Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

Abstract

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.

Fig. 1.

Investigation of genetic substructure. (A) Principle component analysis of UK cultivars and barley progenitor lines (n = 620). Phenotypic combinations for row number (2 = two-row, 6 = six-row) and seasonal growth habit (S, spring; W, winter) are indicated. Varietal membership to the 10 subpopulations identified using STRUCTURE are overlaid. (B) Decay of pair-wise marker LD over increasing genetic distance (cM) before (Left) and after (Right) correction using the mixed linear model. Off-chromosome comparisons are shown at 200 cM. The Bonferroni corrected P = 0.05 significance threshold is indicated. (C) Naïve and corrected GWA analysis of seasonal growth habit illustrating the extent of confounding present. Arrow indicates the significance threshold. (D) Predicted experimental power to detect a trait controlled by 1, 2, and 10 loci (h² = 0.9) over genetic distance (±cM). Power is measured as the proportion of simulations in which at least one causative locus was detected (q value ≤ 0.1). Error bars denote ±1 SE.

Fig. 2.

GWA scans of the 15 traits with significant associations (P < 0.05, Bonferroni corrected; indicated by a dashed line). Barley chromosomes 1H to 7H are shown.

Fig. 3.

Fine-mapping of ANT2. (A) Example of anthocyanin expression in tissues from selected “red” (Retriever) and “white” (Saffron) varieties. (B) The sequenced physical map of the barley ANT2 region aligned to colinear regions of rice, sorghum, and Brachypodium. Barley predicted genes: Expressed Protein 1 (EP1), Acyl-CoA thioesterase (ACA), Conserved Hypothetical Protein (CHP), Basic Helix-loop-helix protein 1 (HvbHLH1), Genetic Modifier (GM), Expressed Protein 2 (EP2), No Apical Meristem (NAM), and Cis-Zeatin O-Glucosyltransferase (Cis-ZOG). Barley BAC clones from left to right are 274B17, 739E22, and 77O02 (GenBank accession HM163343). The positions of BAC end sequences and the transposon-based marker 739E22-1 are indicated by black and green circles, respectively. (C) Haplotype frequencies in “red” and “white” varieties. Awn coloration is illustrated. (D) GWA scan for the trait anthocyanin expression per se. The two peak markers are highlighted in red. (E) Structure of the ANT2 candidate gene, HvbHLH1. Exons are denoted by black rectangles; the region encoding the predicted bHLH domain is highlighted in blue. The position of the premature stop codon in the white allele is indicated by an asterisk. (F) Partial DNA sequence of HvbHLH1 exon 6 illustrating the position of the 16-bp InDel. The 10-bp repeat sequences flanking the deletion are underlined; the probability (p) that these locations are by chance is indicated.