Discovering candidate SNPs for resilience breeding of red clover

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 F_ST 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.