubPhaser program, see method for details).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/77d4f015926d3d97d1824c3c.jpeg"},{"id":82806365,"identity":"2897b130-c819-41b0-843e-c0833ff590c0","added_by":"auto","created_at":"2025-05-15 12:26:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":383961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogeny dating and gene family evolution analyses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Displays the species phylogeny of HY1 and HY2 in references to other citrus species and citrus-related species. The tree topology was obtained using Orthofinder, calibrated at 5 nodes (age annotated in tree) using mcmctree program based on single copy genes. Identified branching groups were highlighted correspondingly. Gene family expansion and contraction at key nodes (numbers) and each taxa (pie charts) were indicated. The percentage of single copy genes, species-specific genes, unassigned genes, and other genes were annotated as bar charts for each taxa. The heatmap represents gene counts for 5 key KEGG pathways: map00900- terpenoid biosynthesis, map00906- carotenoid biosynthesis, map00940- phenylpropanoid biosynthesis, map00941- flavonoid biosynthesis, and map04626- plant-pathogen interaction (Supplementary File S2, S3). \u003cstrong\u003eB\u003c/strong\u003e. Ks distribution of collinear gene pairs of citrus species with the early diverging \u003cem\u003eP. trifoliata\u003c/em\u003e (ZK) species as reference (HWB \u003cem\u003eC. grandis\u003c/em\u003ewanbaiyou, HZYT \u003cem\u003eC. maxima\u003c/em\u003e huazhouyou, SWO \u003cem\u003eC. sinensis\u003c/em\u003e, MSYG \u003cem\u003eC. mangshanensis\u003c/em\u003e, ZGYCC \u003cem\u003eC. ichangensis\u003c/em\u003e, LW \u003cem\u003eC. linwuensis\u003c/em\u003e, HH \u003cem\u003eC. hongheensis\u003c/em\u003e, RL \u003cem\u003eC. medica\u003c/em\u003e, Vv \u003cem\u003eVitis vinifera\u003c/em\u003e). Vitis vinifera was used an outgroup reference. \u003cstrong\u003eC\u003c/strong\u003e. Gene ontology enrichment analysis of HY1-specific genes. GO terms with corrected p-value \u0026lt;0.05 were included as significant. No significant enrichment was identified for HY2-specific genes (Supplementary File S4).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/019e3208e7413d4d4ed53996.jpeg"},{"id":82805172,"identity":"5f4ebe6c-8d90-42e5-abab-ad92d31da100","added_by":"auto","created_at":"2025-05-15 12:18:12","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynteny and genetic variations of HY1, HY2, mandarin, and pummelo genomes\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Display the synteny and structural variations of HY1, HY2, and their ancestral genomes mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e) and pummelo (\u003cem\u003eC. grandis\u003c/em\u003e). Pair-wise sequential whole genome alignments in the order of mandarin-HY1-HY2-pummelo were performed. Synteny (grey) and large structural variations (inversions: orange, translocations: green, duplications: blue) were identified using SyRI. \u003cstrong\u003eB\u003c/strong\u003e. Displays the distribution of the allelic genetic variants (based on SyRI outputs) between HY1 and HY2 and important pathway genes (map00020- citrate cycle, map00195- photosynthesis, map00900- terpenoid biosynthesis, map00906- carotenoid biosynthesis, map00940- phenylpropanoid biosynthesis, map00941- flavonoid biosynthesis).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/70c0e2680d0b867d68f40708.jpeg"},{"id":82805174,"identity":"645c365f-9e1c-4e42-83b3-ed57143b859f","added_by":"auto","created_at":"2025-05-15 12:18:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":657134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional alleic imbalance between HY1 and HY2 in different tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Clustering of the percentages of HY1-preferential (log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(HY1/HY2)\u003c/sup\u003e ≥ 1) and HY2-preferential (log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(HY1/HY2)\u003c/sup\u003e ≤ -1) genes 6 tissues (root, stem, leaf, flower, unripe fruit, ripe fruit) in each chromosome (\u003cstrong\u003eSupplementary File S7\u003c/strong\u003e). Only the collinear genes between HY1 and HY2 were counted. The percentages of HY1 and HY2 preferential genes were calculated relative to the total number of gene in each chromosome. \u003cstrong\u003eB\u003c/strong\u003e. Displays the distribution of HY1 and HY2 preferential genes along each chromosome for the root transcriptome. \u003cstrong\u003eC\u003c/strong\u003e. KEGG enrichment of allelic imbalance collinear genes in root with corrected p-value \u0026lt;1 × 10\u003csup\u003e-5\u003c/sup\u003e were shown. D. KEGG enrichment of allelic imbalance collinear genes in ripe fruit with corrected p-value \u0026lt;1 × 10\u003csup\u003e-5\u003c/sup\u003e were shown.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/64ef21a4c82c844c7fd15d69.jpeg"},{"id":82806553,"identity":"ee2c5ad6-cb97-4949-8958-e305b5d57dea","added_by":"auto","created_at":"2025-05-15 12:34:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1724864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisplays the transcriptome allelic imbalance of HY1 and HY2 for map00906 pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ratios of HY1 and HY2 collinear genes (log2\u003csup\u003e(HY1/HY2)\u003c/sup\u003e) were mapped to KEGG pathway map00906 using TBtools. Transcriptome data in 6 Huyou tissues (root, stem, leaf, flower, unripe fruit and ripe fruit) from a previous study was downloaded and analyzed. Yellow, red, and green highlights indicated neutral, HY1 dominant, and HY2 dominant, respectively. Only the Astaxanthin biosynthesis module of map00906 were displayed.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/dfc5ee4a03cd8717cfc5bef2.jpeg"},{"id":82806366,"identity":"47e6a2ab-1d30-4455-882a-d5e537ed46c9","added_by":"auto","created_at":"2025-05-15 12:26:12","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":442355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CNVs and transcriptome profiles for carotenoid pathway in Huyou.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Displays the CNV in HY1, HY2, and other 8 citrus species included in this study. Gene counts were based on results from Orthofinder analysis in \u003cstrong\u003eFigure 3\u003c/strong\u003e. Genes were assigned to KO orthologs based on kegg annotation. \u003cstrong\u003eB\u003c/strong\u003e. Heatmap displaying the transcription level of each KO ortholog in 6 Huyou tissues. The transcriptome values for multiple genes were summed and normalized (divided by maximum value) across the 6 tissues. \u003cstrong\u003eC\u003c/strong\u003e. Display the metabolic map of the carotenoid pathway. Genes whose copy numbers are highest in Huyou (either HY1 or HY2) were highlighted in yellow. Genes with above median expression in fruit tissues were enclosed in red rectangles.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/6711712f1abb52860239814f.jpeg"},{"id":86185538,"identity":"62a8488e-0e4b-4e51-aaa7-fe79e00c4de1","added_by":"auto","created_at":"2025-07-07 17:23:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7884482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/0b10b110-2505-4b8f-9b87-1d07daa720d0.pdf"},{"id":82805177,"identity":"f8d599e5-eb71-404e-b6f0-36a713426448","added_by":"auto","created_at":"2025-05-15 12:18:12","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9824110,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary File S0. TE annotation for HY1 and HY2.\u003c/p\u003e\n\u003cp\u003eSupplementary File S1. k-mer tracing results of HY1 and HY2.\u003c/p\u003e\n\u003cp\u003eSupplementary File S2. Orthofinder gene matrix for target species in this study.\u003c/p\u003e\n\u003cp\u003eSupplementary File S3. List of Orthogroup IDs identified for key KEGG pathways.\u003c/p\u003e\n\u003cp\u003eSupplementary File S4. List of species-specific gene in HY1 and HY2.\u003c/p\u003e\n\u003cp\u003eSupplementary File S5. SyRI summary for HY1 and HY2 synteny analysis.\u003c/p\u003e\n\u003cp\u003eSupplementary File S6. RNAseq results (quantified TPM and HY1/HY2 ratio) HY1 and HY2 collinear genes in six tissues.\u003c/p\u003e\n\u003cp\u003eSupplementary File S7. Percentage of allelic imbalance genes in each chromosome across 6 tissues.\u003c/p\u003e","description":"","filename":"Supplementarydata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6552063/v1/c601386dcb55176dfd934704.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus Citrus belongs to the Aurantioideae subfamily of Rutaceae and contains a diverse array of species such as sweet orange (\u003cem\u003eC. sinensis\u003c/em\u003e), mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e), pummelo (\u003cem\u003eC. grandis\u003c/em\u003e), citron (\u003cem\u003eC. medica\u003c/em\u003e), and lemon (\u003cem\u003eC. limon\u003c/em\u003e). Their diverse flavors, nutritional benefits, and industrial applications have made them beloved fruits worldwide. Citrus ranks the third largest fruits in term of production volume (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.statista.com/\u003c/span\u003e\u003cspan address=\"https://www.statista.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and hold substantial agricultural, economic, and cultural values globally (Gmitter, \u003cem\u003eet al.\u003c/em\u003e, 2012). Beyond their economic and cultural importance, citrus fruits present a fascinating subject for genomic and evolutionary studies due to their intricate origins and diversification. The genus Citrus is characterized by a reticulate evolutionary history, marked by frequent hybridization and polyploidization events (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2018). This complex evolutionary past has led to various cultivated species or landraces with different heterozygosity levels, making citrus an intriguing subject for genome evolution investigation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCitrus changshan-huyou\u003c/em\u003e Y. B. Chang (Huyou) is a unique Citrus species originated in the Changshan County of the Zhejiang Province in China (Li, \u003cem\u003eet al.