Haplotype-resolved genome assembly of seed hemp (Cannabis sativa) revealed molecular divergence of Y chromosome evolution

Haplotype-resolved genome assembly of seed hemp (Cannabis sativa) revealed molecular divergence of Y chromosome evolution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Haplotype-resolved genome assembly of seed hemp (Cannabis sativa) revealed molecular divergence of Y chromosome evolution Huawei Wei, Zhuqing Yang, Lingling Zhuang, Xueqing Pan, Haifeng Jia, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6374740/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 Seed hemp (Cannabis sativa L.) is an important economic dioecious plant with an XY sex chromosome system, yet its Y chromosome and sex-determining regions (SDR) have not been characterized. Here, we assembled a high-quality haplotype-resolved genome (female: 770.8 Mb; male: 804.7 Mb), and identified an 84 Mb SDR, covering 71% of the Y chromosome, via comparative genomics. X/Y divergence occurred ~37.7 Mya (Ks analysis). Resequencing 35 cannabis accessions revealed higher genetic diversity on the X chromosome. This study provides the first Y chromosome and SDR assembly in seed hemp, offering insights into sex differentiation in dioecious plants. Seed hemp Haplotype genome Sex determining region Whole genome resequencing Evolution of sex chromosomes Figures Figure 1 Figure 2 Introduction Hemp ( Cannabis sativa L.) is an annual dioecious plant with a diploid (2n = 20), comprising 9 pairs of autosomes and a pair of sex chromosomes (XX/XY system). In recent years, due to the agronomic characteristics of hemp and the diversity of renewable resources that it produces, it has attracted more and more attention, yet we still know little about hemp [1, 2]. In general, sex determination of dioecious plants, with separate female and male individuals, is regulated by a heteromorphic sex chromosome or a sex-determining region on homomorphic chromosomes [3]. For example, the sex determination region of asparagus was located within the 132.4 Mb region of chromosome Y [4], and the sex determination region of papaya was located within the 8.1 Mb region of chromosome HSY [5]. Although cannabis has vital economic value, information about its genome is limited. Most importantly, the previously published genomes of medicinal cannabis, wild cannabis, CBD-type cultivar cannabis, and seed hemp have all been sequenced using female plant materials, failing to assemble a complete Y chromosome, which greatly limited the research on gender correlation of cannabis. In the present study, we generated the X and Y chromosomes from male seed hemp by integrating PacBio HiFi and Oxford Nanopore long reads data and using Hi-C mapping technologies via de novo assembly and annotation at independent chromosome-scale. We identified the sex determining region (SDR) of seed hemp used bulk segregation analysis of a mixed pool of female and male data. Further, we identified structural variation in the SDR, predicted the time of differentiation of X and Y, and analyzed the genetic differences between the X and Y chromosomes using resequencing data. This genomic resource will provide fundamental tools for the study of gender and provide new insights for cannabis breeding. Results And Discussion In this study, we generated 26.27 Gb (32× coverage) HiFi reads with the PacBio sequel II platform and 39.57Gb (49× coverage) of ultra-long reads by Oxford Nanopore Technology (ONT) (Table S1). We estimated the genome size of male seed hemp (YSM) by k-mer analysis, which gave a value of 816 Mb with 2.14% heterozygosity (Fig. S1). Because of the high heterozygosity of YSM individual, we performed genome assembly and haplotype phasing, thus generating two haplotypes: YSM1 and YSM2 (Fig. S2, Table S2). The YSM1 assembly was 770 Mb in total length and consisted of 63 scaffolds with an N50 size of 81.3 Mb and the longest scaffold is 89.4 Mb. The YSM2 assembly was 804 Mb and comprising 55 scaffolds with an N50 size of 81.2 Mb and the longest scaffold is 117.6 Mb (Table S3). Benchmarking Universal Single-Copy Orthologs (BUSCO) analyses showed that 98.8% and 95.8% of BUSCO genes are complete in the YSM1 and YSM2, respectively (Table S4). The long terminal repeat Assembly Index (LAI) values of YSM1 and YSM2 seed hemp genomes were estimated to be 23 and 21, respectively, which are much higher than published cannabis genomes (Table S5) [6-8]. Moreover, 92.21% and 92.03% of the expressed transcripts from the seed hemp RNA-seq datasets were mapped in YSM1 and YSM2, respectively (Table S6). Furthermore, comparing the assembled YSM genomes with the previously published genomes of cannabis, Subreads N50, Contig N50, LAI and BUSCO all showed substantial improvements, indicating that higher assembly quality of the genomes (Table S7). Collectively, all these results demonstrated high continuity, accuracy, and completeness of the assembled the male genomes in seed hemp. Using a combination of de novo gene prediction, protein homology searches, and assembly of RNA-Seq reads, we identified 32,519 and 31,465 protein-coding genes in YSM1 and YSM2, respectively (Table S6). Among the predicted protein-coding genes, 31233 and 30235 could be functionally annotated by multiple public databases, respectively (Table S8). BUSCO analysis showed that 98.3% and 96.3% of the predicted genes had full-length sequence information in YSM1 and YSM2 (Table S9). Furthermore, within the two haplotype assemblies, 574.53 Mb (74.53%) and 614.23 Mb (76.33%) of repetitive sequences were identified (Table S10). Because of the sex-determining regions or sex-regulatory genes and the repeated sequences within the so-called sex determining region (SDR), it is difficult to recognize, locate, and define the exact boundaries of sex-specific regions [11]. To identify the SDR in seed hemp, we performed collinear analysis of the two haplotype genomes of YSM1 and YSM2, and found that there was a good collinearity between the autosomes, while the Y and X chromosomes were only partially collinear and occurred at the end (Fig. 1a). To further investigate this SDR, we used bulked DNA pools from 50 female plants (BSA-F) and 50 male plants (BSA-M) collected in the seed hemp to perform BSA. When YSM1 was used as the reference genome, the coverage of all chromosomes was basically the same, while when YSM2 was used as the reference