GWAS for drought resilience traits in red clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is a well-appreciated grassland crop in temperate climates but suffers from increasingly frequent and severe drought periods. Molecular markers for drought resilience (DR) would benefit breeding initiatives for red clover, as would a better understanding of the genes involved in DR. Two previous studies, as follows, have: (1) identified phenotypic DR traits in a diverse set of red clover accessions; and (2) produced genotypic data using a pooled genotyping-by-sequencing (GBS) approach in the same collection. In the present study, we performed genome-wide association studies (GWAS) for DR using the available phenotypic and genotypic data. Single nucleotide polymorphism (SNP) calling was performed using GBS data and the following two red clover genome assemblies: the recent HEN-17 assembly and the Milvus assembly. SNP positions with significant associations were used to delineate flanking regions in both genome assemblies, while functional annotations were retrieved from Medicago truncatula orthologs. GWAS revealed 19 significant SNPs in the HEN-17-derived SNP set, explaining between 5.3 and 23.2% of the phenotypic variation per SNP–trait combination for DR traits. Among the genes in the SNP-flanking regions, we identified candidate genes related to cell wall structuring, genes encoding sugar-modifying proteins, an ureide permease gene, and other genes linked to stress metabolism pathways. GWAS revealed 29 SNPs in the Milvus-derived SNP set that explained substantially more phenotypic variation for DR traits, between 5.3 and 42.3% per SNP–trait combination. Candidate genes included a DEAD-box ATP-dependent RNA helicase gene, a P-loop nucleoside triphosphate hydrolase gene, a Myb/SANT-like DNA-binding domain protein, and an ubiquitin–protein ligase gene. Most accessions in this study are genetically more closely related to the Milvus genotype than to HEN-17, possibly explaining how the Milvus-derived SNP set yielded more robust associations. The Milvus-derived SNP set pinpointed 10 genomic regions that explained more than 25% of the phenotypic variation for DR traits. A possible next step could be the implementation of these SNP markers in practical breeding programs, which would help to improve DR in red clover. Candidate genes could be further characterized in future research to unravel drought stress resilience in red clover in more detail.