\u003c/em\u003e, 2019; Yin-bin, 1991). The earliest cultivation of Huyou has been recorded over 600 years ago, well before its massive planting and production over 100 years as a prominent landrace. Its fruit has a golden color and contains abundant components, such as amino acids, vitamins, naringin, limonin, citrate and other nutrients (Zhang, \u003cem\u003eet al.\u003c/em\u003e, 2012; Guo, \u003cem\u003eet al.\u003c/em\u003e, 2018; Sheng, \u003cem\u003eet al.\u003c/em\u003e, 2017). These components not only invested the fruit unique aroma and taste that combines sweet, sour, and a bit of bitterness, but also health benefits, including lowering blood sugar, hepatoprotective and anti-inflammatory effects (Zhang, \u003cem\u003eet al.\u003c/em\u003e, 2012; Guo, \u003cem\u003eet al.\u003c/em\u003e, 2018; Jiang, \u003cem\u003eet al.\u003c/em\u003e, 2019). These benefits have led to the widespread consumption of Huyou in Zhejiang and neighboring province. The economic importance and scientific values of Huyou are attracting increasing attention. A recent scientific dataset reported the haplotype-resolved genome assembly of a Huyou accession and transcriptome sequencing in 6 tissues (Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024). Despite the high quality Huyou genomic data, the dataset fell short on genomic origin and evolution analysis for this important natural hybrid citrus landrace. The domestication and evolutionary origin of Huyou remains to be characterized.\u003c/p\u003e \u003cp\u003eOver the past decade, numerous citrus species have been sequenced, generating genomic data that has significantly advanced our knowledge of citrus biology and genome evolution. Key milestones include the sequencing and assembly of reference genomes for important citrus species such as sweet orange (\u003cem\u003eC. sinensis\u003c/em\u003e) (Xu, \u003cem\u003eet al.\u003c/em\u003e, 2013), clementine mandarin (\u003cem\u003eC. clementina\u003c/em\u003e) (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2014), and pummelo (\u003cem\u003eC. maxima\u003c/em\u003e) (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2014). These were followed by whole genome resequencing for large citrus germplasm collections that encompass diverse citrus wild, landraces, and cultivars lines (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2018; Wang, \u003cem\u003eet al.\u003c/em\u003e, 2017). All these studies have highlighted a complex history of admixture and hybridization during citrus domestication. The sequencing projects indicate that modern citrus varieties are often the result of crosses between a few ancestral species such as mandarin, pummelo, and citron. Beyond the genomic insights into genome origin and evolution, citrus genome sequencing studies have also led to the identification of genes associated with important agronomic traits, including fruit quality, disease resistance, and stress tolerance. For example, studies have identified genes involved in flavor synthesis in lemon (Bao, \u003cem\u003eet al.\u003c/em\u003e, 2023), disease resistance in trifoliate orange (Peng, \u003cem\u003eet al.\u003c/em\u003e, 2020), and citric acid accumulation in oranges (Huang, \u003cem\u003eet al.\u003c/em\u003e, 2023). Recently, citrus pangenome data for various citrus and citrus related species have been made available (Huang, \u003cem\u003eet al.\u003c/em\u003e, 2023; Liu, \u003cem\u003eet al.\u003c/em\u003e, 2022). These genomic resources allow researchers to delve deeper into the genetic basis of key traits, paving the way for more targeted and efficient breeding strategies (Liu, \u003cem\u003eet al.\u003c/em\u003e, 2022; Humann, \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e \u003cp\u003eHaplotype-phased genome refers to a genome sequence where the two sets of chromosomes inherited from each parent are separately distinguished and assembled. The assembly of highly accurate haplotype-phased genomes is achieved through advanced sequencing technologies such as long-read sequencing (e.g., PacBio HiFi, Nanopore), Hi-C sequencing (chromosome conformation capture). It allows for the identification of allele-specific variations, structural variations, and gene expression patterns that would be obscured in a standard genome assembly. This is particularly crucial for genomic study on heterozygous organisms such as Citrus species, which are marked with frequent hybridization events. Recently, several haplotype-resolved genome assemblies have been generated, including \u003cem\u003eC. limon\u003c/em\u003e (Mario, \u003cem\u003eet al.\u003c/em\u003e, 2021), \u003cem\u003eC. australis\u003c/em\u003e (Nakandala, \u003cem\u003eet al.\u003c/em\u003e, 2023), \u003cem\u003eC. changshanensis\u003c/em\u003e (Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024), \u003cem\u003ePapeda\u003c/em\u003e (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2025), and a haploid genome assembly for pummelo (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2017), offering a more detailed view of their genetic makeup.\u003c/p\u003e \u003cp\u003eIn this study, we performed genome sequencing and assembly of a 120-years-old \u0026ldquo;ancestor tree\u0026rdquo; of Huyou currently still growing in Changshan, Zhejiang Province where Huyou was first discovered. We integrate high coverage Illumina short read and PacBio long read sequencing with Hi-C sequencing technology for the assembly of 2 haplotype-resolved chromosome scale genomes. This is followed by in-depth genome tracing, structural variation, and comparative genomic analysis with previously established citrus ancestor species to fully characterize the unique genomic features of Huyou and to resolve its evolutionary origin. Furthermore, assisted by previously published transcriptome data and the haplotype-resolved genome assemblies in this study, we comprehensively characterized the allelic imbalance across different tissues. Gene family expansion/contraction and transcriptome profiles for key genes from important metabolic pathways related to citrus fruit quality were also analyzed. We highlight the exceptional genome heterozygosity of Huyou compared to known Citrus genomes and the unique tissue-specific allelic imbalance in the root tissue. Our study supports Huyou as a novel genomic resource for citrus breeding and also an interesting model to investigate genome evolution following distant hybridization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and nucleic acid extraction\u003c/h2\u003e \u003cp\u003eFresh leaves, shoots and fruits were collected from a 120-year-old \u003cem\u003eC. changshan-huyou\u003c/em\u003e Y. B. Chang tree in Chengtan Village, Qingshi Town, Changshan County (118\u0026deg;30\u0026prime;E, 28\u0026deg;51\u0026prime;N), Zhejiang Province, China, in the spring of 2022 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. These tissues were quickly frozen in liquid nitrogen and then stored at \u0026minus;\u0026thinsp;80℃ before DNA and RNA extraction. Total genomic DNA was extracted from the leaf tissue, and its quality was assessed by the Qubit 3.0 Fluorometer (Thermo Fisher, USA), while the total RNA from leaves, shoots and fruits was extracted using TRIzol Reagent (Invitrogen). The quantity and quality of the RNA were examined using NanoDrop One spectrophotometer (Thermo Fisher, USA) and an Agilent 2100 Bioanalyser (Agilent Technologies), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome karyotype identification\u003c/h3\u003e\n\u003cp\u003eKaryotype analysis was conducted with fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) method as previously described (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2017; Zhang, \u003cem\u003eet al.\u003c/em\u003e, 2022), and was performed by OMIX Technologies Corporation (Chengdu, China). Briefly, excised young root tips (2-3cm) of Huyou via tissue culture (Li, \u003cem\u003eet al.\u003c/em\u003e, 2017) were treated with nitrous oxide gas for 2h under 1 MPa, then fixed in ice-cold 90% acetic acid for 10 min. After washing in water, the root tips were diced and digested in 1% pectolyase Y23 and 2% cellulase Onozuka R-10 (Yakult Pharmaceutical, Japan) for 1 h at 37℃. After digestion, the root sections were washed in 70% ethanol three times briefly, then broken by using a needle and vortexed at maximum speed in 100% ethanol for 30 sec at room temperature to separate cells from one another. The cells were collected at the bottom of the tube by centrifugation (4000r/min) and resuspended in glacial acetic acid. The cell suspension was dropped onto glass slides in a box lined with wet paper. A telomere-specific repeat probe 5\u0026prime;-(TTTAGGG)\u003csub\u003e6\u003c/sub\u003e-3\u0026prime;) was used to detect the number of intact chromosomes, and 5s rDNA and 18s rDNA repeat sequence probe were used to identify multiple copies of chromosomes. The fluorescence staining of the chromosomes was performed using 4\u0026prime;,6-diamidino-2-phenylindole (DAPI). After DAPI staining, the dispersed metaphase chromosome cells were counted under a fluorescence microscope (Zeiss LSM880, Germany).\u003c/p\u003e\n\u003ch3\u003eGenome size and heterozygosity estimation\u003c/h3\u003e\n\u003cp\u003eFor genome profiling, the DNA library was constructed with insert sizes of ~\u0026thinsp;300 bp using Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA), and subsequently sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), which generated 150 bp pair-end reads. The software FastQC (version: 0.20.1) was used to filter the original reads and discard the low-quality reads. A total of 57.05 Gb clean data were generated by Illumina sequencing and were used to perform the genome survey for Huyou. Reference-free estimation of genome size and heterozygosity were performed using a k-mer frequency approach implemented in GenomeScope 2.0 (Ranallo-Benavidez, \u003cem\u003eet al.\u003c/em\u003e, 2020).