genome, the coverage of the first 84 Mb region of the Y chromosome was significantly lower than that of other regions, and the coverage of BSA-F reads in this region was lower than that of BSA-M reads (Fig. 1b, Fig. S3). Similarly, the coverage depth of resequencing data based on different individuals (11 males and 11 females) with different background varieties in this region is also different, with male samples having higher coverage depth than female samples (Fig. S4). This indicates that the first 84Mb region of Y chromosome is the SDR of seed hemp. Collinearity analysis between X and Y chromosomes based on homologous gene pairs also demonstrated the specificity of the 84 Mb in the Y chromosome (Fig. 1c). In addition, through collinearity analysis between X and Y chromosomes, an inversion was found in the homologous region of both chromosomes. This inversion usually inhibits genetic recombination at the relevant locus and plays a crucial role in the fixing of favorable allelic combinations. The random primer PCR assay targeting the SDR produced specific amplification bands exclusively in male seed hemp (Fig. 1d, Table S11). The results showed that primers in different regions could only amplify specific bands in male seed hemp plants. Based on the above results, we believe that it is first report that the 84 Mb region of Y chromosome is the SDR of seed hemp. The previous study has identified most of the non-recombination regions between the X and Y chromosomes through karyotype analysis of cannabis, and our study is consistent with this result [9]. Although most sex-differentiated plants have small sex-determining regions, there have been the report of large one, such as S. latifolia , where X pericentromeric region (Xpr) accounts for more than 85% of the entire X chromosome [10]. In our study, SDR on Y chromosome accounted for 71% of the whole chromosome, which is related to the presence of a large number of TE insertions. The insertion of TEs and the presence of repetitive sequences have caused the expansion of the Y chromosome. Furthermore, the existence of large inversion in the SDR may have suppressed recombination between the two sex chromosomes, which might be another significant reason for the large size of the SDR [11]. We also observed the high density of repetitive elements specifically accumulated in the SDR of the seed hemp Y chromosome (Fig. 1e). SDR consisted of 91.4% repetitive sequences, mostly in the form of LTR/Gypsy (34.77%). The analysis of high repeat sequences in SDR indicated that the accumulation of repetitive sequences may be contributed to the rapid expansion of SDR in seed hemp. To further study of the SDR and pseudo-autosomal region (PAR) on the Y chromosome and the corresponding regions on the X chromosome, we analyzed the gene content of corresponding regions on SDR, PAR and X chromosome. The results showed that 570 protein-coding genes were annotated in the Y-SDR and 1529 protein-coding genes were annotated in X-SDR. In addition, there were 150 Y/X paired genes in the SDR, 19 Y specific genes, and 34 X specific genes. Similarly, there were 1,897 Y/X paired genes in the PAR, 121 Y specific genes, and 123 X specific genes (Table S12, Fig. S5). To estimate the time since recombination stops between the Y and X across the sex-specific region, we calculated the synonymous substitution rate (Ks) for 150 gene pairs between SDR and X counterpart (Fig. 1f, Table S13). After the removing of outliers, the average Ks for genes within the SDR was 0.46. Using the divergence time between cannabis and mulberry with 63.5 million years ago (Mya) as reference, it is estimated that the divergence of X and Y chromosomes in seed hemp occurred about 37.7 Mya (95% confidence interval: 31.1-44.3 Mya). In addition, we found that there was a giant inversion of the whole SDR, and the encoding sequence pairs 1-65 and 66-150 were inverted relative to X, with sizes of 25.6 Mb and 54.0 Mb, respectively (Fig. 1g, Table S14). This finding might partly explain the larger SDR observed in seed hemp. Our dataset includes 11 newly sequenced male seed hemp genomes and 24 publicly available male cannabis resequenced genomes, including 7 Basic cannabis, 11 Hemp-type, 17 Drug-type feral (Table S15). After mapping to the X chromosome, we identified 1,635,026 high-confidence variants (18.3 variants/kb), including 1,422,211 single nucleotide polymorphisms (SNPs), 98,894 insertions, and 113,921 deletions (a total of 212,815 InDels). After mapping to the Y chromosome, we identified a total of 1,060,233 high-confidence variants (9.0 variants/kb), of which there were 923,502 SNPs, 63,454 insertions and 73,277 deletions (136,731 InDels). Using the X and Y chromosomes as references, we estimated the average nucleotide diversity (pi) to be 0.0034 and 0.0008, respectively, indicating that the X chromosome possesses higher genetic diversity. The Y chromosome is generated after losing many fragments and inserting a large amount of TEs, and TE insertions accumulate in the non-recombining regions of the Y chromosome, and its diversity should be lower than that of the X chromosome [12]. Therefore, we use the X chromosome, which has a higher pi value, as a reference for subsequent analyses. Phylogenetic analysis revealed that these accessions could be clustered into three well-separated genetic groups, corresponding to basic cannabis, hemp-type, and drug-type feral, respectively (Fig. 2a). Principal component analysis (PCA) showed that 35 samples were divided into three groups (Fig. 2b). The population admixture analysis showed that when K=3, three different types of cannabis could be clearly separated (Fig. 2c). The results of phylogenetic classification, PCA and admixture analysis were consistent. However, the samples were divided into four groups in the previous phylogenetic analysis based on the reference of CBDRx genome, which may be caused by different reference genomes and sample differences, but the overall classification results were consistent [13][16]. In addition, nucleotide diversity and population difference analysis showed that there were no significant differences between the three groups (Fig. 2d). Conclusions Our study provides the first high-quality chromosomal-scale male seed hemp genome assembly derived from long-read sequencing. We integrated approach combined collinear analysis, BSA and resequencing