Identification of reference genes and their validation for gene expression analysis in phytopathogenic fungus Macrophomina phaseolina.

Macrophomina phaseolina is a soil-borne pathogenic fungus that infects a wide range of crop species and causes severe yield losses. Although the genome of the fungus has been sequenced, the molecular basis of its virulence has not been determined. Identification of up-regulated genes during fungal infection is important to understand the mechanism involved in its virulence. To ensure reliable quantification, expression of target genes needs to be normalized on the basis of certain reference genes. However, in the case of M. phaseolina, reference genes or their expression analysis have not been reported in the literature. Therefore, the objective of this study was to evaluate 12 candidate reference genes for the expression analysis of M. phaseolina genes by applying three different fungal growth conditions: a) during root and stem infection of soybean, b) in culture media with and without soybean leaf infusion and c) by inoculating a cut-stem. Based on BestKeeper, geNorm and NormFinder algorithms, CYP1 was identified as the best recommended reference gene followed by EF1ß for expression analysis of fungal gene during soybean root infection. Besides Mp08158, CYP1 gene was found suitable when M. phaseolina was grown in potato-dextrose broth with leaf infusion. In the case of cut-stem inoculation, Mp08158 and Mp11185 genes were found to be most stable. To validate the selected reference genes, expression analysis of two cutinase genes was performed. In general, the expression patterns were similar when the target genes were normalized against most or least stable gene. However, in some cases different expression pattern can be obtained when least stable gene is used for normalization. We believe that the reference genes identified and validated in this study will be useful for gene expression analysis during host infection with M. phaseolina.

Three dominant awnless genes in common wheat: Fine mapping, interaction and contribution to diversity in awn shape and length.

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.

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins.

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.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

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.

Comparison of gene expression profiles and responses to zinc chloride among inter- and intraspecific hybrids with growth abnormalities in wheat and its relatives.

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.

Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide with six overlapping epitopes, evolved following polyploidization.

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.

Line differences in Cor/Lea and fructan biosynthesis-related gene transcript accumulation are related to distinct freezing tolerance levels in synthetic wheat hexaploids.

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.

The cuticular wax inhibitor locus Iw2 in wild diploid wheat Aegilops tauschii: phenotypic survey, genetic analysis, and implications for the evolution of 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.

Identification of quantitative trait loci for abscisic acid responsiveness in the D-genome of hexaploid wheat.

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.

Identification of quantitative trait locus for abscisic acid responsiveness on chromosome 5A and association with dehydration tolerance in common wheat seedlings.

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.

A major quantitative trait locus for cold-responsive gene expression is linked to frost-resistance gene Fr-A2 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.

Differential contribution of two Ppd-1 homoeoalleles to early-flowering phenotype in Nepalese and Japanese varieties of common wheat.

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.

Identification of quantitative trait loci controlling grain size and shape in the D genome of synthetic hexaploid wheat lines.

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.

Application of real-time PCR-based SNP detection for mapping of Net2, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and Aegilops tauschii.

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.

Variation in abscisic acid responsiveness of Aegilops tauschii and hexaploid wheat synthetics due to the D-genome diversity.

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.

Identification of chromosomes controlling abscisic acid responsiveness and transcript accumulation of Cor-Lea genes in common wheat seedlings.

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.