Whole-exome sequencing of selected bread wheat recombinant inbred lines as a useful resource for allele mining and bulked segregant analysis

Abstract

Although wheat (Triticum aestivum L.) is the main staple crop in the world and a major source of carbohydrates and proteins, functional genomics and allele mining are still big challenges. Given the advances in next-generation sequencing (NGS) technologies, the identification of causal variants associated with a target phenotype has become feasible. For these reasons, here, by combining sequence capture and target-enrichment methods with high-throughput NGS re-sequencing, we were able to scan at exome-wide level 46 randomly selected bread wheat individuals from a recombinant inbred line population and to identify and classify a large number of single nucleotide polymorphisms (SNPs). For technical validation of results, eight randomly selected SNPs were converted into Kompetitive Allele-Specific PCR (KASP) markers. This resource was established as an accessible and reusable molecular toolkit for allele data mining. The dataset we are making available could be exploited for novel studies on bread wheat genetics and as a foundation for starting breeding programs aimed at improving different key agronomic traits.

Keywords: bulked segregant analysis (BSA); exome capture; recombinant inbred lines; single nucleotide polymorphisms (SNPs); target-enrichment; wheat.

FIGURE 1

(A) Cumulative distribution of coverage depth across target region in 48 wheat individuals. The graph highlights the fraction of bases captured in the target regions covered at a depth ranging between 0x–400x. (B) Per-base depth of coverage for the TraesCS2B01G175300 gene in all individuals. The exon (yellow boxes) -intron (black lines) gene structure is shown above the multi-line graph.

FIGURE 2

Scatter plot for eight Kompetitive Allele-Specifc PCR (KASP) marker assays in 48 wheat varieties. KASP assays showing clustering of individuals on the X-(FAM) and Y-(HEX) axes. Red individuals have the HEX-type allele; blue individuals have the FAM-type allele. In both cases, individuals are homozygous for the reference or alternate allele. Green individuals are heterozygous for the allele. Black dots represent negative control and pink dots uncallable genotypes.