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

Abstract

Improvement of persistency is an important breeding goal in red clover (Trifolium pratense L.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.

Winter survival in red clover: experimental evidence for interactions among stresses

“…4 Principal component analysis of traits recorded in 110 accessions from the EUCLEG red clover panel, grouped into four regions of origin. Canopy height in late October and clover rot resistance expressed during the following winter was recorded in the Norwegian EUCLEG field experiment [ 23 , 27 ], clover rot resistance data are from Frey et al [ 24 ] and freezing tolerance data from Zanotto et al [ 28 ]. Correlation coefficients between traits are given in Supplementary Table 8A …”

“…Larger sets of the EUCLEG red clover collection have been phenotyped in several field experiments [ 27 ] and in experiments under controlled conditions, including phenotyping of clover rot resistance of non-cold acclimated plants [ 24 ] and freezing tolerance of cold acclimated plants [ 28 ]. In the Norwegian field experiment, including 110 of the EUCLEG accessions [ 23 ], shoot growth in the establishment year was recorded with a plate meter at five points per plot in late September and in late October, in 2018.…”

“…Trait variables for which there was a significant effect of population in experiment 1 and 2 were included in correlation and principal component analysis (PCA), including data on shoot growth in the establishment year (two time points as well as the difference between them, which represents late autumn growth) and clover rot resistance as measured in the Norwegian field experiment, as well as freezing tolerance data from Zanotto et al [ 28 ] and clover rot resistance data from Frey et al [ 24 ]. Pearson correlation coefficients were calculated with the CORR procedure in SAS and PCA was performed in MiniTab v21.3.…”

Genome-wide association study for yield-related traits in faba bean (Vicia faba L.)

“…Low LD in faba bean was previously reported ( Skovbjerg et al., 2023 ; Zhang et al., 2023 ) when comparing different faba bean diversity panels. Thus, the EUCLEG collection was the one showing lower LD blocks (higher recombination), an expected outcome in outbreeding species with high genetic diversity ( Pégard et al., 2023 ; Skovbjerg et al., 2023 ; Zanotto et al., 2023 ; Zhang et al., 2023 ). The rapid LD decay observed in this study (within roughly 150 kbp) revealed the variation present in a highly allogamous panmictic population and suggested that the MTAs identified (or the closely linked genes on either flanking side of the significant SNPs) were nearly or completely independent from each other and, hence, were sufficient for association mapping in faba bean.…”

“…Compared with family-based QTL mapping, GWAS significantly increases mapping resolution and enables minor effect genes to be detected ( Abdurakhmonov et al., 2008 ). In recent years, GWAS studies have been reported in a range of legume crops such as soybean ( Hwang et al., 2014 ), pigeon pea ( Varshney et al., 2017 ), common bean ( Raggi et al., 2019 ), chickpea ( Varshney et al., 2019 ), red clover ( Zanotto et al., 2023 ), alfalfa ( Pégard et al., 2023 ), and the model legume Medicago truncatula ( Bonhomme et al., 2014 ). In faba bean, only a few GWAS studies have been reported so far which have identified candidate genes associated with frost tolerance ( Sallam et al., 2016 ); resistance to Ascochyta fabae ( Faridi et al., 2021 ); tolerance to herbicides ( Abou-Khater et al., 2022 ); drought, heat, and freezing tolerance ( Ali et al., 2016 ; Maalouf et al., 2022 ; Gutiérrez et al., 2023 ); agronomic traits ( Skovbjerg et al., 2023 ); and seed size ( Jayakodi et al., 2023 ).…”