Transcripts of Vp-1 homeologues are misspliced in modern wheat and ancestral species.
ABSTRACT: The maize (Zea mays) Viviparous 1 (Vp1) transcription factor has been shown previously to be a major regulator of seed development, simultaneously activating embryo maturation and repressing germination. Hexaploid bread wheat (Triticum aestivum) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting (PHS), a phenomenon that is phenotypically similar to the maize vp1 mutation. Analysis of Vp-1 transcript structure in wheat embryos during grain development showed that each homeologue produces cytoplasmic mRNAs of different sizes. The majority of transcripts are spliced incorrectly, contain insertions of intron sequences or deletions of coding region, and do not have the capacity to encode full-length proteins. Several VP-1-related lower molecular weight protein species were present in wheat embryo nuclei. Embryos of a closely related tetraploid species (Triticum turgidum) and ancestral diploids also contained misspliced Vp-1 transcripts that were structurally similar or identical to those found in modern hexaploid wheat, which suggests that compromised structure and expression of Vp-1 transcripts in modern wheat are inherited from ancestral species. Developing embryos from transgenic wheat grains expressing the Avena fatua Vp1 gene showed enhanced responsiveness to applied abscisic acid compared with the control. In addition, ripening ears of transgenic plants were less susceptible to PHS. Our results suggest that missplicing of wheat Vp-1 genes contributes to susceptibility to PHS in modern hexaploid wheat varieties and identifies a possible route to increase resistance to this environmentally triggered disorder.
Project description:Pre-harvest sprouting (PHS) is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programs. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However, the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 (PM19) genes and the mitogen-activated protein kinase kinase 3 (TaMKK3-A) gene, have been proposed to underlie Phs-A1. To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterization of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A, and not PM19, is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In United Kingdom varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A. Analysis of these haplotypes in a collection of United Kingdom and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the study will help inform the choice of germplasm and breeding strategies for the deployment of Phs-A1 resistance into breeding germplasm.
Project description:Hexaploid wheat (Triticum aestivum) accumulates benzoxazinones (Bxs) as defensive compounds. Previously, we found that five Bx biosynthetic genes, TaBx1-TaBx5, are located on each of the three genomes (A, B, and D) of hexaploid wheat. In this study, we isolated three homoeologous cDNAs of each TaBx gene to estimate the contribution of individual homoeologous TaBx genes to the biosynthesis of Bxs in hexaploid wheat. We analyzed their transcript levels by homoeolog- or genome-specific quantitative RT-PCR and the catalytic properties of their translation products by kinetic analyses using recombinant TaBX enzymes. The three homoeologs were transcribed differentially, and the ratio of the individual homoeologous transcripts to total homoeologous transcripts also varied with the tissue, i.e., shoots or roots, as well as with the developmental stage. Moreover, the translation products of the three homoeologs had different catalytic properties. Some TaBx homoeologs were efficiently transcribed, but the translation products showed only weak enzymatic activities, which inferred their weak contribution to Bx biosynthesis. Considering the transcript levels and the catalytic properties collectively, we concluded that the homoeologs on the B genome generally contributed the most to the Bx biosynthesis in hexaploid wheat, especially in shoots. In tetraploid wheat and the three diploid progenitors of hexaploid wheat, the respective transcript levels of the TaBx homoeologs were similar in ratio to those observed in hexaploid wheat. This result indicates that the genomic bias in the transcription of the TaBx genes in hexaploid wheat originated in the diploid progenitors and has been retained through the polyploidization.
