Genome-Wide Association Mapping of Cold Tolerance Related Traits in White Clover Germplasm Naturalized in the Patagonia Region of South America.

In Mediterranean environments, low temperature stress (5-15°C) causes slow growth rate of White Clover (WC). As a result, WC competitiveness is reduced during early spring and late autumn, thereby, leading to low contribution of WC to the total yield in a grass/WC mixture. A white clover association mapping (WCAM) population comprised of 192 WC genotypes was developed for identifying phenotypic traits and genomic regions (QTLs) controlling the WC cold tolerance. The WCAM population was phenotypically characterized during three growing seasons in three environments that represents a winter cold gradient associated with the altitude: Santa Rosa (SR₁₄₀), Atacalco (AT₆₆₀) and Puente Marchant (PM₁₀₅₀), located at 140, 660, and 1050 m.a.s.l., respectively. The WCAM population was genotyped using a Next Generation Sequencing (NGS) based Capture-Seq technology developed by the RAPID Genomics lab of the University of Florida, FL, USA. Lack of reference genome and tetraploid nature of white clover makes it difficult to identify allele dosage at a given SNP location using NGS techniques. To overcome these challenges, the Medicago truncatula genome was used to perform alignments and SNP calling was carried out using Freebayes variant caller with ploidy function activated. The phenotypic data were fitted in a mixed linear model using lme4 R package. The genome-wide association analyses were performed using a Q + K linear mixed model in the newly GWASpoly R package. All phenotypic traits showed significant genotypic effect and most showed a significant genotype × environment interaction (P<0.05). QTLs were identified for cold tolerance related traits (i.e leaf area, stolon dry matter (DM), spring DM, stolon water soluble carbohydrate, among other). Most of them were located in the chromosome two and seven of M. trucantula.