Global Genomic Diversity of Persian Walnut Reveals A Selective Sweep Underlying Domestication

Global Genomic Diversity of Persian Walnut Reveals A Selective Sweep Underlying Domestication | 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 Global Genomic Diversity of Persian Walnut Reveals A Selective Sweep Underlying Domestication Pat Brown, Mohammad Arab, Abdollatif Sheikhi, Charles Leslie, and 30 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6087036/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Persian walnut (Juglans regia L.) is cultivated globally for its large edible nuts, which have a dehiscent hull that simplifies nut harvest and a papery inner shell or septum that allows easy kernel extraction. Here, we use reduced-representation sequencing to genotype the largest panel of J. regia ever assembled (n = 2,850) and evaluate its evolutionary history and population structure. Haplotype length analysis and genome-wide trait association identify a selective sweep on chromosome 14 associated with the hard septum characteristic, providing new insight into the origin and domestication of J. regia . A YABBY transcription factor in the swept region shows exon skipping that causes frame-shift and deletion of the YABBY domain and is enriched in J. regia hull and shell tissues. These results suggest both a mechanism underlying walnut domestication and a blueprint for domesticating other tree nuts in the Juglandaceae . Biological sciences/Plant sciences/Plant genetics/Plant hybridization Biological sciences/Genetics/Plant genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Persian walnut ( Juglans regia L.) is cultivated globally for food, fuel, timber, and traditional medicine (Vahdati et al., 2019; Costa et al., 2014). Walnut kernels are rich in polyunsaturated fatty acids and bioactive phytochemicals, including phenolic compounds and phytosterols that offer numerous health benefits (Ma et al., 2019; Nguyen and Vu, 2023). Over the last two decades, global production of walnut has surged by more than 200%, accompanied by increasing public recognition of these health benefits. While the genus Juglans contains over twenty species with edible nuts, commercial nut production is largely limited to J. regia and its sister species J. sigillata , which differ from all other species in the genus in two characteristics: a hull that dehisces at maturity, facilitating nut harvest (Figure 1A), and the presence of a papery septum, a modified section of the endocarp (shell) that extends between the kernel halves, facilitating extraction of the kernel (Figure 1B). All other members of the genus have nuts with indehiscent hulls and a hard, shell-like septum that prevents the removal of intact kernels from the shell. Portable and energy-dense, walnuts have long played a significant role in human endeavors, and have long history of cultivation across Asia, the Middle East and Europe (Bernard et al., 2018; Ji et al., 2021). Silk Road trade routes contributed to walnut dispersal, and many ancient trees are still found along these routes in Iran and surrounding regions (Arab et al., 2019; Shahi Shavvon et al., 2023). Recent molecular studies highlight the Himalayan region as a center of diversity (Yan et al., 2024; Ye et al., 2024), and thick-shelled, apparently wild walnuts that persist in this area are often used ceremonially (Avanzato et al., 2014). Identification of crop wild relatives and characterization of genetic diversity across the range of Juglans regia is essential for both efficient germplasm conservation and breeding efforts. Plant domestication frequently results in genetic bottlenecks, selective sweeps near domestication-related loci, and a rise in deleterious variant frequencies, as shown in annual crops such as maize, wheat, rice, and sunflower (Hufford et al., 2012; Huang et al., 2012; Badouin et al., 2017; Moyers et al., 2018; Maccaferri et al., 2019). However, population genetic signatures of domestication are relatively unstudied in woody perennials, which are often clonally propagated, highly heterozygous, and genetically closer to their wild ancestors. Domestication loci in woody perennials control dioecy-to-hermaphroditism transitions in grape (Massonnet et al., 2020) and papaya (Chávez-Pesqueira and Núñez-Farfán, 2017), loss of seed toxicity in almond (Sánchez-Pérez et al., 2019), and the breakdown of self-incompatibility in peach (Yu et al., 2018) and some cacao populations (Cornejo et al., 2018). Despite the importance of walnut in global agriculture, the genetic basis of domestication in J. regia is not well understood. The genus Juglans is separated into three sections, two of which bear nuts with an indehiscent hull and a hard septum: Cardiocaryon, the Asian walnuts, and Rhysocaryon, the New World black walnuts. The third section, Dioscaryon, includes not only J. regia but also J. sigillata, a taxon indigenous to southwestern China that diverged from J. regia approx.0.85 million years ago (MYA) (Ding et al., 2022). Both J. regia and J. sigillata bear nuts with a dehiscent hull and a papery septum (Wambulwa et al. 2022). Widespread genomic introgression of J. regia into J. sigillata ( Yan et al., 2024) supports the hypothesis that these traits arose once during the domestication of J. regia and were subsequently introgressed into J. sigillata , though their genetic control has not been determined. To explore genomic diversity, population structure, and domestication signals in J. regia , we applied reduced representation sequencing to a global sample of 2,893 individuals, including 2,850 J. regia and 43 J. sigillata, and obtained a dataset of 46,582 SNPs. Haplotype length analysis revealed a major selective sweep on chromosome 14 that co-segregates with septum hardness, providing a first step towards understanding the evolutionary genetic basis of domestication in J. regia . Walnuts carrying the minor allele under the sweep display a hard, shell-like septum similar to other Juglans . The major allele is associated with a papery septum and is fixed in J. sigillata samples, offering insight into the relationship between these taxa and the genetic modifications that supported the establishment of J. regia as a major food crop. Results Population Structure of Walnut Population structure in a global sample of unimproved J. regia (n = 2,438) exhibited the expected pattern of isolation by distance (Fig. 2 A-B). Samples from the Tibetan Plateau in China clustered with samples from the Himalayan region of northern Pakistan and India (Fig. 2 A-B), and samples from the Caucasus region between the Black and Caspian Seas showed genetic separation from Iranian, Turkish, and Russian samples. Feral trees collected from the New World predominantly exhibited Western European ancestry (Fig. 2 A). Principal component analysis (PCA) supported the population structure results, with samples from Western Europe and East Asia separating along PC1 and admixed samples clustering near the origin (grey circles; Fig. 2 C). After varying the number of subpopulations from two to ten, we selected five subpopulations (Western Europe, Caucasus, Iran, Himalaya, and China) as the number that maximized the likelihood of membership of the 2,481 unimproved trees (Figure S1 , see Methods). Genetic Diversity, Differentiation, and Linkage Disequilibrium of Walnut Subpopulations Nucleotide diversity (pi) and polymorphism (theta) were highest in the Himalaya and lowest in Western Europe (Table 2 ), whereas genetic differentiation ( F ST ) from the J. sigillata outgroup was lowest in the Himalaya and highest in Western Europe (Table 2 ). Within J. regia , genetic differentiation was greatest when comparing Western Europe with all other groups. Additionally, linkage disequilibrium decay was markedly slower in Europe compared to all other subpopulations (Fig. 2 D). Collectively, these results suggest that the Himalayan subpopulation is ancestral and the Western Europe subpopulation has experienced a bottleneck. Rapid decay of linkage disequilibrium in the Iranian subpopulation may reflect a high long-term effective population size in this center of walnut diversity. Table 2 Nucleotide diversity (Pi), polymorphism (Theta), and F ST within and between walnut populations. Population Pi Theta F ST W. Europe Caucasus China Iran Himalaya J. sigillata W. Europe 0.11 0.08 - 0.12 0.14 0.07 0.17 0.34 Caucasus 0.14 0.10 - - 0.10 0.05 0.09 0.28 China 0.14 0.10 - - - 0.08 0.10 0.27 Iran 0.15 0.12 - - - - 0.07 0.24 Himalaya 0.17 0.15 - - - - - 0.22 Contribution of Walnut Subpopulations to Improved Cultivars To assess subpopulation contribution to modern breeding programs, we defined ancestry informative markers for each subpopulation as the top 2% of F ST values between that subpopulation and the other four subpopulations, and scanned each improved walnut genotype (n = 369) for its proportion of ancestry informative alleles from each subpopulation (Table S1 ). ‘Chandler’, the world’s most popular walnut cultivar, is typical in having predominantly Western European ancestry (72% of alleles) with limited contribution from the Iran (12%), China (10%) Caucasus (5%) and Himalaya (2%) subpopulations (Fig. 2 E). Overall, ancestry informative alleles in the 228 lines from the California breeding program average 61%, 15%, 15%, 7%, and 2% from