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Stevia rebaudiana (Bertoni) Bertoni is a widely grown species in various regions of the world, mainly due to its sweetening properties attributed to steviol glycosides. Despite the fact that Paraguay is the center of origin of S. rebaudiana, genetic characterization of local genotypes has not been studied. Considering that accumulation of steviol glycosides is a complex trait, molecular breeding could be an effective method for its improvement. Therefore, the identification of molecular markers associated with steviol glycoside accumulation is important. The aim of this study was to assess the genetic relationship among eight advanced lines of S. rebaudiana and two varieties selected from local germoplasm and quantify their steviol glycoside content. The stevioside and rebaudioside A content in methanolic extract was determined by high-performance liquid chromatography (HPLC), and the molecular characterization of the ten genotypes of S. rebaudiana was performed using SSR and ISSR markers. A wide range of variability was observed for the metabolites of interest under greenhouse conditions. The variety Eirete presented highest stevioside and rebausiode A content, followed by the variety Katupyry. One of the advanced line presented both metabolite content similar to that of Katupyry. Genotyping of these lines showed two main clusters. Cluster I contained the both varieties and advanced lines with higher stevioside and rebaudioside A content. Our results suggest that S. rebaudiana lines with genetic profile similar to that of cluster I, determined mainly by genotype at the markers SUGMS 35 and ISSR 47, might present higher stevioside and rebaudioside A content.

Corn is one of the most important economic, social and cultural agricultural products in Paraguay, being the main ingredient of several typical foods. Due to the cross-pollination of this plant, it can be contaminated with transgenic maize pollen found in nearby fields. The general objective of this research was to evaluate the presence of transgenic sequences in corn grains marketed in the Departments of Itapúa, Alto Paraná and Canindeyú for the preparation of flour for human consumption. We studied 18 samples of corn kernels marketed for the preparation of flour from the Departments of Itapúa, Alto Paraná and Canindeyú. The DNA was extracted using the Trimethyl Ammonium Bromide (CTAB) protocol. For the detection of the transgenic sequences, the polymerase chain reaction (PCR) method was used. The bands were analyzed visually and using the ImageJ program. The results showed the presence of 5 positive samples for the P-35S-CaMv promoter and 5 samples for the T-nos terminator, none of them presented both sequences sought in the same construction; this indicates that 10 of 18 samples were contaminated with transgenic sequences. These findings show that transgenic products are available in Paraguayan local markets without consumers being aware of this situation.

Alternative splicing produces multiple transcripts from a single gene, and RNA sequencing using next-generation sequencing apparatus has contributed to comprehensive detection of splicing variants in higher plants. Here, we completed a large-scale survey of alternatively spliced transcripts from RNA samples derived from seedling leaves and young spikes of two distinct accessions of wild diploid wheat Aegilops tauschii under normal growth conditions. Alternative splicing for 23,778 loci were analyzed in this study, and at least two splicing variants were found in 4712 genes. The proportion of events for intron retention was much smaller in Ae. tauschii than in other plant species, whereas the proportions of exon skipping and splice donor site events were higher. In at least some genes, alternative splicing patterns were clearly distinct between the two Ae. tauschii accessions. Distinct splicing patterns were transmitted from the parental Ae. tauschii accessions into their synthetic hexaploid wheat lines. Alternative splicing patterns in one of the two synthetic hexaploid wheat lines analyzed appeared to be generally similar to patterns intermediate between the parental lines under normal growth conditions, and seemed to be slightly altered in the other synthetic wheat.

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.

Hybrid necrosis is a well-known reproductive isolation mechanism in plant species, and an autoimmune response is generally considered to trigger hybrid necrosis through epistatic interaction between disease resistance-related genes in hybrids. In common wheat, the complementary Ne1 and Ne2 genes control hybrid necrosis, defined as type I necrosis. Two other types of hybrid necrosis (type II and type III) have been observed in interspecific hybrids between tetraploid wheat and Aegilops tauschii. Another type of hybrid necrosis, defined here as type IV necrosis, has been reported in F1 hybrids between Triticum urartu and some accessions of Triticum monococcum ssp. aegilopoides. In types I, III and IV, cell death occurs gradually starting in older tissues, whereas type II necrosis symptoms occur only under low temperature. To compare comprehensive gene expression patterns of hybrids showing growth abnormalities, transcriptome analysis of type I and type IV necrosis was performed using a wheat 38k oligo-DNA microarray. Defense-related genes including many WRKY transcription factor genes were dramatically up-regulated in plants showing type I and type IV necrosis, similarly to other known hybrid abnormalities, suggesting an association with an autoimmune response. Reactive oxygen species generation and necrotic cell death were effectively inhibited by ZnCl2 treatment in types I, III and IV necrosis, suggesting a significant association of Ca(2+) influx in upstream signaling of necrotic cell death in wheat hybrid necrosis.

