RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat.

Ryo Nishijima,Shigeo Takumi,Kazuhiro Sato,Kohei Sakaguchi,Kentaro Yoshida,Shin-Ichi Yoshimura

doi:10.3390/ijms19123749

Abstract

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 (ΔSNP-index) was higher on the target chromosome than on the other chromosomes. Several SNPs with the highest ΔSNP-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.

Highlights

Wild relatives of common wheat (Triticum aestivum L.), including Aegilops species, constitute important genetic resources for common wheat breeding
The Ae. tauschii strains associated with the origin of common wheat are assumed to be the TauL2 lineage [2,17], and only limited reproductive barriers are thought to exist between tetraploid wheat and many of the TauL2 accessions [19]
Two synthetic hexaploid wheat lines were derived from interspecies crosses of tetraploid wheat cultivar Langdon (Ldn) and two Ae. tauschii accessions (KU-2075 and KU-2025)

Summary

Introduction

Wild relatives of common wheat (Triticum aestivum L.), including Aegilops species, constitute important genetic resources for common wheat breeding. The SNP- and indel-based markers are available for construction of linkage maps in the target chromosomal regions of Ae. tauschii and the D genome of hexaploid wheat including synthetic wheat [26,27]. RNA-seq-based BSA was employed successfully for the development of molecular markers closely linked to target chromosomal genes such as a grain protein content gene (GPC-B1), a yellow rust resistance gene (Yr15), and a powdery mildew resistance gene (Pm4b) in tetraploid and common wheat [36,37,38]. We employed the RNA-seq-based BSA method to develop a molecular marker closely linked to Net2 This process used a mapping population generated from a cross of two synthetic hexaploid wheat lines that shared identical A and B genomes but contained diverse D genomes originating from two distinct pollen parents

Results and Discussion

Materials and Methods

Molecular Marker Development and Genotyping