analysis, and molecular marker validation to successfully pinpointed a core male-specific region of 84 Mb as SDR on the proximal end of the Y chromosome in seed hemp. This resource paved the avenue towards understanding the structural variation of sex chromosomes in seed hemp, and laid the foundation for further studying sex determination mechanism of angiosperms. Furthermore, our study is of great significance for promoting the seed hemp functional genomics and breeding. Methods Sample collection The ‘yushe’ seed hemp was chosen for reference genome sequencing and assembly, were grown at the experimental farm of Fujian Agriculture and Forestry University, Fuzhou, China. Young leaves of male sample were collected to extract high-quality DNA for PacBio HiFi, ONT ultra-long, and Hi-C sequencing. RNA-seq data from different stages and tissues, including root, stem, leaves, flowers, and seeds. Three samples from each tissue were immediately frozen in liquid nitrogen and stored at -80°C until being used for the RNA isolation. Estimation of seed hemp genome size by K-mer analysis The genome size of seed hemp was estimated using K-mer (K=17) analysis. Then using JELLYFISH [14] and Genomescope1.0 [15] for the K-mer countingafter removing potential contaminants and eliminating low-quality reads. Genome size could be determined by the ratio of the total number of K-mers to the peak of their distribution. Hi-C library preparation and sequencing The experimental process of Hi-C sequencing mainly includes cell cross-linking, endonuclease digestion, terminal repair, cyclization, DNA purification and capture, and sequencing. The Hi-C sequencing library was constructed by chromatin extraction and digestion using a standard procedure [16]. The materials were then sequenced on the NovaSeq 5000 Platform. To obtain high quality analysis data, the raw data were filtered with FASTP (software version: 0.20.0) using the default parameters to obtain high-quality clean reads. Then, ALLHiC was used to anchor purged contigs into super-scaffolds with the Hi-C library to complete chromosome level assembly [17]. Finally, the genome of an elite seed hemp cultivar ''yushe'' with 10 pseudo-chromosomes molecules was assembled, and further evaluated for completeness using BUSCO. Genome assembly and assessment The highly accurate HiFi reads were de novo assembled into two haplotypes draft contig genomes using the hifiasm software with default parameters [18]. And then the two haplotype contigs were corrected, grouped, sorted, and anchored to all contigs onto the chromosome with Hi-C reads using 3D-DNA [19] and juicer software [20]. Meanwhile, the ONT long reads were polished by Pilon [21] and then the gaps in the ref-guided pseudochromosomes were filled with TGS-GapCloser [22]. A heatmap of seed hemp genomic interactions was plotted with HiCPlotter software [23]. We used minimap2 to remapped HiFi reads and Illumina reads to the two assembled haplotype genomes to evaluate genomic reliability [24]. In addition, we detect the accuracy and completeness of the assembled two haplotype genomes with a conserved core of 1614 gene sets using BUSCO 4.0.5 [25], while using LTR Assembly Index (LAI) to assessed genome continuity [26]. Gene and repeat annotations Duplicated elements identification and masking are implemented using pipeline EDTA, and the masked genomic sequences is used for further gene prediction [27]. Structures of protein-coding genes in the seed hemp genomes were performed by the braker software [28] using a combination of de novo gene prediction, homology-based gene prediction, and transcript evidence from RNA-seq datasets. Evidence Modeler was used to synthesize the prediction results and conduct the gene model prediction [29]. Genes that reserved more than 100 nucleotides in length and met the criteria of having start and stop codons. Gene function was annotated by eggmapper against NCBI nonredundant protein database (NR) and Swiss-Prot databases. Gene Ontology (GO) and KEGG annotations were obtained using the R package clusterProfiler. Transposable elements (TEs) in the seed hemp genomes were analyzed using the Extensive de novo TE Annotator (EDTA) with default parameters [27]. Identification of repetitive elements We first customized a de novo repeat library of the genome using RepeatModeler (http://www.repeatmasker.org/RepeatModeler/), which can automatically execute two de novo repeat finding programs, including RECON (version 1.08) [30] and RepeatScout (version 1.0.5) [31]. The consensus TE sequences generated above were subject to RepeatMasker (version 4.05) [32] to identify and cluster repetitive elements. Unknown TEs were further classified using TEclass (version 2.1.3) [33]. To identify tandem repeats within the genome, the Tandem Repeat Finder (TRF) package (version 4.07) [34] was used with the modified parameters of “1 1 2 80 5 200 2000 –d -h” to find high order repeats. The insertion time of LTR-RTs was estimated by comparing the sequence divergence of pairwise LTR-RT sequences at both ends of each LTR-RT. Verification of SDR specificity using PCR Four sequences from randomly selected regions within SDR were used to design primers using Primer 5. PCR validation was then conducted using three female and three male individuals from each of the ‘Yushe’ and ‘Bama’ varieties as templates. The results showed that these four primers in different regions could only amplify specific bands in male seed hemp plants. This finding further confirms the specificity of SDR. X and Y chromosome divergence times The blast tool was used to compare the SDR and its corresponding region on the X chromosome, and the homologous gene pairs between the two regions were identified. Based on homologous gene pairs, Ka/Ks_Calculator was used to estimate synonymous substitutions per synonymous site (Ks). The speciation time based on Ks value is converted by the equation T=Ks/2λ, where λ=6.1×10 -9 , and then the divergence time between two sex chromosomes was estimated [35]. Phylogeny, PCA and population structure Eleven male seed hemp samples were selected for sequencing, and the target coverage was 10×. At the same time, the resequencing data of 24 published male samples were downloaded and re-analyzed, and the total sample size of 35 cannabis resequencing samples was obtained (no. PRJNA734114). These cultivars belong to three types (Drug_type