Genome-wide association mapping of frost tolerance in barley (Hordeum vulgare L.)

Abstract

Background: Frost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola (Italy) and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure.

Results: GWAS analyses identified twelve and seven positive SNP associations at Foradada and Fiorenzuola, respectively, using Eigenstrat and six and four, respectively, using kinship. Linkage disequilibrium analyses of the significant SNP associations showed they are genetically independent. In the kinship analysis, two of the significant SNP associations were tightly linked to the Fr-H2 and HvBmy loci on chromosomes 5H and 4HL, respectively. The other significant kinship associations were located in genomic regions that have not previously been associated with cold stress.

Conclusions: Haplotype analysis revealed that most of the significant SNP loci are fixed in the winter or facultative types, while they are freely segregating within the un-adapted spring barley genepool. Although there is a major interest in detecting new variation to improve frost tolerance of available winter and facultative types, from a GWAS perspective, working within the un-adapted spring germplasm pool is an attractive alternative strategy which would minimize statistical issues, simplify the interpretation of the data and identify phenology independent genetic determinants of frost tolerance.

Figure 1

Frost episodes in Foradada (Spain, winter 2004/05) and Fiorenzuola (Italy, winter 2007/08). a. Daily maximum and minimum temperature from sowing to the end of March when cold tolerance was scored. The average mean minimum temperatures were −2.7°C and −1.1°C for Foradada (i) and Fiorenzuola (ii) trials respectively, while the absolute lowest temperatures were −12.7°C and −8.6°C for Foradada and Fiorenzuola trials respectively. An alternation of freezing and thaw periods was observed. b. Differential frost damage in winter barley plots (Foradada, Spain spring 2005). Cold injury was visually estimated on a 0–5 scale: 0. all plants killed; 1. whole plants yellowed and 50% of plant mortality; 2. whole plants yellowed and 20% of plant mortality; 3. fully yellowed basal leaves; 4. half yellowed basal leaves; 5. no damage.

Figure 2

Manhattan plots for frost tolerance in barley in the Foradada (Spain) location where the frost episode was long and severe (A) and in the Fiorenzuola (Italy) location where accessions experienced less severe cold conditions (B). (i) ”Uncorrected” naive analysis. (ii) EIGENSTRAT and (iii) Kinship analysis. The -log₁₀(p-values) from a genome-wide scan are plotted against the position on each of the 7 barley chromosomes. The horizontal line indicates the genome-wide significance threshold (−log₁₀p ≥ 3). Three amongst the top ‘uncorrected’ hits fail to reach the significance threshold in the kinship analysis despite being genetically close to FLT-2L-linked ’frost sensitivity in reproductive tissues’ locus (downward purple arrow), ICE2 regulatory gene (downward green arow) and the Fr-H1 locus (downward red arow), known to be involved with cold tolerance. (red circle) Association hits previously reported in the literature [30]. (down ward light blue arrow) Top hit in the ‘uncorrected’ analysis also significant in the kinship analysis.

Figure 3

Linkage Disequilibrium (LD) amongst significant SNP markers. HAPLOVIEW v.4.2 (http://www.broadinstitute.org/haploview/haploview) pairwise LD values (r²*100) for 22 SNPs based on genotypes of 184 individuals were used to test whether all the SNPs significantly associated with frost tolerance were in strong LD with each other.

Figure 4

QTL haplotype analysis across germplasm clusters. (1) Left figure: Neighbour joining tree of the selected 184 barley cultivars constructed from simple matching distance of 1,307 SNP markers. Lines are coloured according to population structure clusters described in Comadran et al. 2009: a. Northern European springs; b. Turkish; c. Syrian and Jordan; d. South-West Mediterranean accessions; e. Northern European winters. (2) Right figure: QTL and QTL haplotype frequencies within population structure clusters for the eight significant SNPs detected with the kinship analysis in the Foradada and / or the Fiorenzuola field trials (Table 1). Reported allele frequencies correspond to the SNP allele increasing winter survival (blue). The alternative allele is shown in red.