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.

Summary• Common wheat is an allohexaploid species, derived through endoreduplication of an interspecific triploid hybrid produced from a cross between cultivated tetraploid wheat and the wild diploid relative Aegilops tauschii. Hybrid incompatibilities, including hybrid necrosis, have been observed in triploid wheat hybrids. A limited number of A. tauschii accessions show hybrid lethality in triploid hybrids crossed with tetraploid wheat as a result of developmental arrest at the early seedling stage, which is termed severe growth abortion (SGA). Despite the potential severity of this condition, the genetic mechanisms underlying SGA are not well understood.• Here, we conducted comparative analyses of gene expression profiles in crown tissues to characterize developmental arrest in triploid hybrids displaying SGA.• A number of defense-related genes were highly up-regulated, whereas many transcription factor genes, such as the KNOTTED1-type homeobox gene, which function in shoot apical meristem (SAM) and leaf primordia, were down-regulated in the crown tissues of SGA plants. Transcript accumulation levels of cell cycle-related genes were also markedly reduced in SGA plants, and no histone H4-expressing cells were observed in the SAM of SGA hybrid plants.• Our findings demonstrate that SGA shows unique features among other types of abnormal growth phenotypes, such as type II and III necrosis.

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F 2 individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.

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.

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