High-Resolution Genotyping of Wild Barley Introgression Lines and Fine-Mapping of the Threshability Locus thresh-1 Using the Illumina GoldenGate Assay

Abstract

Genetically well-characterized mapping populations are a key tool for rapid and precise localization of quantitative trait loci (QTL) and subsequent identification of the underlying genes. In this study, a set of 73 introgression lines (S42ILs) originating from a cross between the spring barley cultivar Scarlett (Hordeum vulgare ssp. vulgare) and the wild barley accession ISR42-8 (H. v. ssp. spontaneum) was subjected to high-resolution genotyping with an Illumina 1536-SNP array. The array enabled a precise localization of the wild barley introgressions in the elite barley background. Based on 636 informative SNPs, the S42IL set represents 87.3% of the wild barley genome, where each line contains on average 3.3% of the donor genome. Furthermore, segregating high-resolution mapping populations (S42IL-HRs) were developed for 70 S42ILs in order to facilitate QTL fine-mapping and cloning. As a case study, we used the developed genetic resources to rapidly identify and fine-map the novel locus thresh-1 on chromosome 1H that controls grain threshability. Here, the recessive wild barley allele confers a difficult to thresh phenotype, suggesting that thresh-1 played an important role during barley domestication. Using a S42IL-HR population, thresh-1 was fine-mapped within a 4.3cM interval that was predicted to contain candidate genes involved in regulation of plant cell wall composition. The set of wild barley introgression lines and derived high-resolution populations are ideal tools to speed up the process of mapping and further dissecting QTL, which ultimately clears the way for isolating the genes behind QTL effects.

Keywords: Illumina GoldenGate assay; high-resolution mapping populations; threshability locus thresh-1; wild barley introgression lines.

Figure 1

Strategy for developing introgressions lines and high-resolution mapping populations from the wild barley cross Scarlett × ISR42-8. In order to select a set of introgression lines (S42ILs), a strategy combining backcrossing, repeated selfing, and marker-assisted selection (MAS) was applied. In the BC₂S_4:6 generation, the S42ILs have been characterized precisely on the genotype level using Illumina SNPs as described in the present study. Additionally, phenotype data were assessed for a subset of 39 S42ILs in order to validate QTL effects detected in the parental population S42 (Schmalenbach et al. 2008; Schmalenbach et al. 2009; Schmalenbach and Pillen 2009). High-resolution mapping populations (S42IL-HR) were finally developed by backcrossing the S42ILs with the recurrent parent followed by two selfings. They are available for future fine-mapping and cloning of interesting genes.

Figure 2

Graphical genotypes of 73 Hsp introgression lines (S42ILs). The 73 S42ILs, presented vertically, have been characterized with 636 Illumina SNPs, shown horizontally. The order of the S42ILs is in accordance with Table 1, and the marker order is taken from the consensus map of Close et al. (2009). Each S42IL carries one or several homozygous or heterozygous wild barley introgressions (depicted in black and dark gray, respectively) in the genetic background of the elite parent (in light gray). Missing marker data are indicated in white.

Figure 3

Phenotypes of the thresh-1 gene after mechanical threshing. Left image: Difficult to thresh grains from line S42IL-143, homozygous for the recessive Hsp allele thresh-1 where parts of the awns and the rachis remain attached to the grain. Right image: Easy to thresh grains from cultivar Scarlett, homozygous for the dominant Hv allele Thresh-1 without remnants from awns and rachis.

Figure 4

Fine mapping the thresh-1 locus using the high resolution mapping population S42IL-143HR. (A) Screening 49 S42ILs localized the thresh-1 gene to an Hsp introgression on chromosome 1H, which is present in line S42IL-143. (B) Graphical genotypes showing informative recombinants, identified among 91 BC₄S₂ individuals of the S42IL-143HR population, that were used to delimitate the thresh-1 interval based on their threshability phenotype. Lines with missing or ambiguous genotype data were excluded. The numbers below the graphical genotypes refer to the HR line depicted, while the numbers in brackets refer the total number of individuals identified with the same graphical genotype. Progeny tests from HR lines 2, 19, 88, and 89 proved that the elite (Hv) allele Thresh-1 is dominant over the exotic (Hsp) allele thresh-1.