Western Europe, Iran, China, Caucasus, and the Himalaya respectively. The contributions to all 369 improved lines in this study were similar (59%, 17%, 13%, 9%, 3%; Table S1 ). Extended Haplotype Homozygosity Reveals a Selective Sweep on Walnut Chromosome 14 Using haplotype length tests including extended haplotype homozygosity (EHH) and integrated EHH score (IES) to investigate evidence of selection in J. regia , we identified a region on chromosome 14 exhibiting IES scores more than ten times the genome average, indicative of a selective sweep (Fig. 3 A). This sweep is of comparable magnitude in both improved and unimproved groups and across all five subpopulations of J. regia (Figure S2 ), suggesting a potential role in walnut domestication. Notably, we observed similar IES scores in the same genomic region in J. sigillata (Fig. 3 B), and in a combined sample of both J. regia and J. sigillata , indicating that the same haplotype was selected in both species. To elucidate this result, we calculated the average F ST between J. regia and J. sigillata in non-overlapping 100-SNP windows throughout the genome. The four windows with the lowest F ST are all located within the selective sweep region (Figure S3), suggesting possible introgression of the swept allele from J. regia into J. sigillata . The SNP with the highest IES score in all analyses is located at 18,013,474 bp on chromosome 14; the minor allele at this SNP is very rare (minor allele frequency [MAF] = 0.011; 63/5,786 gametes) and is present in only 12 homozygous and 39 heterozygous individuals of J. regia , primarily distributed in northern Pakistan, Iran, and Kyrgyzstan (Table S1 , Figure S4). Homozygotes for the major allele have significantly lower genome-wide nucleotide diversity and polymorphism when summarizing over non-overlapping 100-SNP windows across all chromosomes except chromosome 14 (Figure S5). All samples of J. sigillata are homozygous for the swept, major allele. An additional selective sweep on chromosome 11, found only in improved walnuts and the European subpopulation (Figure S2 ), is likely associated with the dominant allele at the Lateral bearing1 ( Lb1) locus, which controls flower development in axillary buds (Bernard et al., 2020 ). The Minor Allele Under the Selective Sweep is Associated with a Hard, Shell-like Septum To investigate the reason for the selective sweep on chromosome 14, we collected hulled nut samples from 380 trees, including three homozygotes and eleven heterozygotes for the minor allele at the SNP with the highest IES score (Table S2 ). Among these 14 trees, 9 produced nuts with a very hard internal septum (Table 3 ), similar to other Juglans species (Fig. 3 C), while 364 of the 366 remaining trees displayed the papery septum typical of domesticated walnuts. To validate this observation, we used hard/papery septum as a binary trait to conduct a genome-wide association study (GWAS) using a mixed logistic regression model, revealing a cluster of SNPs between 17.9–18.5 Mb on chromosome 14 that strongly associates with the septum phenotype (Fig. 3 D; Table S3). The most significant GWAS SNP is located at 18,485,943 bp, approximately 500 kb from the SNP with the highest IES score, and co-segregates even more strongly with the septum phenotype (Table 3 ). Table 3 Co-segregation of septum phenotype with top SNPs from IES and GWAS Septum Hard Papery Top IES SNP 18,013,474 bp AA 2 364 Aa 6 5 aa 3 0 Top GWAS SNP 18,485,943 bp AA 4 368 Aa 5 0 aa 3 0 Tissue-Biased Exon Skipping in a YABBY2 Gene Expressed in Walnut Shell The interval from 17.7–18.5 Mb on chromosome 14 includes the ten most significant SNPs from both IES and GWAS analyses and contains fourteen predicted protein-coding genes. Expression of these genes was characterized using 40 RNAseq libraries representing four Juglans species and all three sections of Juglans : J. mandshurica (Cardiocaryon), J. nigra (Rhysocaryon), and J. regia and J. sigillata (both Dioscaryon). The 8th gene in the interval (LOC109014004; 18.157–18.165 Mb) shows highly (~ 5.8-fold) elevated expression in shell (n = 20) and hull (n = 8) relative to leaf (n = 12) tissue (Fig. 4 A; Table S4) and is predicted to encode a YABBY2 transcription factor. Splice junctions in the RNAseq data reveal multiple isoforms for this gene, including a shortened exon 4 (41 versus 50 bp) that is fixed in Dioscaryon and a skipped exon 4 that results in a frame-shift eliminating the YABBY domain that is at much higher frequency in Dioscaryon (Fig. 4 B; Table S5). We then quantified the tissue- and species-specificity of this exon skipping event in shell, hull, and leaf transcripts of the four Juglans species, and found that exon skipping is very rare in J. nigra (< 1% of all transcripts), uncommon in J. mandshurica (6%), and common in both J. regia (51%) and J. sigillata (33%; Fig. 4 C, Table S2 ). Exon skipping in J. regia , but not J. sigillata , is significantly enriched in shell and hull relative to leaf tissue (Fig. 4 C; t = -5.5, p = 9e-5). Discussion This study represents the largest genetic sample of Juglans regia reported to date. Although the number of markers utilized is modest compared to studies employing whole-genome sequencing on smaller sample sizes (Stevens et al., 2018 ; Zhang et al., 2019 ; Ji et al., 2021 ), our findings align with previous research identifying the Himalayan region as a center of genetic diversity (Yan et al., 2024; Ye et al., 2024 ). Our results show that Western European walnuts are bottlenecked as previously reported, reflecting their retreat to glacial refugia during the last Ice Age (Aradhya et al., 2017 , Ebrahimi et al., 2017 ; Pollegioni et al., 2017 ). Among the five identified subpopulations, Iran and Pakistan exhibit the highest nucleotide diversity and polymorphism and the lowest levels of linkage disequilibrium. While our sampling from Afghanistan remains limited, the pre-domestication distribution of wild J. regia likely extended from northwestern Iran through Afghanistan and northern Pakistan and potentially further east (Bernard et al., 2018 ). These high-diversity subpopulations are little represented in advanced breeding material. The rare haplotype under the selective sweep on chromosome 14 is essentially confined to these regions, and individuals carrying this haplotype have much higher genome-wide nucleotide diversity, as would be expected for crop wild relatives not subjected to a domestication bottleneck. Since humans would have comparatively little incentive to gather or propagate thick-shelled walnuts, we propose that the selective sweep associated with the papery septum trait underlies the domestication of J. regia . This is the first report of a selective sweep on chromosome 14 of Juglans regia , despite numerous previous genome-wide sequencing studies on diverse walnut germplasm. Several factors could account for this surprising result. First, our sample size is larger than that of previous studies, and the rare haplotype on chromosome 14 is present at a frequency of only 1.1%. Second, this is the first study to test for selection in Juglans using haplotype length statistics, which may offer greater sensitivity to recent selection compared to other methods. Third, a prior study (Ji et al., 2021 ) regarded J. sigillata as the wild progenitor and utilized reductions in nucleotide diversity in J. regia relative to J. sigillata and increases in F ST between the two species as evidence for selection. This approach would be ineffective in identifying a region that has experienced a selective sweep in both J. regia and J. sigillata . The low F ST observed between J. regia and J. sigillata at the locus of the selective sweep is consistent with introgression of a selected allele either from J. regia to J. sigillata or vice versa. However, the Integrated Extended Haplotype (IES) score in this region is higher in J. sigillata compared to J. regia , suggesting more recent introgression into this species. Although the minor (non-selected) allele is only observed in J. regia , our sampling of J. sigillata is limited, and more extensive sampling of wild populations of both J. regia and J. sigillata is needed to confirm this result. Unique characteristics of J. regia and J. sigillata within the genus Juglans include the presence of both a papery septum and an indehiscent hull. Qualitative variation in septum hardness is clearly associated with variation in the swept region, but we did not attempt to measure variation in hull dehiscence, which would be challenging to measure precisely across diverse germplasm with a range of maturity dates. The YABBY2 gene with shell/hull-enriched expression in the swept region has evolved two forms of alternative splicing in Juglans species with papery septa ( J. regia and J. sigillata) . First, the length of exon 4 decreased from 50 to 41 bp, eliminating three amino acids immediately upstream of the YABBY domain; this difference appears fixed across all tissues between Dioscaryon and Rhysocaryon/Cardiocaryon samples. Second, the length reduction in exon 4 is highly correlated with the incidence of exon 4 skipping, which leads to frame-shift and deletion of the YABBY2 domain. In Arabidopsis , the retention of micro-exons shorter than 50 bp requires an RNA-binding protein highly expressed in floral tissues (Wang et al., 2024 ). YABBY2 exon skipping is enriched in shell and hull tissues of J. regia but not J. sigillata , which might reflect selection on additional loci to fine-tune levels of full-length transcript. The control of fruit septum development by YABBY transcription factors has been demonstrated in tomato and other species (Shen et al., 2024 ). We identified two other cases of alternative splicing in protein-coding genes in the swept region, but one (in LOC109014009) does not affect the coding region and the other (in LOC109014007) involves an insertion of two amino acids that is at similar frequency across all three sections of Juglans (Figure S6). Loss-of-function mutations often have undesirable pleiotropic effects, and the observed tissue-biased exon skipping suggests a novel mechanism for tissue-specific modulation of gene function during plant domestication. All transcriptome samples of J. regia and J. sigillata contain full-length transcripts with an intact YABBY domain, with highly variable frequencies of exon skipping. Septum tissue was not sampled, and we hypothesize that a decreased frequency of full-length transcripts during critical stages of septum development may condition the papery septum phenotype. Exon-skipped transcripts are expected to generate truncated proteins with a zinc finger domain but no YABBY domain, which could interfere with functional YABBY proteins by occupying their DNA binding sites. Although the papery septum allele appears recessive (Table 3 ), only three samples homozygous for the minor haplotype were examined, and we cannot exclude the possibility that heterozygotes for the swept allele exhibit changes in septum or hull phenotypes that would have facilitated selection of this allele during the early stages of walnut domestication. While our sparse, reduced representation short-read data were adequate for identifying the selective sweep on chromosome 14 and identifying a candidate gene, the wild haplotype underlying this selective sweep awaits further molecular characterization. Specifically, genome assembly and DNA sequencing of wild individuals of J. regia , followed by transcriptome sequencing across shell, septum, and hull development could be used to identify polymorphisms associated with variation in YABBY2 isoform frequencies in different tissues. Identifying the precise molecular mechanisms responsible for the papery septum phenotype in domesticated J. regia could facilitate the rapid domestication of other Juglans species through gene editing, as well as enhance the breeding of pecan ( Carya illinoinensis ) and related hickory species. On-line Methods Plant Material We sampled over 3,000 adult Juglans regia and J. sigillata trees from 25 countries, retaining 2,850 J. regia and 43 J. sigillata for analysis following DNA extraction, sequencing, and quality control (Table 1 ). Of these, 1,762 consisted of local genotypes collected by the authors or our collaborators. Additional samples were obtained from the US Department of Agriculture National Clonal Germplasm Repository (USDA-NCGR) for Juglans in Winters, California (n = 903); the French National Research Institute for Agriculture, Food and Environment (INRAE) Juglans collections in Bordeaux and Avignon (n = 246); the Nut Application and Research Center (SEKAMER) at Kahramanmaras Sutcu Imam University (n = 63); and the Walnut Improvement Program (WIP) at the University of California, Davis (n = 228). Samples were classified as “Improved” (n = 369) if they resulted from intentional breeding or selection; otherwise, they were classified as “Unimproved” (n = 2,438). Only Unimproved individuals were used for FastStructure analysis, and only Improved individuals were used for testing the proportion of ancestry-informative alleles from different subpopulations. All J. regia individuals, including a small number classified as “Uncertain” (n = 43), were used for haplotype length tests and GWAS. DNA Extraction, Library Construction, and Sequencing Dried leaf segments (~ 1.5 cm long and ~ 1 cm wide) were placed in 96-well plates (1.2 ml, 8-tube strip format) with chrome steel balls (5/32”, Craig Ball Sales) and ground using a GenoGrinder 2000 (Spex CertiPrep, Metuchen, NJ, USA) at 650 strokes/min for 15 to 30 seconds. DNA extractions were performed using 96-well filter plates with silica columns (Epoch Life Science). Genomic DNA was quantified using a Quant-iT PicoGreen dsDNA Kit (Invitrogen, Life Technologies) in 96-well format on a SynergyTM HT plate reader (Bio-Tek Instruments, Winooski, VT, USA) and adjusted to 50 ng/µl. Illumina library preparations were conducted in 96-well plates as described by Poland et al. ( 2012 ), with several modifications. First, to simplify library preparation, we used 7.5 µl of DNA in a 30 µl simultaneous restriction-ligation reaction with HindIII-HF, MseI, and T4 DNA Ligase, applying cycle restriction-ligation to enhance ligation efficiency (Pusch et al., 1997 ). Second, to assist in calling heterozygous genotypes, we added 4 bp unique molecular identifiers (UMIs) to the 5’ end of each A1 adapter immediately before the barcodes. We then utilized the Clumpify tool in BBTools (Bushnell, 2014 ) to remove identical reads, assumed to be PCR duplicates, comprising 20–40% of each lane. Individual barcoded libraries were pooled by 96-well plate, cleaned using Ampure XP beads, PCR amplified, and subsequently cleaned again with Ampure XP beads before loading onto an Agilent Bioanalyzer 2100 in a DNA7500 chip. Libraries were adjusted to 10 nmol and pooled in groups of four (384 individuals per lane) before submission to the UC Davis Genome Center for SR100 sequencing on a HiSeq4000 instrument. Sequence data from 10 Illumina lanes are available at PRJNA1210562. SNP Calling and Imputation: SNP discovery was conducted on individuals of both J. regia and J. sigillata using the TASSEL GBS pipeline (Glaubitz et al., 2014 ). BWA (Li and Durbin, 2009 ) was used to align 64 bp tags to the Chandler v2.0 genome (Marrano et al., 2020 ), retaining only tags that appeared at least 10 times in the dataset with a BWA MAPQ score of at least 20. SNP calls with depth 90% missing data were discarded. Next, indels and SNPs with > 2 alleles, along with SNPs exhibiting > 50% missing data, were discarded, resulting in a final dataset of 2,893 samples and 46,582 SNPs. Beagle 5.0 (Browning et al., 2018 ) was employed for imputation without a reference panel using window and step sizes of 12 and 4, respectively. These filters resulted in a median concordance between replicate samples of 95.9% (mean = 95.5%), a median heterozygous mismatch of 3.9% (mean = 4.2%), and a median homozygous mismatch of 0.2% (mean = 0.3%). Population Structure Analysis Population structure analysis was conducted on J. regia samples classified as “Unimproved” (n = 2,438). First, linkage disequilibrium (LD) pruning was performed using PLINK 1.9 (Purcell et al., citation) to eliminate SNPs with r² > 0.95 in 50-SNP windows, resulting in the removal of 10,608 of the 46,583 variants. Next, FastSTRUCTURE (Raj et al., 2014 ) was applied to the LD-pruned dataset, testing K = 2 through K = 12 with 10 replicate runs at each K level (Raj et al., 2014 ). The optimal level of K was determined to be that which maximized membership in subpopulations; at K = 5, membership in subpopulations was maximized regardless of whether a cutoff of 90%, 95%, or 99% was applied (Figure S1 ). Principal component analysis (PCA) in TASSEL (Bradbury et al., 2007 ) was then conducted on the same dataset, with individual points colored according to the FastSTRUCTURE results. The R package scatterpie (Yu 2018) was employed to visualize the geographic distribution of subpopulations. Genetic Diversity, F ST , Linkage Disequilibrium, and Haplotype Length Analysis TASSEL (Bradbury et al., 2007 ) was used to summarize nucleotide diversity (π), theta (θ), Tajima’s D, and linkage disequilibrium (r²) in 100-SNP windows across the genome for all combined J. regia samples, as well as various subsets of the J. regia dataset (Improved, Unimproved, and Unimproved lines with at > 95% membership in one of the five subpopulations identified using FastSTRUCTURE), and the J. sigillata dataset. A minimum MAF of 0.01 was used for calculating linkage disequilibrium (r²). When comparing groups defined by their genotype at the highest IES SNP, 100-SNP windows on chromosome 14 were excluded. The weir-fst-pop function in VCFtools (Danecek et al., 2011 ) was employed to calculate pairwise F ST values for each SNP between all five J. regia subpopulations identified using FastSTRUCTURE and J. sigillata (six groups total). F ST was also calculated between all individuals of J. regia and J. sigillata , summarized in non-overlapping 100-SNP windows across each chromosome. Pairwise F ST values between each subpopulation and all others were used to define ancestry informative SNPs as the top 2% of pairwise F ST values with MAF > 0.01 (n = 636 in each subpopulation). Each improved line was then assessed for the proportion of ancestry informative SNPs that it carried from each subpopulation; sites heterozygous for an ancestry informative allele were counted as half. The R package rehh (Gautier et al., 2017 ) was used for haplotype length analysis using the phased VCF file as input. Nut Septum GWAS: Nut samples from the