The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6–8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31–43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1–6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31–43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides., The Spanish Ministry of Economy and Competitiveness (Project AGL2013-48946-C3-1-R), the European Regional Development Fund (FEDER), and Junta de Andalucía (Project P11-AGR-7920) supported this work.

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.

The phytohormone abscisic acid (ABA) plays important roles in response to environmental stress as well as in seed maturation and dormancy. In common wheat, quantitative trait loci (QTLs) for ABA responsiveness at the seedling stage have been reported on chromosomes 1B, 2A, 3A, 6D and 7B. In this study, we identified a novel QTL for ABA responsiveness on chromosome 5A using an F2 population derived from a cross between the common wheat cultivar Chinese Spring (CS) and a chromosome substitution line of CS with chromosome 5A of cultivar Hope (Hope5A). This QTL was found in a similar chromosomal region to previously reported QTLs for drought tolerance and seed dormancy. Physiological characterization of the QTL revealed a small effect on dehydration tolerance and seed dormancy. The rate of water loss from leaves during dehydration was lower, and transcript accumulation of the cold responsive (COR)/late embryogenesis abundant (LEA) genes Wrab18 and Wdhn13 tended to be higher under dehydration stress in F2 individuals carrying the Hope allele of the QTL, which also showed higher ABA responsiveness than the CS allele-carrying individuals. Seed dormancy of individuals carrying the Hope allele also tended to be lower than those carrying the CS allele. Our results suggest that variation in ABA responsiveness among common wheat cultivars is at least partly determined by the 5A QTL, and that this QTL contributes to development of dehydration and preharvest sprouting tolerance.

Fusarium head blight (FHB) is a devastating fungal disease of small grain cereals. In wheat, the disease is caused mainly by Fusarium graminearum, but other species may occur such as F. poae, which is able to produce dangerous mycotoxins such as HT-2 toxin, T-2 toxin, nivalenol, and deoxynivalenol (Stenglein et al. 2014). During the months of August and September, between 2013 and 2016, 10 random spikes (100% blighted and nonblighted) each were collected from two cultivars, Itapua 75 and E08075, grown in the Paraguayan Institute of Agricultural Research (IPTA) at Capitan Miranda, Itapua. Randomly selected kernels were surface-sterilized by immersion for 3 min in 50% ethanol and then for 3 min in hypochlorite (6%) and were subsequently washed with sterilized water. The kernels were deposited in Petri dishes containing potato dextrose agar (PDA) and incubated for 7 days at 22 ± 2°C under a 12-h photoperiod. Thirteen of over 450 isolates, representing less than 3%, emitted typical Fusarium poae odor. Of these, the two most representative monosporic strains, named as FP07 and FP013, originating from different spikes, developed a dense, whitish mycelium in PDA at 72 to 96 h. Microconidia were abundant, globose to piriform, zero to one septate, 4 to 10 × 4.5 to 7 μm, and formed on unbranched and branched monophialides. Cultures produced a fruity aroma, in accordance with Leslie and Summerell (2006). The identity of the fungus was confirmed by sequencing a portion of the EF1-α gene using the degenerate primers EF-1 (5′-ATGGGTAAGGAGGACAAGAC-3′) and EF-2 (5′-GGAAGTACCAGTGATCATGTT-3′) (O’Donnell et al. 1998). BLASTn searches of the obtained sequences FP07 (accession no. MG976797) and FP013 (accession no. MG976798) showed a 99% homology with gene sequences of F. poae Schw. in GenBank: accession EU744740 to FP07 and accession MG889924 to FP013. Koch’s postulates were completed for the two isolates under greenhouse conditions. Pathogenicity tests were conducted by drop-inoculating healthy spikelets with 5 ml of microconidia suspension (10⁶ spores/ml) (Stenglein et al. 2014). Control treatment consisted of heads inoculated only with water. After inoculation, plants were placed in a growth chamber with a 12-h photoperiod at 22 ± 2°C and covered with polyethylene bags that were removed after 3 days. Two weeks after inoculations, all spikes of the control treatment were healthy, but the spikes with FP07 and FP013 showed 20 to 30% spike infection; a lower infection rate than the one seen in spikes infected with F. graminearum strains. The fungus was reisolated from infected spikes according to the methods described above and appeared to be morphologically identical to the isolates used to inoculate the plants. This is the first report of F. poae associated with FHB symptoms in wheat in Paraguay, which is considered important owing to its association with nivalenol production.

