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The release of high-quality chromosome-level genome sequences of members of the Triticeae tribe has greatly facilitated genetic and genomic analyses of important crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Due to the large diploid genome size of Triticeae plants (ca. 5 Gbp), transcript analysis is an important method for identifying genetic and genomic differences among Triticeae species. In this review, we summarize our results of RNA-Seq analyses of diploid wheat accessions belonging to the genera Aegilops and Triticum. We also describe studies of the molecular relationships among these accessions and provide insight into the evolution of common hexaploid wheat. DNA markers based on polymorphisms within species can be used to map loci of interest. Even though the genome sequence of diploid Aegilops tauschii, the D-genome donor of common wheat, has been released, the diploid barley genome continues to provide key information about the physical structures of diploid wheat genomes. We describe how a series of RNA-Seq analyses of wheat relatives has helped uncover the structural and evolutionary features of genomic and genetic systems in wild and cultivated Triticeae species.

Dramatic changes occasionally occur in intergenic regions leading to genomic alterations during speciation and will consequently obscure the ancestral species that have contributed to the formation of allopolyploid organisms. The S genome of five species of section Sitopsis of genus Aegilops is considered to be an origin of B-genome in cultivated tetraploid and hexaploid wheat species, although its actual donor is still unclear. Here, we attempted to elucidate phylogenetic relationship among Sitopsis species by performing RNA sequencing of the coding regions of each chromosome. Thus, genome-wide polymorphisms were extensively analyzed in 19 accessions of the Sitopsis species in reference to the tetraploid and hexaploid wheat B genome sequences and consequently were efficiently anchored to the B-genome chromosomes. The results of our genome-wide exon sequencing and resultant phylogenetic analysis indicate that Ae. speltoides is likely to be the direct donor of all chromosomes of the wheat B genome. Our results also indicate that the genome differentiation during wheat allopolyploidization from S to B proceeds at different speeds over the chromosomes rather than at constant rate and recombination could be a factor determining the speed. This observation is potentially generalized to genome differentiation during plant allopolyploid evolution.

Due to large and complex genomes of Triticeae species, skim sequencing approaches have cost and analytical advantages for detecting genetic markers and building linkage maps. Here, we develop a high-density linkage map and identify quantitative trait loci (QTLs) for recombinant inbred lines of Aegilops tauschii, a D-genome donor of bread wheat, using the recently developed genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) system, which facilitates skimming of the large and complicated genome and generates a large number of genetic markers. The deduced linkage groups based on the GRAS-Di genetic markers corresponded to the chromosome number of Ae. tauschii. We successfully identified stable QTLs for flowering time and spikelet shape-related traits. Genotype differences of RILs at the QTL-linked markers were significantly associated with the trait variations. In particular, one of the QTL-linked markers for flowering time was mapped close to VRN3 (also known as FLOWERING LOCUS T), which controls flowering. The GRAS-Di system is, therefore, an efficient and useful application for genotyping and linkage mapping in species with large and complex genomes, such as Triticeae species.

Grain hardness is an important trait for improvement of wheat grain quality, and is mainly controlled by two puroindoline genes, Pina and Pinb, on chromosome 5D. The presence of functional alleles for both PINA and PINB proteins results in a soft grain texture. Here, we report nucleotide sequence variation and novel alleles of Pina and Pinb in a diploid wild wheat relative, Aegilops umbellulata Zhuk. Despite the presence of various alleles, mature grains of all Ae. umbellulata accessions examined had a hard texture. The hard-textured grains could be due to nonsynonymous substitutions in the Pina and Pinb alleles or lack of either PINA or PINB protein accumulation. Synthetic hexaploids with the AABBUU genome, derived from interspecific crosses between durum wheat and the Ae. umbellulata accessions, showed hard-textured grain character due to absent transmission of functional PINA and PINB proteins from the U-genome donors. In addition, increased thickness of the cell wall in the endosperm might contribute to the hard-texture in synthetic hexaploids. The U-genome addition to cultivated tetraploid wheat generally generated hard grains, suggesting that the Ae. umbellulata variation in grain-related traits will be useful for enlargement of grain hardness diversity in hard-textured common wheat.