USDA-NCGR and WIP collections were harvested at hull-split into mesh bags over three years (2022–2024) and dried at room temperature. The internal septum of each nut sample was qualitatively scored as either papery (domesticated type) or hard (wild-type) by cracking open the nuts with a hammer. Phenotype and genotype data from 380 individuals (Table S2 ) were utilized for mixed logistic regression using the milorGWAS package in R (Milet et al., 2020 ). The MLM function in TASSEL yielded similar results (Table S3). Transcriptome analysis: Gzipped fastq files with walnut transcriptome data were downloaded from EMBL-EBI. Reads were aligned to the ChandlerV2 reference genome using STAR 2.7.6a (Dobin et al. 2013 ). Shell/hull-enriched expression in Fig. 4 A is the average of the mean RPKM values in shell and hull divided by the mean RPKM value in leaf across 40 samples (8 hull, 12 leaf, 20 shell; Table S4). The exon skipping index shown in Fig. 4 C reflects the proportion of YABBY2 transcripts that display exon 4 skipping (Table S5). Exon skipping events give rise to splice junctions between exons 3 and 5 (SJ3-5), and non-exon-skipping events give rise to splice junctions both between exons 3 and 4 (SJ3-4) and between exons 4 and 5 (SJ4-5). The exon skipping index is calculated as SJ3-5/(SJ3-5+(SJ3-4 + SJ4-5)/2). Declarations Acknowledgments: We extend our sincere appreciation to our colleagues and collaborators from USA, Iran, France, China, Italy, Spain, Turkey, and other countries who facilitated access to diverse walnut populations. We acknowledge the University of California, Davis, and the University of Tehran for providing essential resources and collaborative opportunities throughout the study. MM Arab and K Vahdati received support from the Iran National Science Foundation (INSF). 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Evol. 36(11): 2451-2461. doi: 10.1093/molbev/msz112. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalFigures.pptx SupplementalTables.xlsx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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\u003cem\u003eJuglans\u003c/em\u003e section Dioscaryon \u003cem\u003e(J. regia and J. sigillata\u003c/em\u003e) compared to other \u003cem\u003eJuglans\u003c/em\u003e species. A. Mature fruit of \u003cem\u003eJ. regia\u003c/em\u003e, demonstrating the dehiscent hull that splits open to release the nut inside. B. Dissected nuts of \u003cem\u003eJ. regia\u003c/em\u003e with the stem end facing upward: i-iii. Shell removal illustrating the orientation of the kernel relative to the seal between the two shell hemispheres; iv. removal of one kernel half revealing the papery septum; v. removal of the septum to display the inner side of the remaining kernel half; vi. visualization of the septum itself.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/6c56f94233c3116a0023b14b.png"},{"id":78961783,"identity":"0263bc4d-34ff-4ab9-8f0b-df9a9e3f2024","added_by":"auto","created_at":"2025-03-21 11:47:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1165933,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation structure in \u003cem\u003eJuglans regia\u003c/em\u003e. A. FastStructure analysis showing results for K = 2-6. Results from K = 5 are used to assign colors in B-E. B. Geographic distribution of subpopulations. C. Principal Component Analysis (PCA); individuals with membership \u0026lt;90% in a subpopulation are classified as mixed and shown in grey. D. Decay of linkage disequilibrium (r\u003csup\u003e2\u003c/sup\u003e) within subpopulations. E. Ancestry informative alleles across the 16 chromosomes of Chandler, the world’s most popular walnut cultivar. Legend shows the proportion of ancestry informative alleles from each subpopulation. Phased haplotypes are shown as lines extending on either side of each chromosome. Homozygosity for an ancestry informative allele is depicted by a line extending from both sides of a chromosome.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/4f44994e7e31693e260eb0ac.png"},{"id":78961781,"identity":"9bcd8103-fd12-4045-bf92-f61839a26f4f","added_by":"auto","created_at":"2025-03-21 11:47:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":545106,"visible":true,"origin":"","legend":"\u003cp\u003eA shared selective sweep on chromosome 14 of \u003cem\u003eJuglans regia \u003c/em\u003eand \u003cem\u003eJ. sigillata\u003c/em\u003e is associated with a papery septum. A-B: Integrated EHH scores (IES) for \u003cem\u003eJ. regia \u003c/em\u003e(A) and \u003cem\u003eJ. sigillata \u003c/em\u003e(B). IES values are divided by 10\u003csup\u003e6\u003c/sup\u003e. The same SNP at 18.01 Mb has the highest IES score in both analyses. C: Shell phenotypes across the three sections of \u003cem\u003eJuglans\u003c/em\u003e, including a \u003cem\u003eJ. regia \u003c/em\u003eheterozygote (left) and homozygote (right) for the major haplotype at the selective sweep. Homozygotes have a papery septum that separates from the rest of the shell. D: GWAS results for the papery septum phenotype (blue) superimposed on IES scores (black) in the swept region on chromosome 14. The GWAS SNP with the lowest p-value is at 18.48 Mb. Dashed vertical lines indicate the region from 17.7-18.5 Mb used for transcriptome analysis.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/3a71211f8c1c81bf402c3aa0.png"},{"id":78962471,"identity":"eebfe38b-e631-48a1-9dcc-8cccd783b8d6","added_by":"auto","created_at":"2025-03-21 11:55:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":284162,"visible":true,"origin":"","legend":"\u003cp\u003eExon skipping in a YABBY2 gene is specific to \u003cem\u003eJuglans\u003c/em\u003especies with papery septa. A. Shell/hull enriched expression of 14 predicted protein-coding genes from 17.7-18.5 Mb on chromosome 14, calculated as the ratio of mean RPKM in shell and hull versus leaf tissue. Gene 8 is a YABBY2 gene (LOC109014004) that shows ~5.8-fold enrichment. B. LOC109014004 isoforms in \u003cem\u003eJuglans\u003c/em\u003e. The fourth exon is 9 bp shorter in \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e(Dioscaryon) compared to \u003cem\u003eJ. mandshurica\u003c/em\u003e (Cardiocaryon) and \u003cem\u003eJ. nigra\u003c/em\u003e(Rhysocaryon). Exon skipping of the shortened fourth exon leads to frame-shift and deletion of the YABBY domain. C. Incidence of exon 4 skipping in LOC109014004 by species and tissue.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/1c6ce077fe6a9e5fca2205be.png"},{"id":78963491,"identity":"ca01dbe5-1f32-4fbe-8dd9-29c5efc9df98","added_by":"auto","created_at":"2025-03-21 12:11:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3877863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/38991fb9-79ef-40fc-b9c3-a9888c5c95d0.pdf"},{"id":78961788,"identity":"9fa93ff0-f700-41b4-a336-d10627200bc3","added_by":"auto","created_at":"2025-03-21 11:47:09","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9938008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementalFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/8a5bd368d51c486299c7955c.pptx"},{"id":78962474,"identity":"56cca196-f35c-4efa-b2f1-5b437c9aa2cf","added_by":"auto","created_at":"2025-03-21 11:55:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8120475,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6087036/v1/a5023ea410b84b08b42d0b26.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Global Genomic Diversity of Persian Walnut Reveals A Selective Sweep Underlying Domestication","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePersian walnut (\u003cem\u003eJuglans regia\u003c/em\u003e L.) is cultivated globally for food, fuel, timber, and traditional medicine (Vahdati et al., 2019; Costa et al., 2014).\u0026nbsp;Walnut kernels are rich in polyunsaturated fatty acids and bioactive phytochemicals, including phenolic compounds and phytosterols that offer numerous health benefits (Ma et al., 2019; Nguyen and Vu, 2023). Over the last two decades, global production of walnut has surged by more than 200%, accompanied by increasing public recognition of these health benefits. While the genus \u003cem\u003eJuglans\u003c/em\u003e contains over twenty species with edible nuts, commercial nut production is largely limited to \u003cem\u003eJ. regia\u003c/em\u003e and its sister species \u003cem\u003eJ. sigillata\u003c/em\u003e, which differ from all other species in the genus in two characteristics: a hull that dehisces at maturity, facilitating nut harvest (Figure 1A), and the presence of a papery septum, a modified section of the endocarp (shell) that extends between the kernel halves, facilitating extraction of the kernel (Figure 1B). All other members of the genus have nuts with indehiscent hulls and a hard, shell-like septum that prevents the removal of intact kernels from the shell.\u003c/p\u003e\n\u003cp\u003ePortable and energy-dense, walnuts have long played a significant role in human endeavors, and have long history of cultivation across Asia, the Middle East and Europe (Bernard et al., 2018; Ji et al., 2021). Silk Road trade routes contributed to walnut dispersal, and many ancient trees are still found along these routes in Iran and surrounding regions (Arab et al., 2019; Shahi Shavvon et al., 2023). Recent molecular studies highlight the Himalayan region as a center of diversity (Yan et al., 2024; Ye et al., 2024), and thick-shelled, apparently wild walnuts that persist in this area are often\u0026nbsp;used\u0026nbsp;ceremonially\u0026nbsp;(Avanzato et al., 2014). Identification of crop wild relatives and characterization of genetic diversity across the range of \u003cem\u003eJuglans regia\u003c/em\u003e is essential for both efficient germplasm conservation and breeding efforts.