Project description:Pre-harvest sprouting frequently occurs in Triticum aestivum (wheat) and Hordeum vulgare (barley) at the end of the maturity period due to high rainfall, particularly in Asian monsoon areas. Seed dormancy is a major mechanism preventing pre-harvest sprouting in these crops.We identified orthologous sequences of the major Hordeum vulgare (barley) seed dormancy gene Qsd1 in hexaploid wheat cv. Chinese Spring by performing genomic clone sequencing, followed by transcript sequencing. We detected 13 non-synonymous amino acid substitutions among the three sub-genomes of wheat and found that the Qsd1 sequence in the B sub-genome is most similar to that in barley. The Qsd1 sequence in A genome diploid wheat is highly similar to that in the hexaploid A sub-genome. Wheat orthologs of Qsd1 showed closer similarities to barley Qsd1 than did those of other accessions in the DNA database. Like barley Qsd1, all three wheat Qsd1s showed embryo-specific gene expression patterns, indicating that barley and wheat Qsd1 share an orthologous origin. The alignment of four hexaploid wheat cultivars indicated that the amino acid sequences of three spring cultivars, Chinese Spring, Haruyo Koi, and Fielder, are exactly the same in each sub-genome. Only Kitahonami has three amino acid substitutions at the B sub-genome.Kitahonami has a longer seed dormancy period than does Chinese Spring. Sequence polymorphisms between Chiniese Spring and Kitahonami in the B sub-genome may underlie the phenotypic differences in seed dormancy between these hexaploid wheat cultivars.
Project description:The DNA sequences of wheat Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, were analyzed with a view to understanding the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Acc-1 and Acc-2 loci from each of the wheats Triticum urartu (A genome), Aegilops tauschii (D genome), Triticum turgidum (AB genome), and Triticum aestivum (ABD genome), as well as two Acc-2-related pseudogenes from T. urartu were sequenced. The 2.3-2.4 Mya divergence time calculated here for the three homoeologous chromosomes, on the basis of coding and intron sequences of the Acc-1 genes, is at the low end of other estimates. Our clock was calibrated by using 60 Mya for the divergence between wheat and maize. On the same time scale, wheat and barley diverged 11.6 Mya, based on sequences of Acc and other genes. The regions flanking the Acc genes are not conserved among the A, B, and D genomes. They are conserved when comparing homoeologous genomes of diploid, tetraploid, and hexaploid wheats. Substitution rates in intergenic regions consisting primarily of repetitive sequences vary substantially along the loci and on average are 3.5-fold higher than the Acc intron substitution rates. The composition of the Acc homoeoloci suggests haplotype divergence exceeding in some cases 0.5 Mya. Such variation might result in a significant overestimate of the time since tetraploid wheat formation, which occurred no more than 0.5 Mya.
Project description:Gene expression levels of newly synthetic triploid wheat (ABD), its chromosome-doubled hexaploid (AABBDD), stable synthetic hexaploid (AABBDD), and their parents, Triticum turgidum (accession KU124, AABB) and Aegilops tauschii (accession KU2074, DD) were compared to understand genome-wide change of gene expressions during the course of amphidiploidization and genome stabilization. Stable synthetic hexaploid which were maintained through self-pollinations for 13 generations using the same combinations of the parents for production of synthetic common wheat. Overall design: Amphidiploidization event between T. turgidum ssp. dicoccum (AABB) and Ae. tauschii ssp. strangulata (DD) was recreated. Gene expression levels of newly synthetic triploid wheat (ABD), its chromosome-doubled hexaploid (AABBDD), stable synthetic hexaploid (AABBDD), and their parents, Triticum turgidum (AABB) and Aegilops tauschii (DD) were compared. Total RNA of each line was extracted from three biological replicates of two leaves seedlings.
Project description:Tetraploid emmer wheat (Triticum turgidum ssp. dicoccon) is a progenitor of the world's most widely grown crop, hexaploid bread wheat (Triticum aestivum), as well as the direct ancestor of tetraploid durum wheat (T. turgidum subsp. turgidum). Emmer was one of the first cereals to be domesticated in the old world; it was cultivated from around 9700 BC in the Levant1,2 and subsequently in south-western Asia, northern Africa and Europe with the spread of Neolithic agriculture3,4. Here, we report a whole-genome sequence from a museum specimen of Egyptian emmer wheat chaff, 14C dated to the New Kingdom, 1130-1000 BC. Its genome shares haplotypes with modern domesticated emmer at loci that are associated with shattering, seed size and germination, as well as within other putative domestication loci, suggesting that these traits share a common origin before the introduction of emmer to Egypt. Its genome is otherwise unusual, carrying haplotypes that are absent from modern emmer. Genetic similarity with modern Arabian and Indian emmer landraces connects ancient Egyptian emmer with early south-eastern dispersals, whereas inferred gene flow with wild emmer from the Southern Levant signals a later connection. Our results show the importance of museum collections as sources of genetic data to uncover the history and diversity of ancient cereals.