The term “plasmon” is used to indicate the whole cytoplasmic genetic system, whereas “genome” refers to the whole nuclear genetic system. Although maternal inheritance of the plasmon is well documented in angiosperms, its genetic autonomy from the coexisting nuclear genome still awaits critical examination. We tested this autonomy in two related studies: One was to determine the persistence of the genetic effect of the plasmon of Aegilops caudata (genome CC) on the phenotype of common wheat, Triticum aestivum strain “Tve” (genome AABBDD), during 63 y (one generation per year) of repeated backcrosses of Ae. caudata and its offspring with pollen of the same Tve wheat, and the second was to reconstruct an Ae. caudata strain from the genome of this strain and its plasmon that had been resident in Tve wheat for 50 generations, and to compare the phenotypic and organellar DNA characteristics between the native and reconstructed strains. Results indicated no change in the effect of Ae. caudata plasmon on Tve wheat during its stay in wheat for more than half a century, and no difference between the native and reconstructed caudata strains in their phenotype and simple sequence repeats in their organellar DNAs, thus demonstrating the prolonged genetic autonomy of the plasmon from the coexisting genomes of wheat and several other species that were used in the reconstruction of Ae. caudata . The relationship between the proven genetic autonomy of the plasmon under changing nuclear conditions and its diversification during evolution of the Triticum – Aegilops complex is discussed.

Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O2. However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O2−) but also singlet oxygen (1O2) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O2− and 1O2 produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants.

Background Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, has been the source of trait improvement in wheat breeding. Intraspecific genetic variation of Ae. umbellulata, however, has not been well studied and the genomic information in this species is limited. Results To develop novel genetic markers distributed over all chromosomes of Ae. umbellulata and to evaluate its genetic diversity, we performed RNA sequencing of 12 representative accessions and reconstructed transcripts by de novo assembly of reads for each accession. A large number of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were obtained and anchored to the pseudomolecules of Ae. tauschii and barley (Hordeum vulgare L.), which were regarded as virtual chromosomes of Ae. umbellulata. Interestingly, genetic diversity in Ae. umbellulata was higher than in Ae. tauschii, despite the narrow habitat of Ae. umbellulata. Comparative analyses of nucleotide polymorphisms between Ae. umbellulata and Ae. tauschii revealed no clear lineage differentiation and existence of alleles with rarer frequencies predominantly in Ae. umbellulata, with patterns clearly distinct from those in Ae. tauschii. Conclusions The anchored SNPs, covering all chromosomes, provide sufficient genetic markers between Ae. umbellulata accessions. The alleles with rarer frequencies might be the main source of the high genetic diversity in Ae. umbellulata. Electronic supplementary material The online version of this article (10.1186/s12870-018-1498-8) contains supplementary material, which is available to authorized users.

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map.

Synthetic hexaploid wheat is an effective genetic resource for transferring agronomically important genes from Aegilops tauschii to common wheat. Wide variation in grain size and shape, one of the main targets for wheat breeding, has been observed among Ae. tauschii accessions. To identify the quantitative trait loci (QTLs) responsible for grain size and shape variation in the wheat D genome under a hexaploid genetic background, six parameters related to grain size and shape were measured using SmartGrain digital image software and QTL analysis was conducted using four F2 mapping populations of wheat synthetic hexaploids. In total, 18 QTLs for the six parameters were found on five of the seven D-genome chromosomes. The identified QTLs significantly contributed to the variation in grain size and shape among the synthetic wheat lines, implying that the D-genome QTLs might be at least partly functional in hexaploid wheat. Thus, synthetic wheat lines with diverse D genomes from Ae. tauschii are useful resources for the identification of agronomically important loci that function in hexaploid wheat.

Construction of high-resolution genetic maps is important for genetic and genomic research, as well as for molecular breeding. Single nucleotide polymorphisms (SNPs) are the predominant class of genetic variation and can be used as molecular markers. Aegilops tauschii, the D-genome donor of common wheat, is considered a valuable genetic resource for wheat improvement. Our previous study implied that Ae. tauschii accessions can be genealogically divided into two major lineages. In this study, the transcriptome of two Ae. tauschii accessions from each lineage, lineage 1 (L1) and 2 (L2), was sequenced, yielding 9435 SNPs and 739 insertion/deletion polymorphisms (indels) after de novo assembly of the reads. Based on 36 contig sequences, 31 SNPs and six indels were validated on 20 diverse Ae. tauschii accessions. Because almost all of the SNP markers were polymorphic between L1 and L2, and the D-genome donor of common wheat is presumed to belong to L2, these markers are available for D-genome typing in crosses between common wheat varieties and L1-derived synthetic wheat. Due to the conserved synteny between wheat and barley chromosomes, the high-density expressed sequence tag barley map and the hypothetical gene order in barley can be applied to develop markers on target chromosomal regions in 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 (&Delta, SNP-index) was higher on the target chromosome than on the other chromosomes. Several SNPs with the highest &Delta, 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.

