Understanding the Complexity of Cold Tolerance in White Clover using Temperature Gradient Locations and a GWAS Approach

White clover (Trifolium repens L.) is the most important grazing perennial forage legume in temperate climates. However, its limited capacity to survive and restore growth after low temperatures during winter constrains the productivity and wide adoption of the crop. Despite the importance of cold tolerance for white clover cultivar development, the genetic basis of this trait remains largely unknown. Hence, in this study, we performed the first genome-wide association study (GWAS) analyses in white clover to identify quantitative trait loci (QTL) for cold-tolerance-related traits. Seeds from 192 divergent genotypes from six populations in the Patagonia region of South America were collected and seed-derived plants were further clonally propagated. Clonal trials were established in three locations representing temperature gradient associated with elevation. Given the allotetraploid nature of the white clover genome, distinct genetic models (diploid and tetraploid) were tested. Only the tetraploid parameterization was able to detect the 53 loci associated with cold-tolerance traits. Out of the 53 single nucleotide polymorphism (SNP) trait associations, 17 controlled more than one trait or were stable across multiple sites. This work represents the first report of QTL for cold-tolerance-related traits, providing insights into its genetic basis and candidate genomic regions for further functional validation studies.