Identification of Loci Controlling Timing of Stem Elongation in Red Clover Using GBS of Pooled Phenotypic Extremes

Abstract

A population of red clover (Trifolium pratense L.), was characterized for timing of stem elongation. Of the 672 plants tested, 52 of the earliest and 52 of the latest were chosen for genetic characterization using genotyping by sequencing (GBS). The 52 individuals from each group were randomly divided into three subgroups and equal amounts of DNA from each individual in each subgroup was combined in a pool, creating a total of six pools, that were subject to GBS. Single nucleotide polymorphisms (SNPs) with allele frequencies that differed significantly between the early and the late group were identified by the use of pairwise F_ST-values for each SNP. At the highest stringency, we found 57 biallelic SNPs with significantly different allele frequencies in the early and the late groups, of which 37 could be mapped to a chromosome and the rest to unplaced scaffolds (P < 0.01, false discovery rate < 0.001). The 37 SNPs corresponded to 22 loci when defining a locus as a ±500 Kb region surrounding a SNP, an interval known to be associated with an almost complete decay of linkage disequilibrium in the studied population. Significant SNPs were distributed among all chromosomes, with the highest number on chromosome 6 and 7.