\u003c/p\u003e\n\u003ch3\u003ePacBio, Hi-C and RNA Sequencing\u003c/h3\u003e\n\u003cp\u003eTo generate long-read sequencing reads for Huyou, DNA libraries were prepared using the SMRTbell Express Template Preparation kit 1.0 following the PacBio 20-kb protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pacb.com/\u003c/span\u003e\u003cspan address=\"https://www.pacb.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and sequenced on the PacBio Sequel II platform (Pacific Biosciences of California, Inc.). After the data quality control, by removing sequencing adaptors and filtering low-quality short read, and correcting errors via SMRT link (v8.0), 26.17 Gb of HIFI reads were obtained (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe generation of a Hi-C library was performed as described previously (Strijk, \u003cem\u003eet al.\u003c/em\u003e, 2021). Briefly, the genomic DNA was random broken into 300\u0026ndash;700 bp fragments via DNA cross-linking, restriction enzyme digestion, cohesive end repair, DNA cyclization, and DNA purification. Then the sample was sequenced on an Illumina NovaSeq 6000 platform (PE 150). After the removal of sequencing adaptors and low-quality reads using fastp software (v 0.21.0) (Chen, \u003cem\u003eet al.\u003c/em\u003e, 2018), 31.77 Gb Hi-C clean reads were obtained.\u003c/p\u003e \u003cp\u003eRNA sequencing (RNA-seq) samples were obtained by mixing equal amounts of RNA extracted from each tissue (leaf, shoot, and fruit) and used for cDNA library construction. After sequencing on the Illumina NovaSeq 6000 platform, approximately 10 Gb of sequencing data was obtained.\u003c/p\u003e\n\u003ch3\u003eDe novo genome assembly and annotation\u003c/h3\u003e\n\u003cp\u003eHaplotype-resolved \u003cem\u003ede novo\u003c/em\u003e genome assembling was performed using hifiasm software (Cheng, \u003cem\u003eet al.\u003c/em\u003e, 2021) with HIFI reads and Hi-C phasing. The default k-mer size was changed 19 to obtain correct estimation heterozygous site coverage. The resulted haplotype-resolved genome contigs were further purged for duplicates using Purge_Dups (Guan, \u003cem\u003eet al.\u003c/em\u003e, 2020) by aligning the HIFI reads back to the assembled genomes using minimap2 tool (Li, 2018). The purged haplotype genomes were scaffolded into chromosome groups using the pipeline implemented at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/WarrenLab/hic-scaffolding-nf\u003c/span\u003e\u003cspan address=\"https://github.com/WarrenLab/hic-scaffolding-nf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, which employs Chromap (Zhang, \u003cem\u003eet al.\u003c/em\u003e, 2021) and YaHS (Zhou, \u003cem\u003eet al.\u003c/em\u003e, 2023) for sequence alignment and Hi-C scaffolding, respectively. Genome statistics were calculated using the assembly-stats tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/sanger-pathogens/assembly-stats\u003c/span\u003e\u003cspan address=\"https://github.com/sanger-pathogens/assembly-stats\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, the juciebox software (v1.11.08) (Durand, \u003cem\u003eet al.\u003c/em\u003e, 2016) was used to draw Hi-C heatmap based on the interaction intensity and relative position between contigs. Following manual correction, the uniquely mapped and valid interaction paired-end reads were used to build the pseudochromosome sequences. The pseudochromosomes were sorted and oriented using C. sinensis as the reference by RagTag (Alonge, \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e \u003cp\u003eThe quality of the assembled genomes was evaluated by Benchmarking Universal Single-Copy Orthologs against the eukaryota_odb10 database (BUSCO v.4.1.4) (Simao, \u003cem\u003eet al.\u003c/em\u003e, 2015). Repeat sequences of the haplotype genomes were searched using an automated pipeline implemented by EarlGrey (Baril, \u003cem\u003eet al.\u003c/em\u003e, 2024). The soft-masked genomes were used for gene model prediction using GeMoMa v1.8 (Keilwagen, \u003cem\u003eet al.\u003c/em\u003e, 2019) based on homology and transcriptome evidence. For homology references, the gene models from three ancestor genomes (C. sinensis, C. ichangensis, C. medica) were used. For transcriptome evidence, the RNA sequencing reads were mapped to the masked genomes using STAR (Dobin, \u003cem\u003eet al.\u003c/em\u003e, 2013) and the resulted alignment files were used as input for GeMoMa. For noncoding RNA annotation, the transfer RNAs (tRNAs) were annotated by tRNAscan-SE (v1.23) (Chan, \u003cem\u003eet al.\u003c/em\u003e, 2021). Other non-coding ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) and microRNAs (miRNAs) were identified using the Infernal toolkits (Nawrocki, \u003cem\u003eet al.\u003c/em\u003e, 2009) based on information from the Rfam database. Gene function annotations were performed using Interproscan for GO annotation and kofam_scan for KEGG annotation. GO and KEGG enrichment analyses for Huyou genome specific genes were performed using TBtools (Chen, \u003cem\u003eet al.\u003c/em\u003e, 2020). The circos v0.69 (Krzywinski, \u003cem\u003eet al.\u003c/em\u003e, 2009) was used to visualize genomic characteristic information, including gene density, GC density, TE density, gene density, and synteny block.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVariants, synteny, and genome origin analyses\u003c/h2\u003e \u003cp\u003eThe genomic variations and synteny across the haplotype genomes of Huyou and their ancestor genomes \u003cem\u003eC. reticulata\u003c/em\u003e (mandarin, NCBI accession ID: ASM325862v1) (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2018) and \u003cem\u003eC. maxima\u003c/em\u003e (pummelo, NCBI accession ID: ASM2964120v1) (Zheng, \u003cem\u003eet al.\u003c/em\u003e, 2023) were analyzed using SyRI software (Goel, \u003cem\u003eet al.\u003c/em\u003e, 2019). Genome alignment was performed using minimap2 (Li, 2018). The ancestral origin of Huyou haplotype genomes were determined using the k-part tool at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/sc-zhang/kPart\u003c/span\u003e\u003cspan address=\"https://github.com/sc-zhang/kPart\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Variations between HY1 and HY2 were counted per 1 Mb windown based on the output from SyRI. For ancestor tracing, genomic data for 3 ancestor genomes (\u003cem\u003eC. reticulata\u003c/em\u003e, \u003cem\u003eC. maxima\u003c/em\u003e, \u003cem\u003eC. medica\u003c/em\u003e) were downloaded and used as references. The ancestor of Huyou genomes were assigned for each 100 kb window using kPart with -k 21 parameter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/sc-zhang/kPart\u003c/span\u003e\u003cspan address=\"https://github.com/sc-zhang/kPart\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The percentages of each ancestor origin were calculated. The haplotype origin for each chromosome was also phased and classified using SubPhaser program with a similar k-mer based method (Jia, \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOrthologs inferences, phylogeny development, and gene family analyses\u003c/h3\u003e\n\u003cp\u003eProtein sequences of Huyou genomes and 19 other species, including \u003cem\u003eAquilegia coerulea\u003c/em\u003e, \u003cem\u003eMangifera indica\u003c/em\u003e, \u003cem\u003eVitis vinifera\u003c/em\u003e, \u003cem\u003eMalus domestica Golden\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eC. sinensis\u003c/em\u003e, \u003cem\u003eC. reticulata\u003c/em\u003e, \u003cem\u003eC. maxima\u003c/em\u003e, \u003cem\u003eC. medica\u003c/em\u003e, \u003cem\u003eC. clementina\u003c/em\u003e, \u003cem\u003ePoncirus trifoliata\u003c/em\u003e, \u003cem\u003eFortunella hindsii\u003c/em\u003e, \u003cem\u003eC. ichangensis\u003c/em\u003e, \u003cem\u003eAtalantia buxfoliata\u003c/em\u003e, and \u003cem\u003eLitchi chinensis Sonn\u003c/em\u003e (downloaded from Citrus Genome Database at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.citrusgenomedb.org/\u003c/span\u003e\u003cspan address=\"https://www.citrusgenomedb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Liu, \u003cem\u003eet al.\u003c/em\u003e, 2022) were used as input for OrthoFinder v2.3.12 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/davidemms/Ortho\u003c/span\u003e\u003cspan address=\"https://github.com/davidemms/Ortho\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Finder) (Emms and Kelly, 2019) for orthologs inferences. For alternative transcripts, only the sequences for the longest transcripts were used. The obtained species tree was used as input for downstream Bayesian estimation of divergence time using mcmctree from PAML v4.9 toolkits (Yang, 2007). The CDS sequences for the identified single copy genes from Orthofinder were extracted. Codon-based sequence alignments were performed using a combination of muscle and seqmagick backtrans-align functions. The concatenated CDS alignment was trimmed using Gblocks before input into mcmctree. Five divergence time points derived from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://timetree.org/\u003c/span\u003e\u003cspan address=\"https://timetree.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e were used to calibrate the species tree. The final obtained species tree from mcmctree and orthologous gene count results from Orthofinder were input into caf\u0026eacute; for gene family expansion/contraction analyses. The gene family changes along the species tree were visualized using tvBOT (Xie, \u003cem\u003eet al.\u003c/em\u003e, 2023) online tool at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/tvbot.html\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/tvbot.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. For gene copy number analysis, the gene counts for corresponding hierarchical orthologoups from Orthofinder outputs were used.\u003c/p\u003e\n\u003ch3\u003eGenome divergence time analyses\u003c/h3\u003e\n\u003cp\u003eThe genome divergence time was assessed by \u003cem\u003eKs\u003c/em\u003e profile analyses using WGDI program (Sun, \u003cem\u003eet al.\u003c/em\u003e, 2022). Each citrus genome was compared to itself following the WGDI instruction. Protein sequences for the primary transcripts of each species were used for blastp using diamond program (Buchfink, \u003cem\u003eet al.