\u003c/p\u003e\n\u003cp\u003ePlant domestication frequently results in genetic bottlenecks, selective sweeps near domestication-related loci, and a rise in deleterious variant frequencies, as shown in annual crops such as maize, wheat, rice, and sunflower (Hufford et al., 2012; Huang et al., 2012; Badouin et al., 2017; Moyers et al., 2018; Maccaferri et al., 2019). However, population genetic signatures of domestication are relatively unstudied in woody perennials, which are often clonally propagated, highly heterozygous, and genetically closer to their wild ancestors. Domestication loci in woody perennials control dioecy-to-hermaphroditism transitions in grape (Massonnet et al., 2020) and papaya (Ch\u0026aacute;vez-Pesqueira and N\u0026uacute;\u0026ntilde;ez-Farf\u0026aacute;n, 2017), loss of seed toxicity in almond (S\u0026aacute;nchez-P\u0026eacute;rez et al., 2019), and the breakdown of self-incompatibility in peach (Yu et al., 2018) and some cacao populations (Cornejo et al., 2018). Despite the importance of walnut in global agriculture, the genetic basis of domestication in \u003cem\u003eJ. regia\u003c/em\u003e is not well understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe genus \u003cem\u003eJuglans\u003c/em\u003e is separated into three sections, two of which bear nuts with an indehiscent hull and a hard septum: Cardiocaryon, the Asian walnuts, and Rhysocaryon, the New World black walnuts. The third section, Dioscaryon, includes not only \u003cem\u003eJ. regia\u003c/em\u003e but also \u003cem\u003eJ. sigillata,\u003c/em\u003e a taxon indigenous to southwestern China that diverged from \u003cem\u003eJ. regia\u003c/em\u003e approx.0.85 million years ago (MYA) (Ding et al., 2022). Both \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e bear nuts with a dehiscent hull and a papery septum (Wambulwa et al. 2022). Widespread genomic introgression of \u003cem\u003eJ. regia\u003c/em\u003e into \u003cem\u003eJ. sigillata\u0026nbsp;\u003c/em\u003e( Yan et al., 2024) supports the hypothesis that these traits arose once during the domestication of \u003cem\u003eJ. regia\u003c/em\u003e and were subsequently introgressed into \u003cem\u003eJ. sigillata\u003c/em\u003e, though their genetic control has not been determined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo explore genomic diversity, population structure, and domestication signals in \u003cem\u003eJ. regia\u003c/em\u003e, we applied reduced representation sequencing to a global sample of 2,893 individuals, including 2,850 \u003cem\u003eJ. regia\u003c/em\u003e and 43 \u003cem\u003eJ. sigillata,\u003c/em\u003e and obtained a dataset of 46,582 SNPs. Haplotype length analysis revealed a major selective sweep on chromosome 14 that co-segregates with septum hardness, providing a first step towards understanding the evolutionary genetic basis of domestication in \u003cem\u003eJ. regia\u003c/em\u003e. Walnuts carrying the minor allele under the sweep display a hard, shell-like septum similar to other \u003cem\u003eJuglans\u003c/em\u003e. The major allele is associated with a papery septum and is fixed in \u003cem\u003eJ. sigillata\u003c/em\u003e samples, offering insight into the relationship between these taxa and the genetic modifications that supported the establishment of \u003cem\u003eJ. regia\u003c/em\u003e as a major food crop.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003ePopulation Structure of Walnut\u003c/h2\u003e \u003cp\u003ePopulation structure in a global sample of unimproved \u003cem\u003eJ. regia\u003c/em\u003e (n = 2,438) exhibited the expected pattern of isolation by distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Samples from the Tibetan Plateau in China clustered with samples from the Himalayan region of northern Pakistan and India (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), and samples from the Caucasus region between the Black and Caspian Seas showed genetic separation from Iranian, Turkish, and Russian samples. Feral trees collected from the New World predominantly exhibited Western European ancestry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Principal component analysis (PCA) supported the population structure results, with samples from Western Europe and East Asia separating along PC1 and admixed samples clustering near the origin (grey circles; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). After varying the number of subpopulations from two to ten, we selected five subpopulations (Western Europe, Caucasus, Iran, Himalaya, and China) as the number that maximized the likelihood of membership of the 2,481 unimproved trees (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, see Methods).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenetic Diversity, Differentiation, and Linkage Disequilibrium of Walnut Subpopulations\u003c/h2\u003e \u003cp\u003eNucleotide diversity (pi) and polymorphism (theta) were highest in the Himalaya and lowest in Western Europe (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas genetic differentiation (\u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e) from the \u003cem\u003eJ. sigillata\u003c/em\u003e outgroup was lowest in the Himalaya and highest in Western Europe (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within \u003cem\u003eJ. regia\u003c/em\u003e, genetic differentiation was greatest when comparing Western Europe with all other groups. Additionally, linkage disequilibrium decay was markedly slower in Europe compared to all other subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these results suggest that the Himalayan subpopulation is ancestral and the Western Europe subpopulation has experienced a bottleneck. Rapid decay of linkage disequilibrium in the Iranian subpopulation may reflect a high long-term effective population size in this center of walnut diversity.\u003c/p\u003e \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNucleotide diversity (Pi), polymorphism (Theta), and F\u003csub\u003eST\u003c/sub\u003e within and between walnut populations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePi\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTheta\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e \u003cp\u003eF\u003csub\u003eST\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW. Europe\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCaucasus\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIran\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHimalaya\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eJ. sigillata\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW. Europe\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaucasus\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIran\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHimalaya\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eContribution of Walnut Subpopulations to Improved Cultivars\u003c/h3\u003e\n\u003cp\u003eTo assess subpopulation contribution to modern breeding programs, we defined ancestry informative markers for each subpopulation as the top 2% of \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e values between that subpopulation and the other four subpopulations, and scanned each improved walnut genotype (n = 369) for its proportion of ancestry informative alleles from each subpopulation (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). ‘Chandler’, the world’s most popular walnut cultivar, is typical in having predominantly Western European ancestry (72% of alleles) with limited contribution from the Iran (12%), China (10%) Caucasus (5%) and Himalaya (2%) subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Overall, ancestry informative alleles in the 228 lines from the California breeding program average 61%, 15%, 15%, 7%, and 2% from Western Europe, Iran, China, Caucasus, and the Himalaya respectively. The contributions to all 369 improved lines in this study were similar (59%, 17%, 13%, 9%, 3%; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eExtended Haplotype Homozygosity Reveals a Selective Sweep on Walnut Chromosome 14\u003c/h3\u003e\n\u003cp\u003eUsing haplotype length tests including extended haplotype homozygosity (EHH) and integrated EHH score (IES) to investigate evidence of selection in \u003cem\u003eJ. regia\u003c/em\u003e, we identified a region on chromosome 14 exhibiting IES scores more than ten times the genome average, indicative of a selective sweep (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This sweep is of comparable magnitude in both improved and unimproved groups and across all five subpopulations of \u003cem\u003eJ. regia\u003c/em\u003e (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting a potential role in walnut domestication. Notably, we observed similar IES scores in the same genomic region in \u003cem\u003eJ. sigillata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and in a combined sample of both \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e, indicating that the same haplotype was selected in both species. To elucidate this result, we calculated the average \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e between \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e in non-overlapping 100-SNP windows throughout the genome. The four windows with the lowest F\u003csub\u003eST\u003c/sub\u003e are all located within the selective sweep region (Figure S3), suggesting possible introgression of the swept allele from \u003cem\u003eJ. regia\u003c/em\u003e into \u003cem\u003eJ. sigillata\u003c/em\u003e. The SNP with the highest IES score in all analyses is located at 18,013,474 bp on chromosome 14; the minor allele at this SNP is very rare (minor allele frequency [MAF] = 0.011; 63/5,786 gametes) and is present in only 12 homozygous and 39 heterozygous individuals of \u003cem\u003eJ. regia\u003c/em\u003e, primarily distributed in northern Pakistan, Iran, and Kyrgyzstan (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Figure S4). Homozygotes for the major allele have significantly lower genome-wide nucleotide diversity and polymorphism when summarizing over non-overlapping 100-SNP windows across all chromosomes except chromosome 14 (Figure S5). All samples of \u003cem\u003eJ. sigillata\u003c/em\u003e are homozygous for the swept, major allele. An additional selective sweep on chromosome 11, found only in improved walnuts and the European subpopulation (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), is likely associated with the dominant allele at the \u003cem\u003eLateral bearing1\u003c/em\u003e (\u003cem\u003eLb1)\u003c/em\u003e locus, which controls flower development in axillary buds (Bernard et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe Minor Allele Under the Selective Sweep is Associated with a Hard, Shell-like Septum\u003c/h3\u003e\n\u003cp\u003eTo investigate the reason for the selective sweep on chromosome 14, we collected hulled nut samples from 380 trees, including three homozygotes and eleven heterozygotes for the minor allele at the SNP with the highest IES score (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Among these 14 trees, 9 produced nuts with a very hard internal septum (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), similar to other \u003cem\u003eJuglans\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), while 364 of the 366 remaining trees displayed the papery septum typical of domesticated walnuts. To validate this observation, we used hard/papery septum as a binary trait to conduct a genome-wide association study (GWAS) using a mixed logistic regression model, revealing a cluster of SNPs between 17.9–18.5 Mb on chromosome 14 that strongly associates with the septum phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table S3). The most significant GWAS SNP is located at 18,485,943 bp, approximately 500 kb from the SNP with the highest IES score, and co-segregates even more strongly with the septum phenotype (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCo-segregation of septum phenotype with top SNPs from IES and GWAS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSeptum\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePapery\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTop IES SNP\u003c/p\u003e \u003cp\u003e18,013,474 bp\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e364\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTop GWAS SNP\u003c/p\u003e \u003cp\u003e18,485,943 bp\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e368\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTissue-Biased Exon Skipping in a YABBY2 Gene Expressed in Walnut Shell\u003c/h3\u003e\n\u003cp\u003eThe interval from 17.7–18.5 Mb on chromosome 14 includes the ten most significant SNPs from both IES and GWAS analyses and contains fourteen predicted protein-coding genes. Expression of these genes was characterized using 40 RNAseq libraries representing four \u003cem\u003eJuglans\u003c/em\u003e species and all three sections of \u003cem\u003eJuglans\u003c/em\u003e: \u003cem\u003eJ. mandshurica\u003c/em\u003e (Cardiocaryon), \u003cem\u003eJ. nigra\u003c/em\u003e (Rhysocaryon), and \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e (both Dioscaryon). The 8th gene in the interval (LOC109014004; 18.157–18.165 Mb) shows highly (~ 5.8-fold) elevated expression in shell (n = 20) and hull (n = 8) relative to leaf (n = 12) tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Table S4) and is predicted to encode a YABBY2 transcription factor. Splice junctions in the RNAseq data reveal multiple isoforms for this gene, including a shortened exon 4 (41 versus 50 bp) that is fixed in Dioscaryon and a skipped exon 4 that results in a frame-shift eliminating the YABBY domain that is at much higher frequency in Dioscaryon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Table S5). We then quantified the tissue- and species-specificity of this exon skipping event in shell, hull, and leaf transcripts of the four \u003cem\u003eJuglans\u003c/em\u003e species, and found that exon skipping is very rare in \u003cem\u003eJ. nigra\u003c/em\u003e (\u0026lt; 1% of all transcripts), uncommon in \u003cem\u003eJ. mandshurica\u003c/em\u003e (6%), and common in both \u003cem\u003eJ. regia\u003c/em\u003e (51%) and \u003cem\u003eJ. sigillata\u003c/em\u003e (33%; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Exon skipping in \u003cem\u003eJ. regia\u003c/em\u003e, but not \u003cem\u003eJ. sigillata\u003c/em\u003e, is significantly enriched in shell and hull relative to leaf tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; t = -5.5, p = 9e-5).\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThis study represents the largest genetic sample of \u003cem\u003eJuglans regia\u003c/em\u003e reported to date. Although the number of markers utilized is modest compared to studies employing whole-genome sequencing on smaller sample sizes (Stevens et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ji et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), our findings align with previous research identifying the Himalayan region as a center of genetic diversity (Yan et al., 2024; Ye et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our results show that Western European walnuts are bottlenecked as previously reported, reflecting their retreat to glacial refugia during the last Ice Age (Aradhya et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Ebrahimi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pollegioni et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the five identified subpopulations, Iran and Pakistan exhibit the highest nucleotide diversity and polymorphism and the lowest levels of linkage disequilibrium. While our sampling from Afghanistan remains limited, the pre-domestication distribution of wild \u003cem\u003eJ. regia\u003c/em\u003e likely extended from northwestern Iran through Afghanistan and northern Pakistan and potentially further east (Bernard et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These high-diversity subpopulations are little represented in advanced breeding material. The rare haplotype under the selective sweep on chromosome 14 is essentially confined to these regions, and individuals carrying this haplotype have much higher genome-wide nucleotide diversity, as would be expected for crop wild relatives not subjected to a domestication bottleneck. Since humans would have comparatively little incentive to gather or propagate thick-shelled walnuts, we propose that the selective sweep associated with the papery septum trait underlies the domestication of \u003cem\u003eJ. regia\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis is the first report of a selective sweep on chromosome 14 of \u003cem\u003eJuglans regia\u003c/em\u003e, despite numerous previous genome-wide sequencing studies on diverse walnut germplasm. Several factors could account for this surprising result. First, our sample size is larger than that of previous studies, and the rare haplotype on chromosome 14 is present at a frequency of only 1.1%. Second, this is the first study to test for selection in \u003cem\u003eJuglans\u003c/em\u003e using haplotype length statistics, which may offer greater sensitivity to recent selection compared to other methods. Third, a prior study (Ji et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) regarded \u003cem\u003eJ. sigillata\u003c/em\u003e as the wild progenitor and utilized reductions in nucleotide diversity in \u003cem\u003eJ. regia\u003c/em\u003e relative to \u003cem\u003eJ. sigillata\u003c/em\u003e and increases in \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e between the two species as evidence for selection. This approach would be ineffective in identifying a region that has experienced a selective sweep in both \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe low \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e observed between \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e at the locus of the selective sweep is consistent with introgression of a selected allele either from \u003cem\u003eJ. regia\u003c/em\u003e to \u003cem\u003eJ. sigillata\u003c/em\u003e or vice versa. However, the Integrated Extended Haplotype (IES) score in this region is higher in \u003cem\u003eJ. sigillata\u003c/em\u003e compared to \u003cem\u003eJ. regia\u003c/em\u003e, suggesting more recent introgression into this species. Although the minor (non-selected) allele is only observed in \u003cem\u003eJ. regia\u003c/em\u003e, our sampling of \u003cem\u003eJ. sigillata\u003c/em\u003e is limited, and more extensive sampling of wild populations of both \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e is needed to confirm this result. Unique characteristics of \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e within the genus \u003cem\u003eJuglans\u003c/em\u003e include the presence of both a papery septum and an indehiscent hull. Qualitative variation in septum hardness is clearly associated with variation in the swept region, but we did not attempt to measure variation in hull dehiscence, which would be challenging to measure precisely across diverse germplasm with a range of maturity dates.