Identification of loci controlling timing of stem elongation in red clover using genotyping by sequencing of pooled phenotypic extremes

Abstract

Main conclusion Through selective genotyping of pooled phenotypic extremes, we identified a number of loci and candidate genes putatively controlling timing of stem elongation in red clover. Abstract We have identified candidate genes controlling the timing of stem elongation prior to flowering in red clover (Trifolium pratense L.). This trait is of ecological and agronomic significance, as it affects fitness, competitivity, climate adaptation, forage and seed yield, and forage quality. We genotyped replicate pools of phenotypically extreme individuals (early and late-elongating) within cultivar Lea using genotyping-by-sequencing in pools (pool-GBS). After calling and filtering SNPs and GBS locus haplotype polymorphisms, we estimated allele frequencies and searched for markers with significantly different allele frequencies in the two phenotypic groups using BayeScan, an FST-based test utilizing replicate pools, and a test based on error variance of replicate pools. Of the three methods, BayeScan was the least stringent, and the error variance-based test the most stringent. Fifteen significant markers were identified in common by all three tests. The candidate genes flanking the markers include genes with potential roles in the vernalization, autonomous, and photoperiod regulation of floral transition, hormonal regulation of stem elongation, and cell growth. These results provide a first insight into the potential genes and mechanisms controlling transition to stem elongation in a perennial legume, which lays a foundation for further functional studies of the genetic determinants regulating this important trait.

A genome-wide association study of freezing tolerance in red clover (Trifolium pratense L.) germplasm of European origin

“…Red clover is known to be a genetically and phenotypically diverse crop ( Nay et al., 2023 ). Ergon et al. (2022) , studying GBS loci of a single cultivar, found an average of 3.6 haplotype variants per HTP, both when Pst I and ApeK I were used as restriction enzymes.…”

“…Haplotype variants within GBS loci were called and their relative frequencies estimated with the SMAP package (Schaumont et al, 2022) as explained in Ergon et al (2022), using the SNP data set achieved when removing only SNPs with more than 20% missing data as input. In the process, haplotype variants with an overall MAF < 0.05 were discarded.…”

“…Therefore, it is important to use GWAS models that can account for this in order to reduce the risk of identifying false positive associations ( Liu et al., 2016 ). In GWAS analysis, haplotype data can be expected to complement single nucleotide polymorphism (SNP) data, as haplotypes may reveal associations that are not detectable when using SNP data only ( Hamblin and Jannink, 2011 ; Bekele et al., 2018 ; Ergon et al., 2022 ).…”

Including marker x environment interactions improves genomic prediction in red clover (Trifolium pratense L.)

“…Pooled genotyping has been used in red clover to identify regions under selection for survival in the field ( Ergon et al., 2019 ), and for genome-wide identification of loci involved in timing of stem elongation and freezing tolerance ( Ergon et al., 2022 ; Zanotto et al., 2023 ). Allele frequency data from pooled samples compared well with those obtained by genotyping individuals from the same population ( Ergon et al., 2019 ).…”

DataSheet_1.zip

“…Pooled genotyping has been used in red clover to identify regions under selection for survival in the field (Ergon et al, 2019), and for genome-wide identification of loci involved in timing of stem elongation and freezing tolerance (Ergon et al, 2022;Zanotto et al, 2023). Allele frequency data from pooled samples compared well with those obtained by genotyping individuals from the same population (Ergon et al, 2019).…”