feral, Hemp-type, Basal cannabis), mainly from Asia and Europe, and detailed information about these varieties is provided in Table S15. Download H. lupulus data from the NCBI website as an outgroup (GenBank accession no. DRR024392). Resequencing data were mapped to X chromosome, and 1,635,026 variants were screened with high confidence for population genomics statistical estimation. Principal component analysis (PCA) was performed using the GCTA software on the filtered 1,422,211 variants. The input Plink binary files are transformed from the filtered VCFs file using VCFtools [36] and PLINK [37], then used the top two principal components to assign the 35 cannabis accessions to PCA clusters. Bi-allelic and polymorphic 1,422,211 SNPs were selected to reconstruct a phylogeny of these accessions using SNPhylo [38]software. ADMIXTUR [39] was used to infer ancestral population stratification, the population with the smallest cross-validation error was selected from K=1~10, and DISTRUCT [40] was used to plot the population stratification results. Declarations Acknowledgements This work was supported by funds from the Natural Science Foundation of Fujian (32472219), Fujian Provincial Natural Science Foundation (2023J01443), the Science and Technology Innovation Project of Fujian Agriculture and Foresty University (KFB23001, KFB24080), and China Agriculture Research System of MOF and MARA (CARS-16). This work was supported by the platforms of Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops, and Fujian International Science and Technology Cooperation Base for Genetic Breeding and Multiple Utilization Development of Southern Economic Crops. Authors’ contributions R.M. and L.Z. jointly supervised the work. H.W. and H.J., performed sequencing, assembly and genome annotation. Z.Y., L.Z., X.P., S.J., Q.L., J.X., A.T., P.F. and J.Q. contributed to data analysis. H.W. wrote the manuscript. R.M. and L.Z. revised the manuscript. L.Z. conceived the project. We thank Hongmei Kang at Shanxi Agricultural University for providing the seeds of ‘yushe’. Data Availability The seed hemp genomic data are available in the China National Center for Bioinformation under accession number PRJCA026839. Conflict of interest The authors declare no competing financial interests. 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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-6374740","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":443493504,"identity":"df8acb38-1171-4f06-92a7-b7886212c39b","order_by":0,"name":"Huawei Wei","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Huawei","middleName":"","lastName":"Wei","suffix":""},{"id":443493505,"identity":"72858331-cfa0-4b4b-b249-b3a12d62e259","order_by":1,"name":"Zhuqing Yang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry 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University","correspondingAuthor":false,"prefix":"","firstName":"Haifeng","middleName":"","lastName":"Jia","suffix":""},{"id":443493509,"identity":"d85615e8-32e0-45c1-8477-66ef344e8452","order_by":5,"name":"Shaolian Jiang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Shaolian","middleName":"","lastName":"Jiang","suffix":""},{"id":443493510,"identity":"aaf1db1c-f18f-4488-ac71-6100b657e170","order_by":6,"name":"Qin Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Li","suffix":""},{"id":443493511,"identity":"f40ba1fa-593e-4764-9161-85db80f3fe44","order_by":7,"name":"Jiantang Xu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jiantang","middleName":"","lastName":"Xu","suffix":""},{"id":443493512,"identity":"c017a473-c8b0-4bea-b1df-e7149707058f","order_by":8,"name":"Aifen Tao","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Aifen","middleName":"","lastName":"Tao","suffix":""},{"id":443493513,"identity":"f67ded87-45e5-4513-9701-72454845ba86","order_by":9,"name":"Pingping Fang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Pingping","middleName":"","lastName":"Fang","suffix":""},{"id":443493514,"identity":"ff97bf67-a393-4fad-9a28-20761287225a","order_by":10,"name":"Jianmin Qi","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jianmin","middleName":"","lastName":"Qi","suffix":""},{"id":443493515,"identity":"28c3b510-0063-4e79-ac66-313f9deca84d","order_by":11,"name":"Ray Ming","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ray","middleName":"","lastName":"Ming","suffix":""},{"id":443493516,"identity":"9febf050-1137-437a-aafe-9bf4d075f547","order_by":12,"name":"Liwu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3QsQqCQBjA8e84uBZr/g4DX6EIrgbDVzGCWq61ORDyFYyC3sLmFqceQHCpxVkhwik6raUhbQy6P5ye8P3gPACd7hfD6jkyAGhUbVnrO4KKsBmAq170S6KWISoCTcTaeunlCtjtxfJ2zovQ6lAgWS4/E7KLhoOuOlgvXhz6gZv01xQo34SfCUVXmKiIiBehabgJUYTRdg1hOL+9iExL4jQSA6Xg2ZOwkkwaCaJcmuUlO6dU8GCWTNeUeLX/YgXzkBdgO9yfppjZyXjve8csryHVFbTvb99kVT9fjhSNIzqdTvfXPQC55UMid+9OnAAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Liwu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-04 09:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6374740/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6374740/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83817473,"identity":"7b93c431-0d7a-4b61-90a6-14e42191d2ec","added_by":"auto","created_at":"2025-06-03 07:55:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":248579,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and characterization of the SDR in dioecious seed hemp with YSM1 and YSM2. (a) The collinearity analysis of YSM1 and YSM2. (b) Differential depth of coverage across Y chromosome in the male genome using BSA data. (c) Synteny plot between the X and Y chromosomes, includingan inversion of the homologous region. (d) The male specific interval of SDR was verified by molecular markers. YS and BM: ‘Yushe’ and ‘Bama’ seed hemp. F: female, M: male. (e) Repeat types and repeat density in Y chromosome. X axis: Y chromosome; Y axis: TE ratio. (f) Ks of homolog gene pairs between X and Y. (g) Comparison between the arrangement of 150 genes on the SDR with homologous copies on the linked colinear region (LCR).