RNA sequencing-based bulked segregant analysis facilitates efficient D-genome marker development for a specific chromosomal region of synthetic hexaploid wheat

Common wheat originated from interspecific hybridization between cultivated tetraploid wheat and its wild diploid relative Aegilops tauschii followed by amphidiploidization. This evolutionary process can be reproduced artificially, resulting in synthetic hexaploid wheat lines. Here we performed RNA sequencing (RNA-seq)-based bulked segregant analysis (BSA) using a bi-parental mapping population of two synthetic hexaploid wheat lines that shared identical A and B genomes but included with D-genomes of distinct origins. This analysis permitted identification of D-genome-specific polymorphisms around the Net2 gene, a causative locus to hybrid necrosis. The resulting single nucleotide polymorphisms (SNPs) were classified into homoeologous polymorphisms and D-genome allelic variations, based on the RNA-seq results of a parental tetraploid and two Ae. tauschii accessions. The difference in allele frequency at the D-genome-specific SNP sites between the contrasting bulks (∆SNP-index) was higher on the target chromosome than on the other chromosomes. Several SNPs with the highest ∆SNP-indices were converted into molecular markers and assigned to the Net2 chromosomal region. These results indicated that RNA-seq-based BSA can be applied efficiently to a synthetic hexaploid wheat population to permit molecular marker development in a specific chromosomal region of the D genome.