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.

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.

The gene pool of Aegilops tauschii, the D-genome donor of common wheat (Triticum aestivum L.), can be easily accessed in wheat breeding, but remains largely unexplored. In our previous studies, many synthetic hexaploid wheat lines were produced through interspecific crosses between the tetraploid wheat cultivar Langdon and various A. tauschii accessions. The synthetic hexaploid wheat lines showed wide variation in many characteristics. To elucidate the genetic basis of variation in flowering-related traits, we analyzed quantitative trait loci (QTL) affecting time to heading, flowering and maturity, and the grain-filling period using four different F2 populations of synthetic hexaploid wheat lines. In total, 10 QTLs located on six D-genome chromosomes (all except 4D) were detected for the analyzed traits. The QTL on 1DL controlling heading time appeared to correspond to a flowering time QTL, previously considered to be an ortholog of Eps-A m 1 which is related to the narrow-sense earliness in einkorn wheat. The 5D QTL for heading time might be a novel locus associated with wheat flowering, while the 2DS QTL appears to be an allelic variant of the photoperiod response locus Ppd-D1. Some of the identified QTLs seemed to be novel loci regulating wheat flowering and maturation, including a QTL controlling the grain filling period on chromosome 3D. The exercise demonstrates that synthetic wheat lines can be useful for the identification of new, agriculturally important loci that can be transferred to, and used for the modification of flowering and grain maturation in hexaploid wheat.

Low temperature induces expression of Cor (cold-responsive)/Lea (late embryogenesis-abundant) gene family members through C-repeat binding factor (CBF) transcription factors in common wheat. However, the relationship between the genetic loci controlling cold-responsive gene expression and freezing tolerance is unclear. In expression quantitative trait locus (eQTL) analysis, accumulated transcripts of Cor/Lea and CBF genes were quantified in recombinant inbred lines derived from a cross between two common wheat cultivars with different levels of freezing tolerance. Four eQTLs controlling five cold-responsive genes were found, and the major eQTL with the greatest effect was located on the long arm of chromosome 5A. At least the 1D and 5A eQTLs played important roles in development of freezing tolerance in common wheat. The chromosomal location of the 5A eQTL, controlling four cold-responsive genes, coincided with a region homoeologous to a frost-tolerance locus (Fr-A m 2) reported as a CBF cluster region in einkorn wheat. The 5A eQTL plays a significant role through Cor/Lea gene expression in cold acclimation of wheat. In addition, our results suggest that one or more CBF copies at the Fr-2 region positively regulate other copies, which might amplify the positive effects of the CBF cluster on downstream Cor/Lea gene activation.

Wheat landraces carry abundant genetic variation in heading and flowering times. Here, we studied flowering-related traits of two Nepalese varieties, KU-4770 and KU-180 and a Japanese wheat cultivar, Shiroganekomugi (SGK). These three wheat varieties showed similar flowering time in a common garden experiment. In total, five significant quantitative trait loci (QTLs) for three examined traits, the heading, flowering and maturation times, were detected using an F2 population of SGK/KU-4770. The QTLs were found at the Ppd-1 loci on chromosomes 2B and 2D and the 2B QTL was also confirmed in another F2 population of SGK/KU-180. The Ppd-D1 allele from SGK and the Ppd-B1 alleles from the two Nepalese varieties might be causal for early-flowering phenotype. The SGK Ppd-D1 allele contained a 2-kb deletion in the 5' upstream region, indicating a photoperiod-insensitive Ppd-D1a allele. Real-time PCR analysis estimating the Ppd-B1 copy number revealed that the two Nepalese varieties included two intact Ppd-B1 copies, putatively resulting in photoperiod insensitivity and an early-flowering phenotype. The two photoperiod-insensitive Ppd-1 homoeoalleles could independently contribute to segregation of early-flowering individuals in the two F2 populations. Therefore, wheat landraces are genetic resources for discovery of alleles useful for improving wheat heading or flowering times.