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/255700e4b8def42dd8961774.png"},{"id":83816191,"identity":"8a75c923-bde7-4f33-89c6-77703636af06","added_by":"auto","created_at":"2025-06-03 07:47:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98150,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation genomic analyses of resequencing 35 cannabis accessions. (a) Phylogenetic relationships among different types of cannabis. (b) PCA analysis between different types of cannabis. (c) Population structure clustering 35 accessions into three subgroups, with optimal clusters as K=3. (d) Nucleotide diversity and population divergence across the three groups. The value within the circle indicates the nucleotide diversity (p) of the population, and the value between the two circles indicates population divergence.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/410f54a06f49c3e87d19c970.png"},{"id":83817799,"identity":"30083730-3027-4df0-b61d-c43f1667e264","added_by":"auto","created_at":"2025-06-03 08:03:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2254966,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/9e4a7d0f-0882-425b-abe2-0f1875a14029.pdf"},{"id":83816192,"identity":"64f3c463-ddf9-423a-bcd1-e8846701e7bb","added_by":"auto","created_at":"2025-06-03 07:47:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72078,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/4efa3a8199adddce39c8bd0b.xlsx"},{"id":83816193,"identity":"d045348e-660f-4c0e-8ae9-4cd544a7873a","added_by":"auto","created_at":"2025-06-03 07:47:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1226877,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/22e27f18af83ffb8e37bdafe.docx"},{"id":83816190,"identity":"1b273c5c-2532-4fd0-bb5c-39cfcc8e1025","added_by":"auto","created_at":"2025-06-03 07:47:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15565,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformationLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6374740/v1/791ea5fa7e75cdb2a4751429.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Haplotype-resolved genome assembly of seed hemp (Cannabis sativa) revealed molecular divergence of Y chromosome evolution","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L.) is an annual dioecious plant with a diploid (2n = 20), comprising 9 pairs of autosomes and a pair of sex chromosomes (XX/XY system). In recent years, due to the agronomic characteristics of hemp and the diversity of renewable resources that it produces, it has attracted more and more attention, yet we still know little about hemp [1, 2].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn general, sex determination of dioecious plants, with separate female and male individuals, is regulated by a heteromorphic sex chromosome or a sex-determining region on homomorphic chromosomes [3]. For example, the sex determination region of asparagus was located within the 132.4 Mb region of chromosome Y [4], and the sex determination region of papaya was located within the 8.1 Mb region of chromosome HSY [5]. Although cannabis has vital economic value, information about its genome is limited. Most importantly, the previously published genomes of medicinal cannabis, wild cannabis, CBD-type cultivar cannabis, and seed hemp have all been sequenced using female plant materials, failing to assemble a complete Y chromosome, which greatly limited the research on gender correlation of cannabis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we generated the X and Y chromosomes from male seed hemp by integrating PacBio HiFi and Oxford Nanopore long reads data and using Hi-C mapping technologies via \u003cem\u003ede novo\u003c/em\u003e assembly and annotation at independent chromosome-scale. We identified the sex determining region (SDR) of seed hemp used bulk segregation analysis of a mixed pool of female and male data. Further, we identified structural variation in the SDR, predicted the time of differentiation of X and Y, and analyzed the genetic differences between the X and Y chromosomes using resequencing data. This genomic resource will provide fundamental tools for the study of gender and provide new insights for cannabis breeding.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eIn this study, we generated 26.27 Gb (32\u0026times; coverage) HiFi reads with the PacBio sequel II platform and 39.57Gb (49\u0026times; coverage) of ultra-long reads by Oxford Nanopore Technology (ONT) (Table S1). We estimated the genome size of male seed hemp (YSM) by k-mer analysis, which gave a value of 816 Mb with 2.14% heterozygosity (Fig. S1). Because of the high heterozygosity of YSM individual, we performed genome assembly and haplotype phasing, thus generating two haplotypes: YSM1 and YSM2 (Fig. S2, Table S2). The YSM1 assembly was 770 Mb in total length and consisted of 63 scaffolds with an N50 size of 81.3 Mb and the longest scaffold is 89.4 Mb. The YSM2 assembly was 804 Mb and comprising 55 scaffolds with an N50 size of 81.2 Mb and the longest scaffold is 117.6 Mb (Table S3). Benchmarking Universal Single-Copy Orthologs (BUSCO) analyses showed that 98.8% and 95.8% of BUSCO genes are complete in the YSM1 and YSM2, respectively (Table S4). The long terminal repeat Assembly Index (LAI) values of YSM1 and YSM2 seed hemp genomes were estimated to be 23 and 21, respectively, which are much higher than published cannabis genomes (Table S5) [6-8]. Moreover, 92.21% and 92.03% of the expressed transcripts from the seed hemp RNA-seq datasets were mapped in YSM1 and YSM2, respectively (Table S6). Furthermore, comparing the assembled YSM genomes with the previously published genomes of cannabis, Subreads N50, Contig N50, LAI and BUSCO all showed substantial improvements, indicating that higher assembly quality of the genomes (Table S7). Collectively, all these results demonstrated high continuity, accuracy, and completeness of the assembled the male genomes in seed hemp.