\u003c/p\u003e\u003cp\u003eThe YABBY2 gene with shell/hull-enriched expression in the swept region has evolved two forms of alternative splicing in \u003cem\u003eJuglans\u003c/em\u003e species with papery septa (\u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata)\u003c/em\u003e. First, the length of exon 4 decreased from 50 to 41 bp, eliminating three amino acids immediately upstream of the YABBY domain; this difference appears fixed across all tissues between Dioscaryon and Rhysocaryon/Cardiocaryon samples. Second, the length reduction in exon 4 is highly correlated with the incidence of exon 4 skipping, which leads to frame-shift and deletion of the YABBY2 domain. In \u003cem\u003eArabidopsis\u003c/em\u003e, the retention of micro-exons shorter than 50 bp requires an RNA-binding protein highly expressed in floral tissues (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). YABBY2 exon skipping is enriched in shell and hull tissues of \u003cem\u003eJ. regia\u003c/em\u003e but not \u003cem\u003eJ. sigillata\u003c/em\u003e, which might reflect selection on additional loci to fine-tune levels of full-length transcript. The control of fruit septum development by YABBY transcription factors has been demonstrated in tomato and other species (Shen et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We identified two other cases of alternative splicing in protein-coding genes in the swept region, but one (in LOC109014009) does not affect the coding region and the other (in LOC109014007) involves an insertion of two amino acids that is at similar frequency across all three sections of \u003cem\u003eJuglans\u003c/em\u003e (Figure S6).\u003c/p\u003e\u003cp\u003eLoss-of-function mutations often have undesirable pleiotropic effects, and the observed tissue-biased exon skipping suggests a novel mechanism for tissue-specific modulation of gene function during plant domestication. All transcriptome samples of \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e contain full-length transcripts with an intact YABBY domain, with highly variable frequencies of exon skipping. Septum tissue was not sampled, and we hypothesize that a decreased frequency of full-length transcripts during critical stages of septum development may condition the papery septum phenotype. Exon-skipped transcripts are expected to generate truncated proteins with a zinc finger domain but no YABBY domain, which could interfere with functional YABBY proteins by occupying their DNA binding sites. Although the papery septum allele appears recessive (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), only three samples homozygous for the minor haplotype were examined, and we cannot exclude the possibility that heterozygotes for the swept allele exhibit changes in septum or hull phenotypes that would have facilitated selection of this allele during the early stages of walnut domestication.\u003c/p\u003e\u003cp\u003eWhile our sparse, reduced representation short-read data were adequate for identifying the selective sweep on chromosome 14 and identifying a candidate gene, the wild haplotype underlying this selective sweep awaits further molecular characterization. Specifically, genome assembly and DNA sequencing of wild individuals of \u003cem\u003eJ. regia\u003c/em\u003e, followed by transcriptome sequencing across shell, septum, and hull development could be used to identify polymorphisms associated with variation in YABBY2 isoform frequencies in different tissues. Identifying the precise molecular mechanisms responsible for the papery septum phenotype in domesticated \u003cem\u003eJ. regia\u003c/em\u003e could facilitate the rapid domestication of other \u003cem\u003eJuglans\u003c/em\u003e species through gene editing, as well as enhance the breeding of pecan (\u003cem\u003eCarya illinoinensis\u003c/em\u003e) and related hickory species.\u003c/p\u003e"},{"header":"On-line Methods","content":"\u003ch2\u003ePlant Material\u003c/h2\u003e\n\u003cp\u003eWe sampled over 3,000 adult \u003cem\u003eJuglans regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e trees from 25 countries, retaining 2,850 \u003cem\u003eJ. regia\u003c/em\u003e and 43 \u003cem\u003eJ. sigillata\u003c/em\u003e for analysis following DNA extraction, sequencing, and quality control (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Of these, 1,762 consisted of local genotypes collected by the authors or our collaborators. Additional samples were obtained from the US Department of Agriculture National Clonal Germplasm Repository (USDA-NCGR) for \u003cem\u003eJuglans\u003c/em\u003e in Winters, California (n\u0026thinsp;=\u0026thinsp;903); the French National Research Institute for Agriculture, Food and Environment (INRAE) \u003cem\u003eJuglans\u003c/em\u003e collections in Bordeaux and Avignon (n\u0026thinsp;=\u0026thinsp;246); the Nut Application and Research Center (SEKAMER) at Kahramanmaras Sutcu Imam University (n\u0026thinsp;=\u0026thinsp;63); and the Walnut Improvement Program (WIP) at the University of California, Davis (n\u0026thinsp;=\u0026thinsp;228). Samples were classified as \u0026ldquo;Improved\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;369) if they resulted from intentional breeding or selection; otherwise, they were classified as \u0026ldquo;Unimproved\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;2,438). Only Unimproved individuals were used for FastStructure analysis, and only Improved individuals were used for testing the proportion of ancestry-informative alleles from different subpopulations. All \u003cem\u003eJ. regia\u003c/em\u003e individuals, including a small number classified as \u0026ldquo;Uncertain\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;43), were used for haplotype length tests and GWAS.\u003c/p\u003e\n\u003ch2\u003eDNA Extraction, Library Construction, and Sequencing\u003c/h2\u003e\n\u003cp\u003eDried leaf segments (~\u0026thinsp;1.5 cm long and ~\u0026thinsp;1 cm wide) were placed in 96-well plates (1.2 ml, 8-tube strip format) with chrome steel balls (5/32\u0026rdquo;, Craig Ball Sales) and ground using a GenoGrinder 2000 (Spex CertiPrep, Metuchen, NJ, USA) at 650 strokes/min for 15 to 30 seconds. DNA extractions were performed using 96-well filter plates with silica columns (Epoch Life Science). Genomic DNA was quantified using a Quant-iT PicoGreen dsDNA Kit (Invitrogen, Life Technologies) in 96-well format on a SynergyTM HT plate reader (Bio-Tek Instruments, Winooski, VT, USA) and adjusted to 50 ng/\u0026micro;l. Illumina library preparations were conducted in 96-well plates as described by Poland et al. (\u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e), with several modifications. First, to simplify library preparation, we used 7.5 \u0026micro;l of DNA in a 30 \u0026micro;l simultaneous restriction-ligation reaction with HindIII-HF, MseI, and T4 DNA Ligase, applying cycle restriction-ligation to enhance ligation efficiency (Pusch et al., \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e). Second, to assist in calling heterozygous genotypes, we added 4 bp unique molecular identifiers (UMIs) to the 5\u0026rsquo; end of each A1 adapter immediately before the barcodes. We then utilized the Clumpify tool in BBTools (Bushnell, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) to remove identical reads, assumed to be PCR duplicates, comprising 20\u0026ndash;40% of each lane. Individual barcoded libraries were pooled by 96-well plate, cleaned using Ampure XP beads, PCR amplified, and subsequently cleaned again with Ampure XP beads before loading onto an Agilent Bioanalyzer 2100 in a DNA7500 chip. Libraries were adjusted to 10 nmol and pooled in groups of four (384 individuals per lane) before submission to the UC Davis Genome Center for SR100 sequencing on a HiSeq4000 instrument. Sequence data from 10 Illumina lanes are available at PRJNA1210562.