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α-, γ-, ω-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ- and α-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω-gliadins and γ-gliadins, and three of these also silenced α-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α- and ω-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars., The Spanish Ministry of Economy and Competitiveness (Project AGL2013-48946-C3-1-R), the European Regional Development Fund (FEDER) and Junta de Andalucía (Project P11-AGR-7920) supported this work.

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 (Triticum aestivum L.) is an allohexaploid that originated from natural hybridization between tetraploid wheat (Triticum turgidum) and diploid Aegilops tauschii. Ae. tauschii is considered one of the potential sources of new genetic variation in abiotic stress tolerance for improving common wheat. Abscisic acid (ABA) plays an important role in plant adaptation to environmental stresses. In this study, ABA responsiveness of 67 Ae. tauschii accessions and their synthetic hexaploid wheat lines, derived from crosses between T. turgidum cv. Langdon and the Ae. tauschii accessions, was evaluated based on growth inhibition by 20 µM ABA. Wide variation was found in ABA responsiveness for both synthetic wheat lines and their parental Ae. tauschii accessions. The variations due to D-genome found at the diploid level were also expressed in a hexaploid genetic background. Two pairs of synthetic wheat lines differing in ABA responsiveness were then selected for gene expression analysis and to test abiotic stress tolerance, because their parental Ae. tauschii accessions similarly exhibited the differential response to ABA. Gene expression of ABA inducible transcription factor, WABI5, and the downstream Cor/Lea genes (Wrab17, Wdhn13 and Wrab18) were analysed. In one pair, the highly responsive line exhibited higher induction of Wrab17 by ABA treatment, but no significant difference in dehydration or salinity tolerance was observed between these lines. In contrast, in the second pair, the highly ABA-responsive line showed higher levels of Wdhn13 expression and dehydration and salinity tolerance. In synthetic wheat lines, the difference in the ABA responsiveness of the lines appeared to be determined by the different sets of D-genome genes. Our findings suggest that highly ABA-responsive Ae. tauschii accessions should be valuable genetic resources for improving the abiotic stress tolerance of common wheat.

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.

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.

For a purpose of better understanding the genome structure of wheat and accelerating the development of DNA markers for gene isolations and breeding, the Japanese research group, as a member of The International Wheat Genome Sequencing Consortium, is now conducting the physical mapping and genomic sequencing of wheat chromosome 6B of ‘Chinese Spring’ (CS). BAC libraries were constructed respectively using the short and long arm-specific DNAs extracted from the flow-sorted chromosome 6BS and 6BL of double ditelosomic 6B lines of CS. With a sequence-based finger printing method and contig assembly, the BAC physical maps for the 6BS and 6BL have been successfully established. For validation and chromosomal landing of the BAC contigs, we have developed a large number of 6B-specific DNA markers using the public resources including available EST databases, and 6B survey sequence. In parallel, three reference maps, a highresolution radiation hybrid map, a gametocidal system-derived deletion map and a genetic linkage map, are also under construction for anchoring the BAC contigs to their specific genomic regions.

For successful speciation through allopolyploidization, normal growth and fertility of interspecific hybrids are essential. However, hybrid plants frequently fail to produce a next generation due to lethality and sterility. Such hybrid incompatabilities are considered a postzygotic reproductive barrier, and play important roles in differentiation and establishment of new genealogical lineages in plants. The Dobzhansky-Muller (DM) model simply explains the process for generating genetic incompatibility in hybrids between two diverging lineages (Bomblies and Weigel 2007). This model proposes that reduction of fitness in hybrids generally occurs due to interaction between at least two epistatic loci derived from divergent parents.

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.

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway.