\u003c/p\u003e\n\u003cp\u003eUsing a combination of de novo gene prediction, protein homology searches, and assembly of RNA-Seq reads, we identified 32,519 and 31,465 protein-coding genes in YSM1 and YSM2, respectively (Table S6). Among the predicted protein-coding genes, 31233 and 30235 could be functionally annotated by multiple public databases, respectively (Table S8). BUSCO analysis showed that 98.3% and 96.3% of the predicted genes had full-length sequence information in YSM1 and YSM2 (Table S9). Furthermore, within the two haplotype assemblies, 574.53 Mb (74.53%) and 614.23 Mb (76.33%) of repetitive sequences were identified (Table S10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause of the sex-determining regions or sex-regulatory genes and the repeated sequences within the so-called sex determining region (SDR), it is difficult to recognize, locate, and define the exact boundaries of sex-specific regions [11]. To identify the SDR in seed hemp, we performed collinear analysis of the two haplotype genomes of YSM1 and YSM2, and found that there was a good collinearity between the autosomes, while the Y and X chromosomes were only partially collinear and occurred at the end (Fig. 1a). To further investigate this SDR, we used bulked DNA pools from 50 female plants (BSA-F) and 50 male plants (BSA-M) collected in the seed hemp to perform BSA. When YSM1 was used as the reference genome, the coverage of all chromosomes was basically the same, while when YSM2 was used as the reference genome, the coverage of the first 84 Mb region of the Y chromosome was significantly lower than that of other regions, and the coverage of BSA-F reads in this region was lower than that of BSA-M reads (Fig. 1b,\u0026nbsp;Fig.\u0026nbsp;S3). Similarly, the coverage depth of resequencing data based on different individuals (11 males and 11 females) with different background varieties in this region is also different, with male samples having higher coverage depth than female samples (Fig. S4). This indicates that the first 84Mb region of Y chromosome is the SDR of seed hemp. Collinearity analysis between X and Y chromosomes based on homologous gene pairs also demonstrated the specificity of the 84 Mb in the Y chromosome (Fig.\u0026nbsp;1c). In addition, through collinearity analysis between X and Y chromosomes, an inversion was found in the homologous region of both chromosomes. This inversion usually inhibits genetic recombination at the relevant locus and plays a crucial role in the fixing of favorable allelic combinations. The random primer PCR assay targeting the SDR produced specific amplification bands exclusively in male seed hemp (Fig.\u0026nbsp;1d,\u0026nbsp;Table S11). \u0026nbsp;The results showed that primers in different regions could only amplify specific bands in male seed hemp plants. Based on the above results, we believe that it is first report that the 84 Mb region of Y chromosome is the SDR of seed hemp. The previous study has identified most of the non-recombination regions between the X and Y chromosomes through karyotype analysis of cannabis, and our study is consistent with this result\u0026nbsp;[9]. Although most sex-differentiated plants have small sex-determining regions, there have been the report of large one, such as \u003cem\u003eS. latifolia\u003c/em\u003e, where X pericentromeric region (Xpr) accounts for more than 85% of the entire X chromosome\u0026nbsp;[10]. In our study, SDR on Y chromosome accounted for 71% of the whole chromosome, which is related to the presence of a large number of TE insertions. The insertion of TEs and the presence of repetitive sequences have caused the expansion of the Y chromosome. Furthermore, the existence of large inversion in the SDR may have suppressed recombination between the two sex chromosomes, which might be another significant reason for the large size of the SDR\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003eWe also observed the high density of repetitive elements specifically accumulated in the SDR of the seed hemp Y chromosome (Fig. 1e). SDR consisted of 91.4% repetitive sequences, mostly in the form of LTR/Gypsy (34.77%). The analysis of high repeat sequences in SDR indicated that the accumulation of repetitive sequences may be contributed to the rapid expansion of SDR in seed hemp. To further study of the SDR and pseudo-autosomal region (PAR) on the Y chromosome and the corresponding regions on the X chromosome, we analyzed the gene content of corresponding regions on SDR, PAR and X chromosome. The results showed that 570 protein-coding genes were annotated in the Y-SDR and 1529 protein-coding genes were annotated in X-SDR. In addition, there were 150 Y/X paired genes in the SDR, 19 Y specific genes, and 34 X specific genes. Similarly, there were 1,897 Y/X paired genes in the PAR, 121 Y specific genes, and 123 X specific genes (Table S12, Fig. S5).\u003c/p\u003e\n\u003cp\u003eTo estimate the time since recombination stops between the Y and X across the sex-specific region, we calculated the synonymous substitution rate (Ks) for 150 gene pairs between SDR and X counterpart (Fig. 1f, Table S13). After the removing of outliers, the average Ks for genes within the SDR was 0.46. Using the divergence time between cannabis and mulberry with 63.5 million years ago (Mya) as reference, it is estimated that the divergence of X and Y chromosomes in seed hemp occurred about 37.7 Mya (95% confidence interval: 31.1-44.3 Mya). In addition, we found that there was a giant inversion of the whole SDR, and the encoding sequence pairs 1-65 and 66-150 were inverted relative to X, with sizes of 25.6 Mb and 54.0 Mb, respectively (Fig. 1g, Table S14). This finding might partly explain the larger SDR observed in seed hemp.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur dataset includes 11 newly sequenced male seed hemp genomes and 24 publicly available male cannabis resequenced genomes, including 7 Basic cannabis, 11 Hemp-type, 17 Drug-type feral (Table S15). After mapping to the X chromosome, we identified 1,635,026 high-confidence variants (18.3 variants/kb), including 1,422,211 single nucleotide polymorphisms (SNPs), 98,894 insertions, and 113,921 deletions (a total of 212,815 InDels). After mapping to the Y chromosome, we identified a total of 1,060,233 high-confidence variants (9.0 variants/kb), of which there were 923,502 SNPs, 63,454 insertions and 73,277 deletions (136,731 InDels). \u0026nbsp;Using the X and Y chromosomes as references, we estimated the average nucleotide diversity (pi) to be 0.0034 and 0.0008, respectively, indicating that the X chromosome possesses higher genetic diversity. The Y chromosome is generated after losing many fragments and inserting a large amount of TEs, and TE insertions accumulate in the non-recombining regions of the Y chromosome, and its diversity should be lower than that of the X chromosome\u0026nbsp;[12]. Therefore, we use the X chromosome, which has a higher pi value, as a reference for subsequent analyses.