The D-genome progenitor of hexaploid wheat, Aegilops tauschii Coss., has a wide natural species range in central Eurasia and possesses wide natural variation in heading and flowering time. Here, we report identification of two Ae. tauschii accessions insensitive to short day length. Similarly to a loss or reduced degree of vernalization requirement, the photoperiod-insensitive mutations were found only in the early flowering sublineage (TauL1b) of Ae. tauschii. Quantitative trait locus (QTL) analyses using two F2 mapping populations showed that a QTL for heading time on the long arm of chromosome 5D was related to the early heading phenotype of the photoperiod-insensitive accessions under short-day conditions. In the photoperiod-insensitive accession, expression patterns of two flowering-related genes were altered under short-day conditions compared with the patterns in photoperiod-sensitive accessions. This study indicates that analysis of natural variations in the Ae. tauschii population is useful to find novel genetic loci controlling agronomically important traits.

The wild wheat Aegilops tauschii Coss. has extensive natural variation available for breeding of common wheat. Drought stress tolerance is closely related to abscisic acid (ABA) sensitivity. In this study, 17 synthetic hexaploid wheat lines, produced by crossing the tetraploid wheat cultivar Langdon with 17 accessions of Ae. tauschii, were used for comparative analysis of natural variation in drought tolerance and ABA sensitivity. Ae. tauschii showed wide natural variation, with weak association between the traits. Drought-sensitive accessions of Ae. tauschii exhibited significantly less ABA sensitivity. D-genome variations observed at the diploid genome level were not necessarily reflected in synthetic wheats. However, synthetic wheats derived from the parental Ae. tauschii accessions with high drought tolerance were significantly more tolerant to drought stress than those from drought-sensitive accessions. Moreover, synthetic wheats with high drought tolerance showed significantly higher ABA sensitivity than drought-sensitive synthetic lines. In the hexaploid genetic background, therefore, weak association of ABA sensitivity with drought tolerance was observed. To study differences in gene expression patterns between stress-tolerant and -sensitive lines, levels of two Cor/Lea and three transcription factor gene transcripts were compared. The more tolerant accession of Ae. tauschii tended to accumulate more abundant transcripts of the examined genes than the sensitive accession under stress conditions. The expression patterns in the synthetic wheats seemed to be additive for parental lines exposed to drought and ABA treatments. However, the transcript levels of transcription factor genes in the synthetic wheats did not necessarily correspond to the postulated levels based on expression in parental lines. Allopolyploidization altered the expression levels of the stress-responsive genes in synthetic wheats.

ABA-responsive element binding protein (AREB) and ABA-responsive element binding factor (ABF), members of the basic region/leucine zipper (bZIP)-type protein family, act as major transcription factors in ABA-responsive gene expression under abiotic stress conditions in Arabidopsis. Barley HvABI5 and rice transcription factor responsible for ABA regulation 1 (TRAB1) are homologues of AREB/ABF and are expressed in drought- and ABA-treated seedlings. However, no direct evidence has shown an association of an AREB/ABF-type transcription factor with stress tolerance in cereals. To understand the molecular basis of abiotic stress tolerance through a cereal AREB/ABF-type transcription factor, a wheat HvABI5 ortholog, Wabi5, was isolated and characterized. Wabi5 expression was activated by low temperature, drought and exogenous ABA treatment, and its expression pattern differed between two wheat accessions with distinct levels of stress tolerance and ABA sensitivity. Wabi5-expressing transgenic tobacco plants showed a significant increase in tolerance to abiotic stresses such as freezing, osmotic and salt stresses and a hypersensitivity to exogenous ABA in the seedling stage compared with wild-type plants. Expression of a GUS reporter gene under the control of promoters of three wheat cold-responsive/late embryogenesis abundant (Cor/Lea) genes, Wdhn13, Wrab18 and Wrab19, was enhanced by ectopic Wabi5 expression in wheat callus and tobacco plants. These results clearly indicated that WABI5 functions as a transcriptional regulator of the Cor/Lea genes in multiple abiotic stress responses in common wheat.

Cold acclimation is an adaptive process for acquiring cold/freezing tolerance in wheat. To clarify the cultivar difference of freezing tolerance, we compared mitochondrial respiration activity and the expression profile of alternative oxidase (AOX) genes under low-temperature conditions using two common wheat cultivars differing in freezing tolerance. During cold acclimation, the respiration capacity of the alternative pathway significantly increased in a freezing-tolerant cultivar compared with a freezing-sensitive cultivar. More abundant accumulation of the AOX and uncoupling protein gene transcripts was also observed under the low-temperature conditions in the tolerant cultivar than in the sensitive cultivar. These results suggest that the mitochondrial alternative pathway might be partly associated with the cold acclimation and freezing tolerance in wheat.