\u003c/em\u003e, 2021). Syntenic blocks were identified using the build-in function in WGDI. Codon-based sequence alignments were performed using muscle (Edgar, 2004) and pal2nal (Suyama, \u003cem\u003eet al.\u003c/em\u003e, 2006) programs. Ks values calculation for collinear genes using yn00 from PAML v4.9 program (Yang, 2007). Ks peaks at p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 were identified and fitted using WGDI. The whole genome duplication event in Vitis vinifera (Jaillon, \u003cem\u003eet al.\u003c/em\u003e, 2007) was used a reference.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analyses\u003c/h2\u003e \u003cp\u003eTranscriptome RNA sequencing data in 6 Huyou tissues (root: SRR28430799, stem: SRR28430800, leaf: SRR28430798, flower: SRR28430801, unripe fruit: SRR28430803, ripe fruit: SRR28430802) were downloaded from a previous study (Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024). Raw reads were trimmed using fastp program with --qualified_quality_phred 20 and --length_required 30 parameters. The resulted clean reads were mapped to merged haplotype genomes using STAR program, then were quantified using RSEM (Li and Dewey, 2011) based on the annotated gene models in this study. The syntenic gene pairs between HY1 and HY2 were identified using MCScanX program (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2012). The comparison of syntenic genes expression was performed by calculating the log2 value of the ratio of HY1 gene expression to HY2 gene expression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGenome assembly and annotation of the 120 years-old \u0026ldquo;ancestral tree\u0026rdquo; of\u003c/strong\u003e \u003cstrong\u003eCitrus changshan-huyou\u003c/strong\u003e \u003cstrong\u003eY. B. Chang\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, a century-old Huyou tree (120 years old as of 2025) currently growing and protected in Chentang Village, Changshan County, after which Huyou was named by Mr. Yunbing Chang in 1991, was selected for genome sequencing (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). This century-old tree was believed by many local Huyou growers and botanists to be the ancestor of most Huyou trees currently cultivated across Zhejiang province and was thus termed as the \u0026ldquo;ancestral tree\u0026rdquo;. Notably, this \u0026ldquo;ancestral tree\u0026rdquo; of Huyou, despite its old age, remains highly vigorous and productive with premium fruits to date (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, photographed in 2024).\u003c/p\u003e\n\u003cp\u003eTo determine the chromosome number and ploidy level of Huyou, high-resolution metaphase chromosome preparation assay was performed using root tip cells from tissue cultured Huyou seedlings. Results showed that the genome of Huyou is composed of 18 chromosomes (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;18, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), which was confirmed by FISH with 5S rDNA and 18S rDNA repeat sequence probes, and consistent with those reported in other citrus species (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2017). \u003cem\u003eK\u003c/em\u003e-mer frequency distribution analysis (\u003cem\u003eK\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21) of Illumina short reads (57.05 Gb, \u0026sim;189 \u0026times; coverage) showed a distinct bimodal profile with 3.07% heterozygosity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), among the highest across all citrus genomes reported to date (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2018; Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003cp\u003eDue to its exceptionally high heterozygosity level, long-read PacBio HIFI sequencing (26.17 Gb, ~ 86 \u0026times;) and the Hi-C sequencing (31.77 Gb, 105 \u0026times;) were performed to obtain high-quality chromosome-scale reference assembly for Huyou. The de novo assembling using Hifiasm with HIFI long reads and Hi-C reads as inputs produced two haplotype genomes HY1 (324 contigs, total size at 326.65 Mb with contig N50 of 21.15 Mb) and HY2 (235 contigs, total size at 322.79 Mb with contig N50 of 18.77 Mb) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). GC contents for HY1 (56.6%) and HY2 (55.02%) were comparable with each other. The largest contigs for HY1 and HY2 were at 46.45 Mb and 34.26 Mb, respectively. The assembled contigs were anchored into 9 pairs of chromosomes with Hi-C scaffolding. Distinct chromosomal groupings on HI-C interaction heatmap were observed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, indicating the high quality of genome anchoring. The chromosomes were further ordered and named using \u003cem\u003eC. sinensis\u003c/em\u003e (v3.0 from the Citrus Genome Database \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://citrus.hzau.edu.cn/download.php\u003c/span\u003e\u003c/span\u003e) as reference. Particularly, 91.8% and 94.4% of the contigs for HY1 and HY2, respectively, could be assigned to chromosomes. Using the eukaryota_odb10 database as reference, the Benchmarking Universal Single-Copy Orthologs (BUSCO) revealed completeness of 98.1% and 97.7% for HY1 and HY2, respectively. Using HY1 as the reference, genome alignment of HY1 and HY2 revealed overall well-conserved synteny between the 2 haplotype genomes, albeit some large structural variations were observed mainly in the central region of each chromosome, such as a large insertion in chr4 and large inversions in chr8 and chr9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Despite the overall synteny, the mosaic pattern and gaps in the genomic alignment plot indicated significant genetic variations between HY1 and HY2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE), consistent with the exceptional high heterozygosity in Huyou observed in this study.\u003c/p\u003e\n\u003cp\u003eRepetitive sequences were identified in the two haplotype genomes HY1 (181.5 Mb or 55.56%) and HY2 (179.4 Mb or 55.58%) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In both genomes, LTR represented the largest TE family, followed by DNA, LINE, Rolling Circle, sequentially (\u003cstrong\u003eSupplementary File S0\u003c/strong\u003e). Despite a relatively smaller genome size and smaller total TE content, HY2 displayed higher number of TEs in all TE classes than that in HY1 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The repeat sequences were masked before gene model prediction using both homology search (\u003cem\u003eC. sinensis\u003c/em\u003e, \u003cem\u003eC. ichangensis\u003c/em\u003e, \u003cem\u003eC. medica\u003c/em\u003e) and RNA sequencing data (10 Gb), which led to the identification of 42091 and 42050 protein-coding genes for HY1 and HY2, respectively, with comparable mean length for transcripts, CDS, exon, and average exon number per gene (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). HY1 displayed relatively higher number of micro-RNA (161 miRNA) and tRNA (1003) but relatively lower number of small nuclear RNA (1393 snoRNA) than that in HY2 (150 miRNA, 790 tRNA, 1463 snoRNA). However, the number of ribosomal RNA in HY2 (2686 rRNA) was much higher than that in HY1 (1590 rRNA). The distribution of TEs, genes, and non-coding RNAs in HY1 and HY2 were displayed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. Overall, we observed higher gene density in the distal regions of each chromosome compared to the central region, whereas TEs tend to be enriched in the central regions. In summary, we obtained 2 haplotype-resolved Huyou assemblies with high quality and completeness.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eThe statistics for genome assembly and gene annotation of Huyou.\u003c/strong\u003e HY1 and HY2 refers the two haplotype genomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSequencing platform\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIllumina, PacBio, Hi-C\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eAssembly\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHY1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHY2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChromosome number\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal genome size\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e326.65 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e322.79 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSize anchored to chromosome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e299.73 Mb / 91.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e304.81 Mb / 94.4%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNumber of contigs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e324\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e235\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN50 contigs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21.15 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.77 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLargest contig\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.45 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.26 Mb\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eComplete BUSCOs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e98.1%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e97.7%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGC content\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.60%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.02%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eGenome annotation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNumber of protein-coding genes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42091\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42050\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMean mRNA length\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2570 bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2554 bp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMean CDS length\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1245 bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1245 bp\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMean exons per gene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMean exon length\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e216.