\u003c/p\u003e\n\u003ch2\u003eSNP Calling and Imputation:\u003c/h2\u003e\n\u003cp\u003eSNP discovery was conducted on individuals of both \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e using the TASSEL GBS pipeline (Glaubitz et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). BWA (Li and Durbin, \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) was used to align 64 bp tags to the Chandler v2.0 genome (Marrano et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), retaining only tags that appeared at least 10 times in the dataset with a BWA MAPQ score of at least 20. SNP calls with depth\u0026thinsp;\u0026lt;\u0026thinsp;5 were set to missing, and taxa with \u0026gt;\u0026thinsp;90% missing data were discarded. Next, indels and SNPs with \u0026gt;\u0026thinsp;2 alleles, along with SNPs exhibiting\u0026thinsp;\u0026gt;\u0026thinsp;50% missing data, were discarded, resulting in a final dataset of 2,893 samples and 46,582 SNPs. Beagle 5.0 (Browning et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) was employed for imputation without a reference panel using window and step sizes of 12 and 4, respectively. These filters resulted in a median concordance between replicate samples of 95.9% (mean\u0026thinsp;=\u0026thinsp;95.5%), a median heterozygous mismatch of 3.9% (mean\u0026thinsp;=\u0026thinsp;4.2%), and a median homozygous mismatch of 0.2% (mean\u0026thinsp;=\u0026thinsp;0.3%).\u003c/p\u003e\n\u003ch2\u003ePopulation Structure Analysis\u003c/h2\u003e\n\u003cp\u003ePopulation structure analysis was conducted on \u003cem\u003eJ. regia\u003c/em\u003e samples classified as \u0026ldquo;Unimproved\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;2,438). First, linkage disequilibrium (LD) pruning was performed using PLINK 1.9 (Purcell et al., citation) to eliminate SNPs with r\u0026sup2; \u0026gt; 0.95 in 50-SNP windows, resulting in the removal of 10,608 of the 46,583 variants. Next, FastSTRUCTURE (Raj et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) was applied to the LD-pruned dataset, testing K\u0026thinsp;=\u0026thinsp;2 through K\u0026thinsp;=\u0026thinsp;12 with 10 replicate runs at each K level (Raj et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The optimal level of K was determined to be that which maximized membership in subpopulations; at K\u0026thinsp;=\u0026thinsp;5, membership in subpopulations was maximized regardless of whether a cutoff of 90%, 95%, or 99% was applied (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Principal component analysis (PCA) in TASSEL (Bradbury et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e) was then conducted on the same dataset, with individual points colored according to the FastSTRUCTURE results. The R package \u003cem\u003escatterpie\u003c/em\u003e (Yu 2018) was employed to visualize the geographic distribution of subpopulations.\u003c/p\u003e\n\u003ch2\u003eGenetic Diversity, F\u003csub\u003eST\u003c/sub\u003e, Linkage Disequilibrium, and Haplotype Length Analysis\u003c/h2\u003e\n\u003cp\u003eTASSEL (Bradbury et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e) was used to summarize nucleotide diversity (\u0026pi;), theta (\u0026theta;), Tajima\u0026rsquo;s D, and linkage disequilibrium (r\u0026sup2;) in 100-SNP windows across the genome for all combined \u003cem\u003eJ. regia\u003c/em\u003e samples, as well as various subsets of the \u003cem\u003eJ. regia\u003c/em\u003e dataset (Improved, Unimproved, and Unimproved lines with at \u0026gt;\u0026thinsp;95% membership in one of the five subpopulations identified using FastSTRUCTURE), and the \u003cem\u003eJ. sigillata\u003c/em\u003e dataset. A minimum MAF of 0.01 was used for calculating linkage disequilibrium (r\u0026sup2;). When comparing groups defined by their genotype at the highest IES SNP, 100-SNP windows on chromosome 14 were excluded. The \u003cem\u003eweir-fst-pop\u003c/em\u003e function in VCFtools (Danecek et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e) was employed to calculate pairwise F\u003csub\u003eST\u003c/sub\u003e values for each SNP between all five \u003cem\u003eJ. regia\u003c/em\u003e subpopulations identified using FastSTRUCTURE and \u003cem\u003eJ. sigillata\u003c/em\u003e (six groups total). F\u003csub\u003eST\u003c/sub\u003e was also calculated between all individuals of \u003cem\u003eJ. regia\u003c/em\u003e and \u003cem\u003eJ. sigillata\u003c/em\u003e, summarized in non-overlapping 100-SNP windows across each chromosome. Pairwise F\u003csub\u003eST\u003c/sub\u003e values between each subpopulation and all others were used to define ancestry informative SNPs as the top 2% of pairwise F\u003csub\u003eST\u003c/sub\u003e values with MAF\u0026thinsp;\u0026gt;\u0026thinsp;0.01 (n\u0026thinsp;=\u0026thinsp;636 in each subpopulation). Each improved line was then assessed for the proportion of ancestry informative SNPs that it carried from each subpopulation; sites heterozygous for an ancestry informative allele were counted as half. The R package \u003cem\u003erehh\u003c/em\u003e (Gautier et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used for haplotype length analysis using the phased VCF file as input.\u003c/p\u003e\n\u003ch2\u003eNut Septum GWAS:\u003c/h2\u003e\n\u003cp\u003eNut samples from the USDA-NCGR and WIP collections were harvested at hull-split into mesh bags over three years (2022\u0026ndash;2024) and dried at room temperature. The internal septum of each nut sample was qualitatively scored as either papery (domesticated type) or hard (wild-type) by cracking open the nuts with a hammer. Phenotype and genotype data from 380 individuals (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e) were utilized for mixed logistic regression using the milorGWAS package in R (Milet et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The MLM function in TASSEL yielded similar results (Table S3).\u003c/p\u003e\n\u003ch2\u003eTranscriptome analysis:\u003c/h2\u003e\n\u003cp\u003eGzipped fastq files with walnut transcriptome data were downloaded from EMBL-EBI. Reads were aligned to the ChandlerV2 reference genome using STAR 2.7.6a (Dobin et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Shell/hull-enriched expression in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA is the average of the mean RPKM values in shell and hull divided by the mean RPKM value in leaf across 40 samples (8 hull, 12 leaf, 20 shell; Table S4). The exon skipping index shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC reflects the proportion of YABBY2 transcripts that display exon 4 skipping (Table S5). Exon skipping events give rise to splice junctions between exons 3 and 5 (SJ3-5), and non-exon-skipping events give rise to splice junctions both between exons 3 and 4 (SJ3-4) and between exons 4 and 5 (SJ4-5). The exon skipping index is calculated as SJ3-5/(SJ3-5+(SJ3-4\u0026thinsp;+\u0026thinsp;SJ4-5)/2).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere appreciation to our colleagues and collaborators from USA, Iran, France, China, Italy, Spain, Turkey, and other countries who facilitated access to diverse walnut populations. We acknowledge the University of California, Davis, and the University of Tehran for providing essential resources and collaborative opportunities throughout the study. MM Arab and K Vahdati received support from the Iran National Science Foundation (INSF). J Liu was funded by the National Natural Science Foundation of China (32170398) and CAS \u0026ldquo;Light of West China\u0026rdquo; Program.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArab, M.M., A. Marrano, R. Abdollahi-Arpanahi, C.A. Leslie, H. 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Evol. 36(11): 2451-2461. doi: 10.1093/molbev/msz112.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6087036/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6087036/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePersian walnut \u003cem\u003e(Juglans regia L.)\u003c/em\u003e is cultivated globally for its large edible nuts, which have a dehiscent hull that simplifies nut harvest and a papery inner shell or septum that allows easy kernel extraction. Here, we use reduced-representation sequencing to genotype the largest panel of \u003cem\u003eJ. regia\u003c/em\u003e ever assembled (n = 2,850) and evaluate its evolutionary history and population structure. Haplotype length analysis and genome-wide trait association identify a selective sweep on chromosome 14 associated with the hard septum characteristic, providing new insight into the origin and domestication of \u003cem\u003eJ. regia\u003c/em\u003e. A YABBY transcription factor in the swept region shows exon skipping that causes frame-shift and deletion of the YABBY domain and is enriched in \u003cem\u003eJ. regia\u003c/em\u003e hull and shell tissues. These results suggest both a mechanism underlying walnut domestication and a blueprint for domesticating other tree nuts in the \u003cem\u003eJuglandaceae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Global Genomic Diversity of Persian Walnut Reveals A Selective Sweep Underlying Domestication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 11:47:03","doi":"10.21203/rs.3.rs-6087036/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2c788378-dfc0-4759-a02a-c874c7650c6b","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":45531049,"name":"Biological sciences/Plant sciences/Plant genetics/Plant hybridization"},{"id":45531050,"name":"Biological sciences/Genetics/Plant genetics"}],"tags":[],"updatedAt":"2025-03-21T11:47:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-21 11:47:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6087036","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6087036","identity":"rs-6087036","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}