In crop species such as wheat, abiotic stresses and preharvest sprouting reduce grain yield and quality. The plant hormone abscisic acid (ABA) plays important roles in abiotic stress tolerance and seed dormancy. In previous studies, we evaluated ABA responsiveness of 67 Aegilops tauschii accessions and their synthetic hexaploid wheat lines, finding wide variation that was due to the D-genome. In this study, quantitative trait locus (QTL) analysis was performed using an F2 population derived from crosses of highly ABA-responsive and less-responsive synthetic wheat lines. A significant QTL was detected on chromosome 6D, in a similar location to that reported for ABA responsiveness using recombinant inbred lines derived from common wheat cultivars Mironovskaya 808 and Chinese Spring. A comparative map and physiological and expression analyses of the 6D QTL suggested that this locus involved in line differences among wheat synthetics is different from that involved in cultivar differences in common wheat. The common wheat 6D QTL was found to affect seed dormancy and the regulation of cold-responsive/late embryogenesis abundant genes during dehydration. However, in synthetic wheat, we failed to detect any association of ABA responsiveness with abiotic stress tolerance or seed dormancy, at least under our experimental conditions. Development of near-isogenic lines will be important for functional analyses of the synthetic wheat 6D QTL.

Segregation distortion of molecular markers is closely related to hybrid incompatibility in progeny from intraspecific crosses. Recent reports in higher plants have demonstrated that hybrid sterility results in segregation distortion at the causal gene regions in progeny of intraspecific crosses. Ne1 and Ne2 complementary loci are known to control hybrid necrosis in intraspecific crosses of common wheat cultivars. Here, we examine the effect of a weak necrosis allele Ne1(w) on the segregation ratio of molecular markers in recombinant inbred lines (RILs) of common wheat. Some RILs showed accelerated cell death in the leaves at the heading stage due to the epistatic interaction between two quantitative trait loci (QTL) on chromosomes 5B and 2B. Chromosomal localization of these QTL corresponding to Ne1(w) and Ne2 showed distorted segregation ratios of assigned markers having oppositely biased direction. Although the Ne1(w) and Ne2 interaction had no obvious effect on seed fertility, Ne1(w) reduced completion of grain development under the Ne2-homozygous background. This reduction might be one of causes that induces segregation distortion in the 5B and 2B chromosomal regions of RILs. The present study demonstrated that weak hybrid necrosis has limited phenotypic effects; it causes segregation distortion in progeny from intraspecific crosses.

Wheat landraces carry abundant genetic variation in heading and flowering times. Here, we studied flowering-related traits of two Nepalese varieties, KU-4770 and KU-180 and a Japanese wheat cultivar, Shiroganekomugi (SGK). These three wheat varieties showed similar flowering time in a common garden experiment. In total, five significant quantitative trait loci (QTLs) for three examined traits, the heading, flowering and maturation times, were detected using an F2 population of SGK/KU-4770. The QTLs were found at the Ppd-1 loci on chromosomes 2B and 2D and the 2B QTL was also confirmed in another F2 population of SGK/KU-180. The Ppd-D1 allele from SGK and the Ppd-B1 alleles from the two Nepalese varieties might be causal for early-flowering phenotype. The SGK Ppd-D1 allele contained a 2-kb deletion in the 5′ upstream region, indicating a photoperiod-insensitive Ppd-D1a allele. Real-time PCR analysis estimating the Ppd-B1 copy number revealed that the two Nepalese varieties included two intact Ppd-B1 copies, putatively resulting in photoperiod insensitivity and an early-flowering phenotype. The two photoperiod-insensitive Ppd-1 homoeoalleles could independently contribute to segregation of early-flowering individuals in the two F2 populations. Therefore, wheat landraces are genetic resources for discovery of alleles useful for improving wheat heading or flowering times.

Low temperature causes serious losses in agricultural productivity of wheat. The short-term exposure of plants to low, nonfreezing temperatures leads to an increase in freezing tolerance. This adaptive process called as cold acclimation involves drastic changes in gene expression. A correlation between cold-responsive gene expression patterns and freezing tolerance has been reported in various studies. Natural variations in gene expression between accessions are considered more sensitive to molecular diversity than phenotypic differences. Quantitative trait loci (QTLs) controlling differences in transcript accumulation levels are generally called expression QTLs (eQTLs). Our eQTL analysis of five cold-responsive genes demonstrated to identify two major freezing tolerance loci using recombinant inbred lines of common wheat. The identified eQTLs are corresponding to previously identified QTL for freezing tolerance, and play important roles in development of freezing tolerance. Therefore, eQTL analysis is a powerful tool for genetic dissection of complex traits such as abiotic stress tolerance in common wheat.