Project description:Untimely rains in wheat fields during harvest season can cause pre-harvest sprouting (PHS), which deteriorates the yield and quality of wheat crop. Metabolic homeostasis of the embryo plays a role in seed dormancy, determining the status of the maturing grains either as dormant (PHS-tolerant) or non-dormant (PHS-susceptible). Very little is known for direct measurements of global metabolites in embryonic tissues of dormant and non-dormant wheat seeds. In this study, physiologically matured and freshly harvested wheat seeds of PHS-tolerant (cv. Sukang, dormant) and PHS-susceptible (cv. Baegjoong, non-dormant) cultivars were water-imbibed, and the isolated embryos were subjected to high-throughput, global non-targeted metabolomic profiling. A careful comparison of identified metabolites between Sukang and Baegjoong embryos at 0 and 48 h after imbibition revealed that several key metabolic pathways [such as: lipids, fatty acids, oxalate, hormones, the raffinose family of oligosaccharides (RFOs), and amino acids] and phytochemicals were differentially regulated between dormant and non-dormant varieties. Most of the membrane lipids were highly reduced in Baegjoong compared to Sukang, which indicates that the cell membrane instability in response to imbibition could also be a key factor in non-dormant wheat varieties for their untimely germination. This study revealed that several key marker metabolites (e.g., RFOs: glucose, fructose, maltose, and verbascose), were highly expressed in Baegjoong after imbibition. Furthermore, the data showed that the key secondary metabolites and phytochemicals (vitexin, chrysoeriol, ferulate, salidroside and gentisic acid), with known antioxidant properties, were comparatively low at basal levels in PHS-susceptible, non-dormant cultivar, Baegjoong. In conclusion, the results of this investigation revealed that after imbibition the metabolic homeostasis of dormant wheat is significantly less affected compared to non-dormant wheat. The inferences from this study combined with proteomic and transcriptomic studies will advance the molecular understanding of the pathways and enzyme regulations during PHS.
Project description:Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating foliar disease. The Ug99 race group has combined virulence to most stem rust (Sr) resistance genes deployed in wheat and is a threat to global wheat production. Here we identified a coiled-coil, nucleotide-binding leucine-rich repeat protein (NLR) completely linked to the Ug99 resistance gene Sr21 from Triticum monococcum. Loss-of-function mutations and transgenic complementation confirmed that this gene is Sr21. Sr21 transcripts were significantly higher at high temperatures, and this was associated with significant upregulation of pathogenesis related (PR) genes and increased levels of resistance at those temperatures. Introgression of Sr21 into hexaploid wheat resulted in lower levels of resistance than in diploid wheat, but transgenic hexaploid wheat lines with high levels of Sr21 expression showed high levels of resistance. Sr21 can be a valuable component of transgenic cassettes or gene pyramids combining multiple resistance genes against Ug99.
Project description:Wild emmer wheat, Triticum dicoccoides, is the progenitor of modern tetraploid and hexaploid cultivated wheats. Our objective was to map domestication-related quantitative trait loci (QTL) in T. dicoccoides. The studied traits include brittle rachis, heading date, plant height, grain size, yield, and yield components. Our mapping population was derived from a cross between T. dicoccoides and Triticum durum. Approximately 70 domestication QTL effects were detected, nonrandomly distributed among and along chromosomes. Seven domestication syndrome factors were proposed, each affecting 5-11 traits. We showed: (i) clustering and strong effects of some QTLs; (ii) remarkable genomic association of strong domestication-related QTLs with gene-rich regions; and (iii) unexpected predominance of QTL effects in the A genome. The A genome of wheat may have played a more important role than the B genome during domestication evolution. The cryptic beneficial alleles at specific QTLs derived from T. dicoccoides may contribute to wheat and cereal improvement.
Project description:Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat.Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression.Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.