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e215.87\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNumber of TEs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14097\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15545\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLINE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6762\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7428\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSINE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e248\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e269\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLTR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50487\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56301\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTotal size of TEs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e181.5 Mb / 55.56%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e179.4Mb / 55.58%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNumber of non-coding RNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emiRNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e150\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etRNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e790\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003erRNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1590\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2686\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esnoRNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1393\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1463\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eAncestral tracing of Huyou haplotype genomes HY1 and HY2\u003c/h2\u003e\n\u003cp\u003eDue to the high heterozygosity level and significant structural variations between the haplotype-resolved genomes, the ancestral origin of each block (100 kb) of the HY1 and HY2 genomes were determined using a k-mer based tracing approach. The distribution of differential k-mers of homologous chromosomes indicated that HY1 and HY2 could be clearly separated into 2 distinct progenitors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), except for chr9 in HY1 which displayed a mixture origin and was clustered with HY2. Using the citrus ancestor genomes mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e), pummelo (\u003cem\u003eC. maxima\u003c/em\u003e), and citron (\u003cem\u003eC. medica\u003c/em\u003e) as references, the genomic origin of HY1 and HY2 were traced and quantified. Results showed that 87.8% of HY1 genome originated from mandarin, followed by 7.3% from pummelo, 0.2% from citron, and 4.7% unknown source (could not be explicitly assigned) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cstrong\u003eSupplementary File S1\u003c/strong\u003e). In contrast, 85.0% of HY2 genome was derived from pummelo, followed by 2.9% from mandarin, 0.3% from citron, and 11.9% from unknown source (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results strongly suggested that Huyou may have originated from a hybridization between mandarin (HY1) and pummelo (HY2). At the chromosome level, chr1-chr7 of HY1 were predominantly from mandarin, whereas chr8 (11.9% pummelo) and chr9 (54.1% pummelo) of HY1 contained significant components from pummelo, which may have resulted from chromosome recombination. Mapping the genomic origin to the chromosomes indicated clear evidence of chromosome recombination in HY1 genome, whereby one distal region of chr8 and the central chr9 were replaced by pummelo genome (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). For the HY2 genome, all chromosomes were predominantly from pummelo, with minor introgression of mandarin on chr1 (3.3% mandarin), chr3 (7.6%), chr5 (4.8%), chr8 (1.2%), and chr9 (3.6%) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast to HY1 which retained clear segmental recombination boundary, the genome introgressions in HY2 were generally mosaic along the chromosomes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), which may provide further clue on the evolution history of Huyou following the hybridization of its pummelo and mandarin parents. Furthermore, the LTR insertion age analysis also indicated distinct profiles for HY1 and HY2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), providing additional support for two divergent progenitors. Specifically, most of the LRTs in HY1 displayed concentrated age with a single peak of insertion time, whereas the insertion time of LRTs in HY2 was relatively dispersed and contained an additional minor peak at a much older age (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eThe ancestral origin of each chromosome of haplotype-resolved Huyou.\u003c/strong\u003e Genome tracing was implemented using a k-mer-based approach by mapping sequencing data of three reference ancestral genomes mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e), pummelo (\u003cem\u003eC. maxima\u003c/em\u003e), and citron (\u003cem\u003eC. medica\u003c/em\u003e) to HY1 and HY2 using 100kb window. See the method section and \u003cstrong\u003eSupplementary File S1\u003c/strong\u003e for details.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eChromosome\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLength (Mb)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e໿Percentage (%) of sequences from ancestor species\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eOrigin tracing\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ecitron\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eUnknown\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eClosest\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAncestor\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHY1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eclementina\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eclementina\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eclementina\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29.54\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.68\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esinensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50.35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ereticulata\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esinensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.88\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esinensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esinensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emandarin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH1chr9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esinensis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e299.73\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e7.34\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.15\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e87.78\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e4.73\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eHY2\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emaxima\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24.99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28.27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eH2chr9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003egrandis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epummelo\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e304.81\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e84.95\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.30\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.90\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u003cstrong\u003e11.86\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003ePhylogeny dating and gene family analyses of HY1 and HY2\u003c/h2\u003e\n\u003cp\u003eTo deduce the divergence time of HY1 and HY2, single copy genes (2754 in total, \u003cstrong\u003eSupplementary File S2\u003c/strong\u003e) for 13 previously published citrus genomes and 6 citrus-related genomes, together with HY1 and HY2 were identified and used for phylogeny development. Five calibration points (annotated in tree in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA: 3.49\u0026thinsp;~\u0026thinsp;7.39, 3.92\u0026thinsp;~\u0026thinsp;11.0, 12.8\u0026thinsp;~\u0026thinsp;13.0, 7.57\u0026thinsp;~\u0026thinsp;25.92, and 13.5\u0026thinsp;~\u0026thinsp;30.0 Mya) were applied to estimate the divergence time. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, the 6 citrus-related species were resolved as basal branches (group F), followed by the early diverging citrus species \u003cem\u003eP. trifoliata\u003c/em\u003e (group G). The rest citrus species could be divided into 5 major groups (A-E highlighted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Three of those corresponded to the well-recognized citrus ancestors: mandarin (A: \u003cem\u003eC. reticulata\u003c/em\u003e), pumelo (C: \u003cem\u003eC. maxima\u003c/em\u003e), and citron (E: \u003cem\u003eC. medica\u003c/em\u003e). Consistent with the genome tracing results, HY1 and HY2 were grouped with mandarin and pumelo, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The divergence time of HY1 was estimated at 2.0 Mya, earlier than the split between \u003cem\u003eC. clementina\u003c/em\u003e and \u003cem\u003eC. reticulata\u003c/em\u003e but after the divergence of \u003cem\u003eC. sinensis\u003c/em\u003e, while HY2 was estimated to diverge at 2.18 Mya, before the split between \u003cem\u003eC. grandis\u003c/em\u003e and \u003cem\u003eC. maxima\u003c/em\u003e and after the branching of \u003cem\u003eC. hongheensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Therefore, HY1 and HY2 represented 2 novel genomes that has not been reported before. Using the earliest diverging \u003cem\u003ePoncirus trifoliata\u003c/em\u003e as reference, ks peak fitting showed that HY1, HY2, and other Citrus species displayed a single ks peaks at similar position (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), implying no additional large scale whole genome duplication events. Gene family analyses based on the developed phylogeny revealed more than 3-fold expansion (+\u0026thinsp;345) than contraction (-102) in the branch leading to HY1. In contrast, slightly more expansion (+\u0026thinsp;255) than contraction (-162) was observed in the branch leading to HY2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The percentage of single copy genes in HY1 and HY2 (6.5%) were relatively lower than that in other citrus species (8.1% ~ 12.1%), probably caused by genome annotation bias (bar plot in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Relatively higher percentage of HY2-specific genes (0.6% or 261) was identified than that in HY1 (0.4% or 158). The numbers of genes for 5 important metabolic pathways (map00900: terpenoid biosynthesis, map00906: carotenoid biosynthesis, map00940: phenylpropanoid biosynthesis, map00941: flavonoid biosynthesis, and map04626: plant-pathogen interaction) related to fruit quality were counted (heatmap in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA; \u003cstrong\u003eSupplementary File S3\u003c/strong\u003e). Overall, we observed a general expansion of gene members in all target pathways for citrus species, compared to the basal citrus-related species and the early diverging species \u003cem\u003eP. trifoliata\u003c/em\u003e, implying a potential human selection impact. Particularly, we noticed that group A and C encompassing HY1 and HY2 contained the most expanded gene members compared to other citrus species, except for \u003cem\u003eC. maxima\u003c/em\u003e Huazhouyou instead displayed relatively contracted gene families in these pathways. Further analysis is needed to investigate if this is caused by genome annotation bias or not. Noteworthy, a total of 158 and 261 species-specific genes were identified for HY1 and HY2, respectively (\u003cstrong\u003eSupplementary File S4\u003c/strong\u003e). Enrichment analyses showed that gene ontology (GO) terms related to photosynthesis, signal transduction, and response to stimulus were significantly enriched in the HY1-specific genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). In contrast, no significantly enriched GO term could be detected in the HY2-specific genes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eSynteny and structural variations analysis of HY1 and HY2\u003c/h2\u003e\n\u003cp\u003eThe synteny and structural variations of HY1 and HY2 were inferred in reference to their ancestral genomes: mandarin and pummelo, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Overall, we observed much less large structural variations between mandarin and HY1, compared to that between HY1 and HY2, consistent with our ancestor tracing results that HY1 was derived from mandarin. For example, the large inversions identified between HY1 and HY2 on chromosomes chr6, chr7, and chr8 were not observed between mandarin and HY1, providing direct evidence that HY1 may have originated from a mandarin ancestor. Similar patterns could be found for other chromosomes and other structural variations as well. In contrast to the strong synteny conservation between HY1 and its ancestor genome mandarin, we observed significant large structural variations between HY2 and the pummelo genome (\u003cem\u003eC. grandis\u003c/em\u003e L. Osbeck.cv. Wanbaiyou) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Noteworthy, these large structural variations such as the inversions on chromosomes chr1, chr5, and chr9 were specific to the pummelo genome used in this study but not present between HY1 and HY2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), implying that these large structural variations between HY2 and pummelo were mainly due to genome re-arrangement in \u003cem\u003eC. grandis\u003c/em\u003e L. Osbeck.cv. Wanbaiyou but not in the pummelo ancestor. Further analyses are needed to investigate if these large genome re-arrangements are conserved in other pummelo genomes or not.\u003c/p\u003e\n\u003cp\u003eThe genomic variations between HY1 and HY2 were examined specifically to investigate the allelic diversity in Huyou. We identified 1,996,631 SNPs, 150,487 insertions (1.66 Mb), and 139,519 deletions (1.57 Mb) (\u003cstrong\u003eSupplementary File S5\u003c/strong\u003e). These variations led to the classification of 25,503 highly diverged regions, accounting for (~\u0026thinsp;163.49 Mb or 50%) of the HY1 genome, consistent with heterozygosity. Despite these divergent regions, majority of the genome (252.08 Mb or 77.2%) were found conserved in syntenic regions, implying a generally conserved synteny between the ancestral genomes for HY1 and HY2 before their hybridization that resulted in Huyou. For structural variations, we identified a total of 73 inversions (21.04 Mb), 592 translocations (6.35 Mb), and 938 duplications (8.14 Mb), which were mainly located in the central region of each chromosome (\u003cstrong\u003eSupplementary File S5\u003c/strong\u003e). Three large inversions between HY1 and HY2 were observed in the middle regions of chr9 and chr8, followed by a few moderate inversions on chr1, chr2, chr3, chr4 and chr6. In addition, a genomic region in the middle of chr5 in HY2 seems to be expanded through significant duplications, which may have contributed to the chromosome extension of chr5 in HY2 compared to chr5 in HY1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Lastly, the number of genetic variations between HY1 and HY2 were calculated per 1 Mb window along each chromosome. The obtained variation distribution profile revealed a relatively higher-level of genetic diversity in the distal regions of each chromosome compared to the central regions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Mapping the genes from the 6 important metabolic pathways (map00020- Citrate cycle, map00195- Photosynthesis, map00900- terpenoid biosynthesis, map00906- carotenoid biosynthesis, map00940- phenylpropanoid biosynthesis, map00941- flavonoid biosynthesis) to the genome showed that many of these genes were located within the identified divergent regions, suggesting a high allelic diversity for important fruit quality characters in Huyou.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eAllelic imbalance of gene expression profiles in different Huyou tissues\u003c/h2\u003e\n\u003cp\u003eAs the most heterozygous citrus genome to date, Huyou provides an invaluable opportunity to study genome evolution. To investigate the allelic imbalance post ancient hybridization, we retrieved public transcriptome data of 6 different tissues (root, stem, leaf, flower, unripe fruit, ripe fruit) in Huyou from a previous study (Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024) and compared the expression levels of collinear genes (26,092 in total) between HY1 and HY2. The percentages of genes preferentially expressed in HY1 (HY1higher (log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(HY1/HY2)\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;1) and HY2 (HY2higher log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(HY1/HY2)\u003c/sup\u003e \u0026le; -1) were calculated across each chromosome and tissue (\u003cstrong\u003eSupplementary File S6\u003c/strong\u003e). Overall, we observed relatively higher numbers of HY2-preferential genes than HY1-preferential genes across most tissues and chromosomes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), implying a clear transcriptional bias for HY2 genes following hybridization. Noteworthy, we found that the HY2 allelic dominance was exceptionally prominent in root tissue (HY2higher at 12.5% versus HY1higher at 7.24%) compared to that in the other tissues (leaf, stem, flower, unripe fruit, ripe fruit), which only displayed slight HY2 allelic dominance (11.67% versus 11.10% on average), implying a strong tissue-specific pattern. The exceptional HY2 allelic dominance in root tissue across the 9 chromosomes could be seen in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, relatively more evident on chromosomes chr1-7 and relatively mild on chr8 and chr9. This might be caused by the large segmental replacements observed on chr8 and chr9. Indeed, at the chromosome level, chr9 displayed the lowest level of total allelic divergence (HY1higher\u0026thinsp;+\u0026thinsp;HY2higher\u0026thinsp;=\u0026thinsp;16.77%) and followed by chr8 (21.29%) and other chromosomes (22.30-24.27%).\u003c/p\u003e\n\u003cp\u003eDue to the exceptional HY2 allelic dominance in the root tissue, the collinear genes with transcriptional preference from HY1 (1354 HY1higher genes) and HY2 (2432 HY2higher genes) were used for KEGG pathway enrichment analysis (corrected p-value\u0026thinsp;\u0026le;\u0026thinsp;1-e5). Interestingly, these two sets of genes shared some commonly enriched pathways that are mainly related to antioxidants biosynthesis, including flavonoid, phenylpropanoid, isoflavonoid, carotenoid, benzoxazinoid, flavone, flavonol, alkaloid, ascorbate, and steroid biosynthesis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). It is well-known that antoxidant biosynthesis plays critical role in plants\u0026rsquo; adaptation to various biotic and abiotic stresses. The allelic imbalance of genes within these pathways in the root tissue may have important implication in Huyou\u0026rsquo;s environmental adaptation following its ancient genome hybridization. In addition to these common pathways, HY1 preferential genes were uniquely enriched with amino acid and linoleic acid metabolism and the biosynthesis of cutin, wax, diterpenoid, triterpenoid, sesquiterpenoid, stilbenoid, diarylheptanoid, and gingerol which are closely linked to environmental adaptation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, HY2 preferential genes were uniquely enriched with protein families related to signaling and cellular processes, including transporter genes, sphingolipid metabolism, and genes responsible for fructose, mannose, pentose, glucuronate interconversions, glycerolphospholipid, peptidoglycan metabolism. In addition, the biosynthesis of anthocyanin, folate, and various secondary metabolites were also enriched in HY2 preferential genes but not in HY1 preferential genes.