Low temperature induces expression of Cor (cold-responsive)/Lea (late embryogenesis-abundant) gene family members through C-repeat binding factor (CBF) transcription factors in common wheat. However, the relationship between the genetic loci controlling cold-responsive gene expression and freezing tolerance is unclear. In expression quantitative trait locus (eQTL) analysis, accumulated transcripts of Cor/Lea and CBF genes were quantified in recombinant inbred lines derived from a cross between two common wheat cultivars with different levels of freezing tolerance. Four eQTLs controlling five cold-responsive genes were found, and the major eQTL with the greatest effect was located on the long arm of chromosome 5A. At least the 1D and 5A eQTLs played important roles in development of freezing tolerance in common wheat. The chromosomal location of the 5A eQTL, controlling four cold-responsive genes, coincided with a region homoeologous to a frost-tolerance locus (Fr-A (m) 2) reported as a CBF cluster region in einkorn wheat. The 5A eQTL plays a significant role through Cor/Lea gene expression in cold acclimation of wheat. In addition, our results suggest that one or more CBF copies at the Fr-2 region positively regulate other copies, which might amplify the positive effects of the CBF cluster on downstream Cor/Lea gene activation.

Abiotic stresses, such as cold, drought or high salinity, seriously affect plant growth and reduce yield in crop species including common wheat (Triticum aestivum L.). The phytohormone ABA plays important roles in plant adaptation to abiotic stress. We compared responsiveness to exogenous ABA, based on root growth inhibition by ABA, among three common wheat cultivars. Seedlings of the cultivars Cheyenne (Cnn) and Hope showed higher ABA responsiveness and higher levels of Cor (cold-responsive)–Lea (late embryogenesis abundant) gene expression than seedlings of Chinese Spring (CS). The chromosomes involved in the regulation of ABA responsiveness and Cor–Lea expression were identified using chromosome substitution lines, in which a chromosome pair of CS was substituted for the corresponding homologous pair of Cnn or Hope. In the CS–Cnn substitution lines, chromosomes 3A, 5A, 5D and 7A increased the ABA responsiveness of CS. Chromosomes 3A and 5A were also involved in the regulation of Cor–Lea gene expression and stomatal response during leaf dehydration. Substitution of CS chromosomes 3A or 5A with the respective homologous pair from Hope also enhanced ABA responsiveness and Cor–Lea expression. In addition, the factors present on chromosomes 4D and 7B of highly responsive cultivars increased Wrab17 expression but had little or no effect on ABA responsiveness. Cultivar differences in ABA responsiveness appear to be determined by genes present on these specific chromosomes in common wheat.

Background Cuticular wax production on plant surfaces confers a glaucous appearance and plays important roles in plant stress tolerance. Most common wheat cultivars, which are hexaploid, and most tetraploid wheat cultivars are glaucous; in contrast, a wild wheat progenitor, Aegilops tauschii, can be glaucous or non-glaucous. A dominant non-glaucous allele, Iw2, resides on the short arm of chromosome 2D, which was inherited from Ae. tauschii through polyploidization. Iw2 is one of the major causal genes related to variation in glaucousness among hexaploid wheat. Detailed genetic and phylogeographic knowledge of the Iw2 locus in Ae. tauschii may provide important information and lead to a better understanding of the evolution of common wheat. Results Glaucous Ae. tauschii accessions were collected from a broad area ranging from Armenia to the southwestern coastal part of the Caspian Sea. Linkage analyses with five mapping populations showed that the glaucous versus non-glaucous difference was mainly controlled by the Iw2 locus in Ae. tauschii. Comparative genomic analysis of barley and Ae. tauschii was then used to develop molecular markers tightly linked with Ae. tauschii Iw2. Chromosomal synteny around the orthologous Iw2 regions indicated that some chromosomal rearrangement had occurred during the genetic divergence leading to Ae. tauschii, barley, and Brachypodium. Genetic associations between specific Iw2-linked markers and respective glaucous phenotypes in Ae. tauschii indicated that at least two non-glaucous accessions might carry other glaucousness-determining loci outside of the Iw2 locus. Conclusion Allelic differences at the Iw2 locus were the main contributors to the phenotypic difference between the glaucous and non-glaucous accessions of Ae. tauschii. Our results supported the previous assumption that the D-genome donor of common wheat could have been any Ae. tauschii variant that carried the recessive iw2 allele. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0246-y) contains supplementary material, which is available to authorized users.