13,347 high-confidence SNPs were discovered through transcriptome sequencing of Aegilops tauschii, which are useful for genomic analysis and molecular breeding of hexaploid wheat. In organisms with large and complex genomes, such as wheat, RNA-seq analysis is cost-effective for discovery of genome-wide single nucleotide polymorphisms (SNPs). In this study, deep sequencing of the spike transcriptome from two Aegilops tauschii accessions representing two major lineages led to the discovery of 13,347 high-confidence (HC) SNPs in 4,872 contigs. After removing redundant SNPs detected in the leaf transcriptome from the same accessions in an earlier study, 10,589 new SNPs were discovered. In total, 5,642 out of 5,808 contigs with HC SNPs were assigned to the Ae. tauschii draft genome sequence. On average, 732 HC polymorphic contigs were mapped in silico to each Ae. tauschii chromosome. Based on the polymorphic data, we developed markers to target the short arm of chromosome 2D and validated the polymorphisms using 20 Ae. tauschii accessions. Of the 29 polymorphic markers, 28 were successfully mapped to 2DS in the diploid F2 population of Ae. tauschii. Among ten hexaploid wheat lines, which included wheat synthetics and common wheat cultivars, 25 of the 43 markers were polymorphic. In the hexaploid F2 population between a common wheat cultivar and a synthetic wheat line, 23 of the 25 polymorphic markers between the parents were available for genotyping of the F2 plants and 22 markers mapped to chromosome 2DS. These results indicate that molecular markers that developed from polymorphisms between two distinct lineages of Ae. tauschii might be useful for analysis not only of the diploid, but also of the hexaploid wheat genome.

Triploid wheat hybrids between tetraploid wheat and Aegilops tauschii sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat APETALA1-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes SPLs, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/SPLs module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.

Hybrid necrosis has been observed in many interspecific hybrids from crosses between tetraploid wheat and the wheat D-genome donor Aegilops tauschii. Type II necrosis is a kind of hybrid incompatibility that is specifically characterized by low-temperature induction and growth suppression. Two complementary genes, Net1 on the AB genome and Net2 on the D genome, putatively control type II necrosis in ABD triploids and synthetic hexaploid wheat. Toward map-based cloning of Net2, a fine map around the Net2 region on 2DS was constructed in this study. Using the draft genome sequence of Ae. tauschii and the physical map of the barley genome, the Net2 locus was mapped within a 0.6 cM interval between two closely linked markers. Although local chromosomal rearrangements were observed in the Net2-corresponding region between the barley/Brachypodium and Ae. tauschii genomes, the two closely linked markers were significantly associated with type II necrosis in Ae. tauschii. These results suggest that these markers will aid efficient selection of Net2 non-carrier individuals from the Ae. tauschii population and intraspecific progeny, and could help with introgression of agriculturally important genes from Ae. tauschii to common wheat.