\u003c/p\u003e\n\u003cp\u003eIn addition to the root tissue, the allelic imbalance genes from HY1 (2053HY1higher genes) and HY2 (2084 HY2higher genes) in ripe fruit tissue were also investigated. Overall, we observed a strikingly similar enrichment profiles with that in the root tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting that the allelic imbalance at the gene functional level may be conserved across tissues despite the variation in gene numbers. In details, like that in the root tissue, the commonly enriched pathways from HY1 and HY2 were also prominently related to antioxidants biosynthesis such as flavonoid, phenylpropanoid, isoflavonoid, flavone, flavonol, isoquinoline alkaloid, carotenoid, benzoxazinoid, and glucosinolate. Other common pathway terms included lipid, cofactors and vitamin, amino acid, porphyrin and chlorophyll metabolism which were also detected in the root tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). The unique pathway terms in HY1 (linolenic acid metabolism and the biosynthesis of cutin, suberine, wax, stilbenoid, diarylheptanoid, gingerol, sesquiterpenoid, triterpenoid) and HY2 (tryptophan, ascorbate, aldarate, sphingolipid, pentose, glucuronate, terpenoids metabolisms and the biosynthesis of secondary metabolites, anthocyanin, folate, peptidoglycan) in ripe fruit tissue were also similar to that in the root tissue (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eTissue-specific allelic imbalance in key pathways\u003c/h2\u003e\n\u003cp\u003eCarotenoid biosynthesis pathway (map00906) plays a critical role in citrus fruit quality, controlling fruit color and flavor. To investigate the genomic characteristics of key Huyou candidate genes in this pathway, we first investigated the allelic imbalance by mapping the ratio of HY1 and HY2 collinear gene transcription to this pathway. Whilst the major components of the pathway displayed no allelic preference, we observed a clear tissue-specific allelic imbalance for some key genes responsible for the astaxanthin biosynthesis specifically (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). For instance, we found one enzyme (EC 1.14.15.24) encoding beta-carotene 3-hydroxylase displayed a consistent HY2 dominance in the root tissue but a HY1 dominance in the stem, flower, unripe fruit, and ripe fruit tissue and no imbalance in leaf. Another enzyme (EC 1.23.5.1) encoding violaxanthin de-epoxidase displayed HY1 dominance in root, leaf, ripe fruit but no imbalance in the other tissue. Conversely, the enzyme (EC 1.2.3.14) encoding abscisic-aldehyde oxidase display HY2 dominance in stem, leaf, and unripe fruit but not in other tissue. These observations provided direct support of various allelic imbalance present in Huyou genome and shed valuable insights into genome evolution following ancient hybridization.\u003c/p\u003e\n\u003cp\u003eTo characterize the complete profile of the carotenoid biosynthesis pathway in Huyou, we further extracted the gene copy number information of key genes (represented by 39 KEGG orthologs) in HY1, HY2, and another 8 citrus species and explored their transcriptional profiles in 6 Huyou tissues. Overall, we observed conserved gene presence and copy number pattern across different citrus species, with cytochrome P450 (CYP701, CYP97C1, CYP707A, CYP97A3, CYP82G1) as the most expanded protein families within the carotenoid pathway. Copy number variations (CNVs) were observed in all KO orthologs except for 2 single copy genes K15744 (zeta-carotene isomerase) and K17911 (beta-carotene isomerase) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, HY1 and HY2 displayed varied copy numbers in 33 out of the 39 KO orthologs. Noteworthy, Huyou (HY1 or HY2) displayed the highest copy numbers in 21 out of the 39 KO orthologs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). At the gene expression level, we observed varied transcriptional profiles across the 6 tissues investigated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Interestingly, we found that over half of the KO orthologous genes in Huyou displayed the highest expression in the flower tissue, followed by the root tissue. Instead, out of the 39 KO orthologs, we found 17 KOs displayed above median transcription in fruit tissues (either unripe or ripe), of which 6 KOs (FDFT1, LCYB, LCYE, CCS1, AOG, DWARF27) displayed the highest expression in fruit tissues (highlighted in red rectangles in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC), which require more attention for their potential critical roles in fruit development, despite that some of these genes may not be expanded in Huyou (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). At the pathway map level, we found most of the genes leading to the terpenoid biosynthesis (C30, C15, C5, C10, C20) were found expanded compared to other species, whilst this pattern was not prominent for the carotenoid biosynthesis (C40) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). Taken together, CNV and transcriptome analyses revealed unique genomic characteristics for the carotenoid biosynthesis pathway in Huyou which may have contributed to its distinct fruit flavor against other citrus species.\u003c/p\u003e\n\u003cp\u003eThe ratios of HY1 and HY2 collinear genes (log2\u003csup\u003e(HY1/HY2)\u003c/sup\u003e) were mapped to KEGG pathway map00906 using TBtools. Transcriptome data in 6 Huyou tissues (root, stem, leaf, flower, unripe fruit and ripe fruit) from a previous study was downloaded and analyzed. Yellow, red, and green highlights indicated neutral, HY1 dominant, and HY2 dominant, respectively. Only the Astaxanthin biosynthesis module of map00906 were displayed.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a unique natural hybrid citrus landrace first discovered and widely grown in Zhejiang province in China, Huyou holds important agricultural, economic, and medicinal values due to its premium fruit quality characteristics such as a golden skin, abundant bioactive components, extended storage life. In this study, we leverage PacBio long-read and Hi-C sequencing technologies and presented 2 high-quality haplotype-resolved genome assemblies HY1 and HY2 of 120 years old Huyou \u0026ldquo;ancestral tree\u0026rdquo;. Our in-depth ancestor tracing, phylogeny dating, genome structure, and transcriptome analyses offered an unprecedent view of Huyou\u0026rsquo;s genetic makeup and evolutionary origin. The explicit determination of Huyou\u0026rsquo;s genome origin from a mandarin and pummelo hybridization event solved a long biological mystery that has puzzled local Huyou growers and botanists (Li, \u003cem\u003eet al.\u003c/em\u003e, 2019; Chen, \u003cem\u003eet al.\u003c/em\u003e, 2002; Chen, \u003cem\u003eet al.\u003c/em\u003e, 2006; Xu, \u003cem\u003eet al.\u003c/em\u003e, 2006). Based on whole genome survey of 58 worldwide diverse citrus germplasms, hybrid citrus species derived from 2 or more ancestral species have been found to have generally high genome heterozygosity at 1.5\u0026ndash;2.4% (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2018). Here, we highlight the exceptional genome heterozygosity of Huyou at 3.07%, among the highest across known citrus genomes, which will make Huyou an interesting subject for genome evolution studies by broad citrus researchers in the future. The haplotype genomes HY1 and HY2 obtained in this study essentially represent 2 novel ancestral citrus genomes not reported before, thus would significantly contribute to the currently limited citrus genetic pool. This study will enable citrus breeders to harness the genetic potential of Huyou more effectively, potentially accelerating the development of improved citrus varieties with unique traits.\u003c/p\u003e \u003cp\u003eThe explicit resolution of Huyou\u0026rsquo;s genome origin in this study highlights the power of haplotype-resolved genomes in dissecting the complex evolutionary origin of citrus species. In the citrus genus, there are a total of 30 species, most of which have been derived from the hybridization across 3 ancestral species: mandarin (\u003cem\u003eC. reticulata\u003c/em\u003e), pummelo (\u003cem\u003eC. maxima\u003c/em\u003e), citron (\u003cem\u003eC. medica\u003c/em\u003e). The ancient hybrids were subjected to further reticulate hybridization during citrus domestication, leading to a complex history of admixture in current citrus cultivars and landraces characterized by high heterozygosity (Wu, \u003cem\u003eet al.\u003c/em\u003e, 2018). Tracing the evolution and domestication history of citrus species has been extremely challenging in traditional citrus genome studies based on short read sequencing, which often produced a mosaic genome assembly and failed to account for allelic variants, leading to incomplete or inaccurate representations of the genome. By leveraging a k-mer-based tracing approach on the haplotype-resolved genomes, we are able to resolve and quantify the ancestral components of Huyou to specific genomic regions. In addition, the haplotype genome tracing also allows us to dissect the segmental insertions, deletions, inversions, recombinations and other structural variations between HY1 and HY2, particularly the recombination events on chr8 and chr9 in HY2. These unprecedented genomic insights are critical for the accurate interpretation of the genomic and phenotypic features of Huyou. Our study was inspired by a recent study in which the haplotype-resolved lemon genome was traced to be an admixture of 3 ancestor genomes of citron, mandarin, and pummelo (Bao, \u003cem\u003eet al.\u003c/em\u003e, 2023). Notably, numerous studies highlight the increasing trend of generating chromosome-level and haplotype-resolved genome assemblies across the plant kingdom, such as potato, lychee, tea, and strawberry. The advantages of haplotype-resolved genome for citrus genome are being increasingly appreciated in several recent citrus genome research including \u003cem\u003eC. limon\u003c/em\u003e (Mario, \u003cem\u003eet al.