The wheat (Triticum aestivum L.) CBF gene family is assumed to play important roles in development of low-temperature and freezing tolerance through activation of the downstream Cor/Lea genes. However, no direct evidence shows association of the wheat CBF genes with stress tolerance or any interaction between wheat CBF transcription factors and Cor/Lea gene activation. Here, we introduced Wcbf2, one of the wheat CBF genes, into the tobacco (Nicotiana tabacum L.) genome. Expression of Wcbf2 significantly increased the level of freezing tolerance in the transgenic tobacco plants without phenotypic retardation, and altered the expression patterns of tobacco genes, including cold-responsive genes. A transgenic tobacco plant expressing Wcbf2 was crossed to other transgenic plants expressing a GUS reporter gene under control of the wheat Cor/Lea gene promoter. Analysis of the F1 plants showed that the WCBF2 protein positively regulated at least the expression of Wdhn13 and Wrab17. These results strongly indicate that WCBF2 functions as a transcription factor in the development of freezing tolerance in common wheat.

Background A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. Results We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91 % of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87 % of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. Conclusions We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1803-y) contains supplementary material, which is available to authorized users.

Abscisic acid (ABA) plays important role in mediating stress responses and in acquiring desiccation tolerance and dormancy of plant seeds. To study roles of ABA in cold acclimation and freezing tolerance in wheat, expression profiles of Cor/Lea and their putative transcription factor (TF) genes were analysed using a dominant mutation line of common wheat EH47-1 lacking seed dormancy. The mutant line was less sensitive to exogenous ABA than the original line as judged by the magnitude of ABA inhibition of seedling growth. Expression analysis of Cor/Lea and TF genes however, showed that more transcripts were present in ABA-treated seedlings of the mutant line. In developing caryopses, the same tendency was observed. The mutant line showed no changes in the cold acclimation ability, but it showed a higher level of freezing tolerance than the original line without cold acclimation. No significant differences were observed in the expression profiles of Cor/Lea and TF genes during cold acclimation between the two lines. Our results imply the presence of an unknown ABA-dependent cold responsive pathway, which enhances the basal level of freezing tolerance by a dominant mutation in EH47-1.

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Vrn-1/Fr-1 chromosomal regions of common wheat possess major QTLs for both winter hardiness (Fr )a nd vernalization requirement (Vrn). The Vrn-1/Fr-1 intervals are assigned to long arms of the homoeologous group 5 chromosomes. To investigate the role of the Vrn-1/Fr-1 intervals on the low-temperature (LT) inducibility of wheat Cor/Lea genes and its putative transcription factor gene Wcbf2, LT response of these genes was monitored using near-isogenic lines (NILs) for the Vrn-1 loci. The Wcbf2 transcript accumulated rapidly after LT treatment and remained at a high level in lines without any dominant Vrn-1 alleles. By contrast, the Wcbf2 transcript level was greatly reduced in lines carrying the Vrn-1 alleles. The Vrn-1 NILs accumulated much lower amounts of Cor/Lea transcripts and COR/LEA proteins than the non-carrier line. The observed patterns and levels of gene expression, particularly in the Vrn-A1 NIL, agreed with the higher sensitivity to freezing damage in this line than in the noncarrier line. Up-regulation of the expression of the WAP1 gene, a candidate of the Vrn-1 loci, was much delayed in the non-carrier line than all the NILs carrying the Vrn-1 loci. Neither positive nor negative relationships were found between the WAP1 expression and the Cbf2/Cor/ Lea expression. These results support the intimate relationship between the Cbf2/Cor/Lea expression and the level of freezing tolerance, and suggest that a functional Fr-A1 allele linked to the vrn-A1 allele, instead of the vernalization gene itself, plays a major role in regulating the CBF-mediated Cor/lea gene expression in wheat.