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis revealed that these accessions could be clustered into three well-separated genetic groups, corresponding to basic cannabis, hemp-type, and drug-type feral, respectively (Fig. 2a). Principal component analysis (PCA) showed that 35 samples were divided into three groups (Fig. 2b). The population admixture analysis showed that when K=3, three different types of cannabis could be clearly separated (Fig. 2c). The results of phylogenetic classification, PCA and admixture analysis were consistent. However, the samples were divided into four groups in the previous phylogenetic analysis based on the reference of CBDRx genome, which may be caused by different reference genomes and sample differences, but the overall classification results were consistent [13][16]. In addition, nucleotide diversity and population difference analysis showed that there were no significant differences between the three groups (Fig. 2d).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study provides the first high-quality chromosomal-scale male seed hemp genome assembly derived from long-read sequencing. We integrated approach combined collinear analysis, BSA and resequencing analysis, and molecular marker validation to successfully pinpointed a core male-specific region of 84 Mb as SDR on the proximal end of the Y chromosome in seed hemp. This resource paved the avenue towards understanding the structural variation of sex chromosomes in seed hemp, and laid the foundation for further studying sex determination mechanism of angiosperms. Furthermore, our study is of great significance for promoting the seed hemp functional genomics and breeding.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026lsquo;yushe\u0026rsquo; seed hemp was chosen for reference genome sequencing and assembly, were grown at the experimental farm of Fujian Agriculture and Forestry University, Fuzhou, China. Young leaves of male sample were collected to extract high-quality DNA for PacBio HiFi, ONT ultra-long, and Hi-C sequencing. RNA-seq data from different stages and tissues, including root, stem, leaves, flowers, and seeds. Three samples from each tissue were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C until being used for the RNA isolation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of seed hemp genome size by K-mer analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome size of seed hemp was estimated using K-mer (K=17) analysis. Then using JELLYFISH [14] and Genomescope1.0 [15] for the K-mer countingafter removing potential contaminants and eliminating low-quality reads. Genome size could be determined by the ratio of the total number of K-mers to the peak of their distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHi-C library preparation and sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental process of Hi-C sequencing mainly includes cell cross-linking, endonuclease digestion, terminal repair, cyclization, DNA purification and capture, and sequencing. The Hi-C sequencing library was constructed by chromatin extraction and digestion using a standard procedure\u0026nbsp;[16]. The materials were then sequenced on the NovaSeq 5000 Platform. To obtain high quality analysis data, the raw data were filtered with FASTP (software version: 0.20.0) using the default parameters to obtain high-quality clean reads. Then, ALLHiC was used to anchor purged contigs into super-scaffolds with the Hi-C library to complete chromosome level assembly\u0026nbsp;[17]. Finally, the genome of an elite seed hemp cultivar \u0026apos;\u0026apos;yushe\u0026apos;\u0026apos; with 10 pseudo-chromosomes molecules was assembled, and further evaluated for completeness using BUSCO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome assembly and assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highly accurate HiFi reads were de novo assembled into two haplotypes draft contig genomes using the hifiasm software with default parameters [18]. And then the two haplotype contigs were corrected, grouped, sorted, and anchored to all contigs onto the chromosome with Hi-C reads using 3D-DNA [19] and juicer software [20]. Meanwhile, the ONT long reads were polished by Pilon [21] and then the gaps in the ref-guided pseudochromosomes were filled with TGS-GapCloser [22].\u003c/p\u003e\n\u003cp\u003eA heatmap of seed hemp genomic interactions was plotted with HiCPlotter software [23]. We used minimap2 to remapped HiFi reads and Illumina reads to the two assembled haplotype genomes to evaluate genomic reliability [24]. In addition, we detect the accuracy and completeness of the assembled two haplotype genomes with a conserved core of 1614 gene sets using BUSCO 4.0.5 [25], while using LTR Assembly Index (LAI) to assessed genome continuity [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene and repeat annotations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuplicated elements identification and masking are implemented using pipeline EDTA, and the masked genomic sequences is used for further gene prediction [27]. Structures of protein-coding genes in the seed hemp genomes were performed by the braker software [28] using a combination of de novo gene prediction, homology-based gene prediction, and transcript evidence from RNA-seq datasets. Evidence Modeler was used to synthesize the prediction results and conduct the gene model prediction [29]. Genes that reserved more than 100 nucleotides in length and met the criteria of having start and stop codons. Gene function was annotated by eggmapper against NCBI nonredundant protein database (NR) and Swiss-Prot databases. Gene Ontology (GO) and KEGG annotations were obtained using the R package clusterProfiler. Transposable elements (TEs) in the seed hemp genomes were analyzed using the Extensive de novo TE Annotator (EDTA) with default parameters [27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of repetitive elements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first customized a de novo repeat library of the genome using RepeatModeler (http://www.repeatmasker.org/RepeatModeler/), which can automatically execute two de novo repeat finding programs, including RECON (version 1.08) [30] and RepeatScout (version 1.0.5) [31]. The consensus TE sequences generated above were subject to RepeatMasker (version 4.05) [32] to identify and cluster repetitive elements. Unknown TEs were further classified using TEclass (version 2.1.3) [33]. To identify tandem repeats within the genome, the Tandem Repeat Finder (TRF) package (version 4.07) [34] was used with the modified parameters of \u0026ldquo;1 1 2 80 5 200 2000 \u0026ndash;d -h\u0026rdquo; to find high order repeats. The insertion time of LTR-RTs was estimated by comparing the sequence divergence of pairwise LTR-RT sequences at both ends of each LTR-RT. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification of SDR specificity using PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour sequences from randomly selected regions within SDR were used to design primers using Primer 5. PCR validation was then conducted using three female and three male individuals from each of the \u0026lsquo;Yushe\u0026rsquo; and \u0026lsquo;Bama\u0026rsquo; varieties as templates. \u0026nbsp;The results showed that these four primers in different regions could only amplify specific bands in male seed hemp plants. This finding further confirms the specificity of SDR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX and Y chromosome divergence times\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blast tool was used to compare the SDR and its corresponding region on the X chromosome, and the homologous gene pairs between the two regions were identified. Based on homologous gene pairs, Ka/Ks_Calculator was used to estimate synonymous substitutions per synonymous site (Ks). The speciation time based on Ks value is converted by the equation T=Ks/2\u0026lambda;, where \u0026lambda;=6.1\u0026times;10\u003csup\u003e-9\u003c/sup\u003e, and then the divergence time between two sex chromosomes was estimated [35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogeny, PCA and population structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEleven male seed hemp samples were selected for sequencing, and the target coverage was 10\u0026times;. At the same time, the resequencing data of 24 published male samples were downloaded and re-analyzed, and the total sample size of 35 cannabis resequencing samples was obtained (no. PRJNA734114). These cultivars belong to three types (Drug_type feral, Hemp-type, Basal cannabis), mainly from Asia and Europe, and detailed information about these varieties is provided in Table S15. Download \u003cem\u003eH. lupulus\u003c/em\u003e data from the NCBI website as an outgroup (GenBank accession no. DRR024392). \u0026nbsp; Resequencing data were mapped to X chromosome, and 1,635,026 variants were screened with high confidence for population genomics statistical estimation. Principal component analysis (PCA) was performed using the GCTA software on the filtered 1,422,211 variants. The input Plink binary files are transformed from the filtered VCFs file using VCFtools [36] \u0026nbsp;and PLINK [37], then used the top two principal components to assign the 35 cannabis accessions to PCA clusters. Bi-allelic and polymorphic 1,422,211 SNPs were selected to reconstruct a phylogeny of these accessions using SNPhylo [38]software. ADMIXTUR [39] was used to infer ancestral population stratification, the population with the smallest cross-validation error was selected from K=1~10, and DISTRUCT [40] was used to plot the population stratification results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funds from the Natural Science Foundation of Fujian (32472219), Fujian Provincial Natural Science Foundation (2023J01443), the Science and Technology Innovation Project of Fujian Agriculture and Foresty University (KFB23001, KFB24080), and China Agriculture Research System of MOF and MARA (CARS-16). This work was supported by the platforms of Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops, and Fujian International Science and Technology Cooperation Base for Genetic Breeding and Multiple Utilization Development of Southern Economic Crops.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.M. and L.Z. jointly supervised the work. H.W. and H.J., performed sequencing, assembly and genome annotation. Z.Y., L.Z., X.P., S.J., Q.L., J.X., A.T., P.F. and J.Q. contributed to data analysis. H.W. wrote the manuscript. R.M. and L.Z. revised the manuscript. L.Z. conceived the project. We thank Hongmei Kang at Shanxi Agricultural University for providing the seeds of\u0026nbsp;\u0026lsquo;yushe\u0026rsquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seed hemp genomic data are available in the China National Center for Bioinformation under accession number PRJCA026839.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndre CM, Hausman JF, Guerriero G: \u003cstrong\u003e\u003cem\u003eCannabis sativa\u003c/em\u003e: The Plant of the Thousand and One Molecules.\u003c/strong\u003e \u003cem\u003eFront Plant Sci \u003c/em\u003e2016, \u003cstrong\u003e7:\u003c/strong\u003e19.\u003c/li\u003e\n\u003cli\u003eBarth M, Carus M: \u003cstrong\u003eCarbon Footprint and Sustainability of Different Natural 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region, Whole genome resequencing, Evolution of sex chromosomes","lastPublishedDoi":"10.21203/rs.3.rs-6374740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6374740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Seed hemp (Cannabis sativa L.) is an important economic dioecious plant with an XY sex chromosome system, yet its Y chromosome and sex-determining regions (SDR) have not been characterized. Here, we assembled a high-quality haplotype-resolved genome (female: 770.8 Mb; male: 804.7 Mb), and identified an 84 Mb SDR, covering 71% of the Y chromosome, via comparative genomics. X/Y divergence occurred ~37.7 Mya (Ks analysis). Resequencing 35 cannabis accessions revealed higher genetic diversity on the X chromosome. This study provides the first Y chromosome and SDR assembly in seed hemp, offering insights into sex differentiation in dioecious plants.","manuscriptTitle":"Haplotype-resolved genome assembly of seed hemp (Cannabis sativa) revealed molecular divergence of Y chromosome evolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 07:47:51","doi":"10.21203/rs.3.rs-6374740/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":"21cd20f6-f0a6-4967-b8e5-942aff832071","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T07:47:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 07:47:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6374740","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6374740","identity":"rs-6374740","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}