\u003c/em\u003e, 2021), \u003cem\u003eC. australis\u003c/em\u003e (Nakandala, \u003cem\u003eet al.\u003c/em\u003e, 2023), \u003cem\u003eC. changshanensis\u003c/em\u003e (Miao, \u003cem\u003eet al.\u003c/em\u003e, 2024), \u003cem\u003ePapeda\u003c/em\u003e (Wang, \u003cem\u003eet al.\u003c/em\u003e, 2025). However, we noticed that only the collapsed genome sequence was reported and analyzed in several haplotype-resolved genome studies, which in our opinion should be avoided, particularly for those organisms with high heterozygosity such as Citrus species. We expect an increasingly more haplotype-resolved citrus genomes to be generated and published, which is positioned to revolutionize the citrus genome evolution and domestication research.\u003c/p\u003e \u003cp\u003eAllelic imbalance (AI) refers to the unequal expression of alleles from a heterozygous locus, leading to differential gene expression. This is another critical insight gained from haplotype-resolved genome assembly. In this study, based on gene expression analysis of collinear genes between HY1 and HY2, we observed clear evidence of AI (HY2 dominant over HY1) across all 6 tissues investigated, suggesting AI may be a prevalent phenomenon in genome evolution in the plant kingdom. One of the most notable findings in this study is that we observed AI tends to be imbalanced across tissues, i.e the HY2 allelic dominance was much more prominent in some tissues such as root over the others. Metabolic pathway enrichment analyses of AI genes in Huyou root tissue revealed a strong over-representation of genes related to various antioxidant production, such as ໿flavonoid, phenylpropanoid, isoflavonoid, carotenoid, benzoxazinoid, flavone, flavonol, alkaloid, ascorbate, and steroid biosynthesis. These antioxidants have well-known biological functions in plants\u0026rsquo; adaptation to various biotic and abiotic stresses. The allelic imbalance of genes within these pathways in the root tissue may have important implication in Huyou\u0026rsquo;s environmental adaptation following its ancient genome hybridization. It is interesting to observe that these antioxidant biosynthesis terms were over-represented in both HY1-higher and HY2-higher genes, suggesting an overall evolutionary preference of AI genes related to environmental adaptation. At the plant tissue level, we observed overall conservation of AI functions across tissues such as root and ripe fruit, despite a significantly higher degree of AI in root. This novel insight needs to be verified in future analysis in other species. In plants, especially those with high heterozygosity, AI plays a significant role in genome evolution, influencing traits such as disease resistance, growth patterns, and adaptation to environmental changes (Jang, \u003cem\u003eet al.\u003c/em\u003e, 2024; Djari, \u003cem\u003eet al.\u003c/em\u003e, 2024; Li, \u003cem\u003eet al.\u003c/em\u003e, 2023). The impact of AI on plants\u0026rsquo; genome evolution and environmental adaption has been well recognized in several recent haplotype-resolved genome studies. For example, a study in \u003cem\u003ePopulus tomentosa\u003c/em\u003e identified heterozygous alleles associated with adaptive traits, highlighting the role of AI in evolutionary processes (Li, \u003cem\u003eet al.\u003c/em\u003e, 2023). Similarly, haplotype-resolved assemblies grapevine identified structural variations between haplotypes associated with allelic expression linked to disease resistance and fruit quality (Djari, \u003cem\u003eet al.\u003c/em\u003e, 2024). Another recent study in \u003cem\u003ePinus densiflora\u003c/em\u003e revealed the functional contributions of AI to flowering and abiotic stress-related traits (Jang, \u003cem\u003eet al.\u003c/em\u003e, 2024). In addition to the overlapping AI functions, haplotype-specific AI functions were also observed. The biological implications of our observed AI patterns remain to be characterized, particularly for citrus genome research. As far as the authors are concerns, our study is the first to demonstrate the presence of strong AI in citrus hybrid genome. Based on our observation of AI in Huyou, we reason that AI should be prevalent across the various citrus genomes due to the presence of frequent hybridization events. Given the still limited haplotype resolved genome assemblies for citrus species, the importance of AI in citrus genome evolution and its interplay with phenotypic diversity in citrus deserves much more attention in future research.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe sequenced a 120-year-old \"ancestral tree\" of \u003cem\u003eC. changshan\u003c/em\u003e Y. B. Chang (Huyou), a unique natural hybrid citrus landrace first discovered in Changshan, Zhejiang province, China. Huyou displayed genome heterozygosity at 3.07%, among the highest across known citrus genomes. We presented two high-quality haplotype-resolved genomes HY1 and HY2 and determined that Huyou had originated from a mandarin and pummelo hybridisation event, resolving a mystery that has puzzled local Huyou growers and botanists. HY1 and HY2 had diverged at 2.0 Mya and 2.18 Mya, earlier than the split of \u003cem\u003eC. clementina\u003c/em\u003e and \u003cem\u003eC. reticulata\u003c/em\u003e, and the split of \u003cem\u003eC. grandis\u003c/em\u003e and \u003cem\u003eC. maxima\u003c/em\u003e, respectively. Haplotype-based transcriptome analyses also demonstrated the presence of strong tissue-specific allelic imbalance in Huyou\u0026rsquo;s genome. The haplotype genomes HY1 and HY2 represent two novel ancestral citrus genomes not previously reported, contributing to the limited citrus genetic pool and enabling citrus breeders to harness Huyou's genetic potential. The explicit resolution of Huyou\u0026rsquo;s genome origin highlights the power of haplotype-resolved genomes in dissecting the complex evolutionary origin of citrus species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe acknowledge the relevant citrus research communication for making the citrus genomic data available to the public, particularly the recently published Huyou transcriptome data\u0026nbsp;(Miao\u003cem\u003e, et al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eYJ, CL, ZZ designed and supervised the study. ZZ, YJ, TX, YL, HH, LL, BG, PZ, CT, TQ, ZY, ZC performed data analysis. YJ, ZZ, XH, YL wrote the manuscript. TX, CL, LW edited the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe raw sequencing data has been deposited at NCBI with project ID PRJNA1090545 (release upon acceptance). The genome assemblies and gene annotations are available at https://figshare.com/s/ba2b2081a2512770c31a\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32301872), the Natural Science Foundation of Zhejiang Province (LQ23C130003), and Interdisciplinary Research Project of Hangzhou Normal University (2025JCXK01).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u003cb\u003eGmitter, FG, Chen, C, Machado, MA, de Souza, AA, Ollitrault, P, Froehlicher, Y, Shimizu, T\u003c/b\u003e. \u003cb\u003e2012\u003c/b\u003e. 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Genomic insights into selection for heterozygous alleles and woody traits in. \u003cem\u003ePlant Biotechnology Journal\u003c/em\u003e, \u003cb\u003e21\u003c/b\u003e: 2002\u0026ndash;2018.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Citrus changshan-huyou Y. B. Chang, evolutionary origin, haplotype-resolved genome, Hi-C, highly heterozygous genome, allelic imbalance","lastPublishedDoi":"10.21203/rs.3.rs-6552063/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6552063/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCitrus is an interesting model for plant genome evolution study due to its reticulate evolutionary history with frequent hybridization. \u003cem\u003eCitrus changshan\u003c/em\u003e Y. B. Chang (Huyou) is a unique landrace first discovered in Zhejiang Province, China with premium fruit quality. The evolutionary origin of Huyou has puzzled local botanists. Here, we sequenced a 120-years-old “ancestral tree” of Huyou and assembled two haplotype-resolved genomes HY1 and HY2. Huyou displayed 3.07% genome heterozygosity level, the highest in published citrus genomes. K-mer-based genome tracing revealed that HY1 contained 87.8% mandarin, 7.3% pummelo, 0.2% citron origin, whereas HY2 had 85.0% pummelo, 2.9% mandarin, 0.3% citron, implying a clear hybridization origin. Phylogeny dating showed that HY1 (2.0Mya) and HY2 (2.18Mya) had diverged earlier than the split of \u003cem\u003eCitrus clementina\u003c/em\u003e and \u003cem\u003eCitrus reticulata\u003c/em\u003e, and the split of \u003cem\u003eCitrus grandis\u003c/em\u003e and \u003cem\u003eCitrus maxima\u003c/em\u003e, respectively. Further transcriptome analyses revealed a strong allelic dominance of HY2 over HY1 in root tissue and moderately in stem, leaf, flower, and fruits. We found that genes related to antioxidants biosynthesis and lipid metabolisms were most significantly affected by allelic imbalance. This first report of allelic imbalance in citrus species support Huyou as an interesting model to investigate genome evolution following distant hybridization.\u003c/p\u003e","manuscriptTitle":"Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 12:18:07","doi":"10.21203/rs.3.rs-6552063/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ce4d6ec-a97a-48c1-810e-98ebdc4342b9","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48444397,"name":"Biological sciences/Evolution/Molecular evolution"},{"id":48444398,"name":"Biological sciences/Computational biology and bioinformatics/Classification and taxonomy"}],"tags":[],"updatedAt":"2025-07-07T17:15:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-15 12:18:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6552063","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6552063","identity":"rs-6552063","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}