Interaction between nucleus and cytoplasm has essential roles in plant development, including that of floral organs. We isolated a wheat homolog Whlp of Arabidopsis HUELLENLOS PARALOG (HLP) gene encoding a mitochondrial (mt) ribosomal protein L14. Transient expression analysis using the green fluorescent protein (GFP) fusion protein showed that 50 amino residues located on the N-terminal of the wheat HLP homolog (WHLP) protein acted as a mt-targeting signal (MTS). Expression patterns of the Whlp gene were compared among floral organs of alloplasmic lines of wheat, in which intrinsic cytoplasms were replaced by the cytoplasm of a wild relative Aegilops crassa. In these alloplasmic lines, pistillody (homeotic transformation of stamens into pistil-like organs) is induced by the alien cytoplasm in the absence of nuclear restorer genes. The Whlp transcripts preferentially accumulated in stamens compared with pistils, leaves, and roots. The expression level of Whlp in the pistillate stamens of the alloplasmic lines was similar to that in genuine pistils of both euplasmic lines and fertile alloplasmic lines. The result suggested that the elevated expression of the Whlp gene plays a role in aiding the development of male reproductive organ but not in the determination of its whorl identity. A comparable expression pattern was observed in another nuclear-encoded mt ribosomal protein gene but not in a mt-encoded gene. The different expression patterns of different mt ribosomal protein genes suggest that the abundance of mt ribosomal proteins is differentially regulated in the organ/tissue development in wheat.

The ability to cause reproductive isolation often varies among individuals within a plant species. We addressed whether such polymorphism influenced speciation of the allopolyploid common wheat (Triticum aestivum L., AABBDD genome) by evaluating the expression of pre-pollination (outcrossing potential) and post-pollination (crossability) barriers in Aegilops tauschii Coss. (the D genome progenitor). In total, 201 Ae. tauschii accessions representing the entire natural habitat range of the species were used for anther length measurement and artificial crosses with a Triticum turgidum L. (the AB genome progenitor) tester. Intraspecific comparisons showed that both barriers were more strongly expressed in the TauL1 lineage than in the TauL2 lineage. The ability of Ae. tauschii to cause reproductive isolation in the hybridisation with T. turgidum might have markedly influenced common wheat’s speciation by inducing lineage-associated patterns of gene flow. The TauL2 accessions with high potential for natural hybridisation with T. turgidum clustered in the southern coastal Caspian region. This provided phenotypic support for the derivation of the D genome of common wheat from southern Caspian populations. The present study underscored the importance of approaches that incorporate the genealogical and geographic structure of the parental species’ reproductive isolation in understanding the mechanism of plant allopolyploid speciation.

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.

The awn is a long needle-like structure formed at the tip of the lemma in the florets of some grass species. It plays a role in seed dispersal and protection against animals, and can contribute to the photosynthetic activity of spikes. Three main dominant inhibitors of awn development (Hd, B1 and B2) are known in hexaploid wheat, but the causal genes have not been cloned yet and a genetic association with awn length diversity has been found only for the B1 allele. To analyze the prevalence of these three awning inhibitors, we attempted to predict the genotypes of 189 hexaploid wheat varieties collected worldwide using markers tightly linked to these loci. Using recombinant inbred lines derived from two common wheat cultivars, Chinese Spring and Mironovskaya 808, both with short awns, and a high-density linkage map, we performed quantitative trait locus analysis to identify tightly linked markers. Because this linkage map was constructed with abundant array-based markers, we converted the linked markers to PCR-based markers and determined the genotypes of 189 hexaploids. A significant genotype-phenotype correlation was observed at the Hd and B1 regions. We also found that interaction among these three awning inhibitors is involved in development of a membranous outgrowth at the base of awn resembling the Hooded mutants of barley. For the hooded awn phenotype, presence of the Hd dominant allele was essential but not sufficient, so B2 and other factors appear to act epistatically to produce the ectopic tissue. On the other hand, the dominant B1 allele acted as a suppressor of the hooded phenotype. These three awning inhibitors largely contribute to the genetic variation in awn length and shape of common wheat.

The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F1 hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T . turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae . tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae . tauschii ’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.

A mitochondrial (mt) transcriptional unit, nad3-orf156, was studied in the nucleus-cytoplasm hybrids of wheat with D/D2 plasmons from Aegilops species and their parental lines. A comparative RFLP analysis and sequencing of the random PCR clones revealed the presence of seven sequence types and their polymorphic sites were mapped. All the hybrids possessed the paternal copies besides the maternal copies. More paternal copies were present in the D2 plasmon hybrids, whereas more maternal copies were present in the D plasmon hybrids. Two major copies were present with different stoichiometries in the maternal Aegilops parents. However, only a major D plasmon copy was detected in the hybrids, irrespective of their plasmon types. The hexaploid wheat parent (AABBDD genome) possessed the major D plasmon copy in ~5% stoichiometry, while no D plasmon-homologous copies were detected in the tetraploid wheat parent (AABB genome). The results suggest that the observed mtDNA heteroplasmy is due to paternal contribution of mtDNA. The different copy stoichiometry suggests differential amplification of the heteroplasmic copies among the hybrids and the parental lines. All editing sites and their editing frequencies were conserved among the lines, and only the maternal pattern of editing occurred in the hybrids.

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100–115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.