Genomic structure and ex situ conservation of the North American grapevine <em>Vitis labrusca</em>

Vitis labrusca, a North American wild grapevine, is an important source of disease resistance and climate resilience traits for grape breeding, yet its genomic diversity is incompletely represented in ex situ germplasm collections. We genotyped 314 accessions, which included material conserved at the USDA germplasm collection and newly sampled wild individuals. Accessions were genotyped using genotyping-by-sequencing, and after imputation and filtering, we identified a total of 44,701 SNPs. Within the accessions genotyped, we identified extensive mislabelling and hybridization, with approximately one-third of accessions classified as putative hybrids. We also detected genetically distinct populations from Virginia and North Carolina that are not currently conserved. These results reveal significant geographic and genomic gaps in ex situ conservation of V. labrusca and highlight priority regions for future sampling to better safeguard this species for breeding and research. Methods A total of 314 unique samples were sequenced, although 11 of these were sequenced twice. Sequencing data were processed and single nucleotide polymorphisms (SNPs) identified using the Tassel 5 GBS v2 Pipeline v5.2.94 (Bradbury et al., 2007). During this process, replicate samples with the same IDs were automatically merged. Default parameters were used for all steps except the initial GBSSeqToTagDBPlugin() step, where a minimum quality score (-mnQS) of 20 was required. Reads were aligned to the V. labrusca reference genome for GREM4, version 1 (Li & Gschwend, 2023) using BWA v0.7.18 (Li & Durbin, 2009). A minimum minor allele frequency (MAF) of 0.01 was set for SNP discovery using Tassel, and a total of 154,638 SNPs were identified. These SNPs were filtered using VCFTools v0.1.16 to remove indels, retain only biallelic sites, and ensure a MAF threshold of 0.01 (Danecek et al., 2011), resulting in 145,155 SNPs across the 314 accessions. Imputation was performed using LinkImputeR (Money et al., 2017), with input filters of MAF 0.01, a maximum missingness by position of 70%, and a Hardy-Weinberg equilibrium filter of p < 0.0001. Parameters tested included depths of 2 to 6 (increasing by 1) and maximum missingness per SNP and sample of 0.2 to 0.6 (increasing by 0.1). The selected imputation case applied the filters of PositionMiss(0.6) and SampleMiss(0.6), resulting in an accuracy of 0.935 and a correlation value of 0.8091. After imputation, 271 accessions and 46,916 SNPs remained. VCFTools (v0.1.16) (Danecek et al., 2011) was used to remove SNPs located on unassembled contigs in the V. labrusca genome from the SNP set, resulting in a final filtered dataset of 44,701 SNPs. PLINK v2.00a5.8 (Chang et al., 2015; Purcell & Chang, 2025) was used to prune the imputed SNP table for linkage disequilibrium (LD). LD-pruning was performed by considering a window of 10 SNPs, removing one SNP from a pair if the r2 was >0.5 then shifting the window by 3 SNPS and repeating the procedure (--indep-pairwise 10 3 0.5). The resulting dataset contained 271 accessions and 32,267 SNPs. Accessions with >0.90 ancestry proportion assignment to Cluster 2 (n = 183) were retained as putative “pure” V. labrusca. These accessions were kept using PLINK (v2.00a5.8), followed by a filter for MAF 0.01, resulting in 27,071 SNPs remaining (Chang et al., 2015; Purcell & Chang, 2025). LD-pruning was repeated as described above and the final dataset contained 18,849 SNPs and 183 accessions. References Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y. & Buckler, E.S. (2007). TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics, 23(19), pp. 2633–2635. Chang, C.C., Chow, C.C., Tellier, L.C., Vattikuti, S., Purcell, S.M. & Lee, J.J. (2015). Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience, 4(1), pp. s13742-015-0047–8 https://doi.org/10.1186/s13742-015-0047-8. Danecek, P., Auton, A., Abecasis, G., Albers, C.A., Banks, E., DePristo, M.A., Handsaker, R.E., Lunter, G., Marth, G.T., Sherry, S.T., McVean, G., Durbin, R. & Genomes Project Analysis, G. (2011). The variant call format and VCFtools. Bioinformatics, 27(15), pp. 2156–8 https://doi.org/10.1093/bioinformatics/btr330. Li, B. & Gschwend, A.R. (2023). Vitis labrusca genome assembly reveals diversification between wild and cultivated grapevine genomes. Frontiers in Plant Science, 14 https://doi.org/10.3389/fpls.2023.1234130. Li, H. & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25(14), pp. 1754–60 https://doi.org/10.1093/bioinformatics/btp324. Money, D., Migicovsky, Z., Gardner, K. & Myles, S. (2017). LinkImputeR: user-guided genotype calling and imputation for non-model organisms. BMC Genomics, 18(1), p. 523 https://doi.org/10.1186/s12864-017-3873-5. Purcell, S. & Chang, C. (2025). PLINK v2.00a5.8 www.cog-genomics.org/plink/2.0/.圆叶葡萄（Vitis labrusca）是一种北美野生葡萄藤，作为葡萄育种中抗病与气候适应性性状的重要种质资源，其基因组多样性在迁地种质资源库（ex situ germplasm collections）中的覆盖度仍不完善。本研究对314份种质进行了基因分型，其中涵盖了美国农业部（USDA）种质资源库保存的材料以及新采集的野生个体。本研究采用测序分型（genotyping-by-sequencing, GBS）技术对所有种质进行基因分型，经基因型填充（imputation）与过滤后，共鉴定得到44701个单核苷酸多态性位点（single nucleotide polymorphisms, SNPs）。在完成基因分型的种质中，我们发现了大量的标签错误与杂交现象，约三分之一的种质被归类为疑似杂交种。此外，我们还检测到来自弗吉尼亚州与北卡罗来纳州的遗传分化种群，此类种群目前尚未被迁地保存。本研究结果揭示了圆叶葡萄迁地保护中存在的显著地理与基因组缺口，并明确了未来采集的优先区域，以更好地保护该物种用于育种与研究工作。 方法 本研究共对314份独有的样本进行了测序，其中11份样本被重复测序两次。测序数据的处理与单核苷酸多态性（single nucleotide polymorphisms, SNPs）的鉴定采用Tassel 5 测序分型v2流程（Tassel 5 GBS v2 Pipeline）v5.2.94版本（Bradbury等，2007）完成。在此流程中，带有相同编号的重复样本将被自动合并。除初始的GBSSeqToTagDBPlugin()步骤需设置最低质量分数（-mnQS）为20外，其余所有步骤均采用默认参数。利用BWA v0.7.18（Li & Durbin, 2009）将测序读段比对至GREM4版本1的圆叶葡萄参考基因组（Li & Gschwend, 2023）。本研究通过Tassel进行SNP鉴定时，设置最小次要等位基因频率（minor allele frequency, MAF）为0.01，共鉴定得到154638个SNPs。随后使用VCFTools v0.1.16（Danecek等，2011）对这些SNPs进行过滤，剔除插入缺失位点（insertions-deletions, indels），仅保留双等位基因位点，并维持MAF阈值为0.01，最终在314份种质中共得到145155个SNPs。 基因型填充（imputation）采用LinkImputeR（Money等，2017）完成，输入过滤条件设置为MAF 0.01、位点最大缺失率70%以及哈迪-温伯格平衡（Hardy-Weinberg equilibrium）过滤阈值p<0.0001。本研究测试的参数包括测序深度2至6（步长为1）、每个SNP和样本的最大缺失率0.2至0.6（步长为0.1）。最终选定的填充方案采用了PositionMiss(0.6)与SampleMiss(0.6)过滤条件，其填充准确率为0.935，相关系数为0.8091。基因型填充完成后，共保留271份种质与46916个SNPs。随后再次使用VCFTools v0.1.16（Danecek等，2011）移除比对至圆叶葡萄基因组中未组装重叠群（contigs）的SNP位点，最终得到经过过滤的数据集，包含44701个SNPs。 使用PLINK v2.00a5.8（Chang等，2015; Purcell & Chang, 2025）对填充后的SNP数据集进行连锁不平衡（linkage disequilibrium, LD）修剪。LD修剪的具体步骤为：以10个SNPs为窗口，若SNP对的r²>0.5则移除其中一个SNP，随后将窗口滑动3个SNPs并重复该过程（--indep-pairwise 10 3 0.5）。最终得到的数据集包含271份种质与32267个SNPs。 将祖先组分比例归属至簇2（Cluster 2，n=183）且比例>0.90的种质保留为疑似“纯种”圆叶葡萄。通过PLINK v2.00a5.8筛选得到此类种质，随后设置MAF过滤阈值为0.01，最终保留27071个SNPs（Chang等，2015; Purcell & Chang, 2025）。按照前述步骤再次进行LD修剪，最终得到的数据集包含18849个SNPs与183份种质。 参考文献 Bradbury, P.J.、Zhang, Z.、Kroon, D.E.、Casstevens, T.M.、Ramdoss, Y. 与 Buckler, E.S.（2007）。TASSEL：用于多样本复杂性状关联定位的软件。《生物信息学》，23(19)，第2633-2635页。 Chang, C.C.、Chow, C.C.、Tellier, L.C.、Vattikuti, S.、Purcell, S.M. 与 Lee, J.J.（2015）。第二代PLINK：应对更大更丰富的数据集挑战。《GigaScience》，4(1)，第s13742-015-0047-8页，DOI: 10.1186/s13742-015-0047-8。 Danecek, P.、Auton, A.、Abecasis, G.、Albers, C.A.、Banks, E.、DePristo, M.A.、Handsaker, R.E.、Lunter, G.、Marth, G.T.、Sherry, S.T.、McVean, G.、Durbin, R. 与基因组项目分析组（2011）。变异调用格式与VCFTools。《生物信息学》，27(15)，第2156-2158页，DOI: 10.1093/bioinformatics/btr330。 Li, B. 与 Gschwend, A.R.（2023）。圆叶葡萄基因组组装揭示野生与栽培葡萄基因组的分化。《植物科学前沿》，14，DOI: 10.3389/fpls.2023.1234130。 Li, H. 与 Durbin, R.（2009）。基于Burrows-Wheeler变换的快速准确短读段比对。《生物信息学》，25(14)，第1754-1760页，DOI: 10.1093/bioinformatics/btp324。 Money, D.、Migicovsky, Z.、Gardner, K. 与 Myles, S.（2017）。LinkImputeR：面向非模式生物的用户引导型基因型调用与填充工具。《BMC基因组学》，18(1)，第523页，DOI: 10.1186/s12864-017-3873-5。 Purcell, S. 与 Chang, C.（2025）。PLINK v2.00a5.8，网址：www.cog-genomics.org/plink/2.0/。