Table_1_Discovering candidate SNPs for resilience breeding of red clover.xlsx

Red clover is a highly valuable crop for the ruminant industry in the temperate regions worldwide. It also provides multiple environmental services, such as contribution to increased soil fertility and reduced soil erosion. This study used 661 single nucleotide polymorphism (SNP) markers via targeted sequencing using seqSNP, to describe genetic diversity and population structure in 382 red clover accessions. The accessions were selected from NordGen representing red clover germplasm from Norway, Sweden, Finland and Denmark as well as from Lantmännen, a Swedish seed company. Each accession was represented by 10 individuals, which was sequenced as a pool. The mean Nei’s standard genetic distance between the accessions and genetic variation within accessions were 0.032 and 0.18, respectively. The majority of the accessions had negative Tajima’s D, suggesting that they contain significant proportions of rare alleles. A pairwise FST revealed high genetic similarity between the different cultivated types, while the wild populations were divergent. Unlike wild populations, which exhibited genetic differentiation, there was no clear differentiation among all cultivated types. A principal coordinate analysis revealed that the first principal coordinate, distinguished most of the wild populations from the cultivated types, in agreement with the results obtained using a discriminant analysis of principal components and cluster analysis. Accessions of wild populations and landraces collected from southern and central Scandinavia showed a higher genetic similarity to Lantmännen accessios. It is therefore possible to link the diversity of the environments where wild populations were collected to the genetic diversity of the cultivated and wild gene pools. Additionally, least absolute shrinkage and selection operator (LASSO) models revealed associations between variation in temperature and precipitation and SNPs within genes controlling stomatal opening. Temperature was also related to kinase proteins, which are known to regulate plant response to temperature stress. Furthermore, the variation between wild populations and cultivars was correlated with SNPs within genes regulating root development. Overall, this study comprehensively investigated Nordic European red clover germplasm, and the results provide forage breeders with valuable information for further selection and development of red clover cultivars.

红车轴草（Red Clover）是全球温带地区反刍动物产业极具经济价值的作物，同时可提供多种生态系统服务，例如提升土壤肥力、减轻土壤侵蚀。本研究借助seqSNP靶向测序技术，采用661个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）标记，对382份红车轴草种质资源的遗传多样性与群体结构展开解析。供试种质源自北欧基因资源中心（NordGen）保存的挪威、瑞典、芬兰、丹麦红车轴草种质，以及瑞典种子公司兰塔曼能（Lantmännen）的种质资源。每份种质包含10个个体，以混合样本的形式完成测序。供试种质间的平均奈氏标准遗传距离（Nei's standard genetic distance）与种质内的遗传变异分别为0.032与0.18。绝大多数种质的塔伊马D统计量（Tajima's D）为负值，表明其携带比例可观的稀有等位基因。两两固定指数（Fixation Index, FST）分析结果显示，不同栽培类型间遗传相似性较高，而野生种群则呈现显著分化。与存在遗传分化的野生种群不同，所有栽培类型间未出现清晰的遗传分化。主坐标分析（Principal Coordinate Analysis, PCoA）表明，第一主坐标可将多数野生种群与栽培类型区分开来，这与主成分判别分析（Discriminant Analysis of Principal Components, DAPC）及聚类分析所得结果一致。源自斯堪的纳维亚南部与中部的野生种群及地方品种种质，与兰塔曼能种质的遗传相似性更高。据此可将野生种群采集环境的多样性与栽培及野生基因库的遗传多样性建立关联。此外，最小绝对收缩和选择算子（Least Absolute Shrinkage and Selection Operator, LASSO）模型分析显示，温度与降水的变异与调控气孔开放的基因内的SNP位点存在显著关联。温度还与已知可调控植物温度胁迫响应的激酶蛋白密切相关。进一步分析发现，野生种群与栽培品种间的遗传变异与调控根系发育的基因内的SNP位点存在相关性。总体而言，本研究对北欧地区红车轴草种质资源进行了全面系统的解析，研究结果可为牧草育种工作者开展红车轴草品种的进一步选育与培育提供极具价值的参考依据。