100 Gy 60 Co γ-ray induced novel mutations in tetraploid wheat.
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ABSTRACT:
SUBMITTER: Yang C
PROVIDER: S-EPMC4055256 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with (60)Co-?-rays at dosages of 100, 200, and 300 Gy. The M? and M? generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M? plants. In all, 39 M? plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M? plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained ?-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.
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Project description:Targeted re-sequencing of NLRs in tetraploid wheat
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Project description:Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.
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Project description:Among cereals, triticale (×Trititcoseale Wittmack ex A. Camus) represents a number of advantages such as high grain yield even in marginal environments, tolerance to drought, cold and acid soils, as well as lower production costs. Together with high biomass of grain and straw, triticale is also considered as an industrial energy crop. As an artificial hybrid, it has not evolved naturally, which is reflected in narrow genetic diversity causing a resistance collapse in recent years. Here, we describe a novel, synthetic tetraploid triticale, which was developed by the crossing of rye (Secale cereale L.) with einkorn wheat (Triticum monococcum spp. monococcum), which possess Sr35 stem rust resistance gene. Three subsequent generations of alloploids were obtained by chromosome doubling followed by self-pollination. The cytogenetic analyses revealed that the amphiploids possess a set of 28 chromosomes (14 of Am-genome and 14 of R-genome). The values of the most important yield-shaping traits for these tetraploid triticale form, including thousand-grain weight, plant height and stem length were higher compared to parental genotypes, as well as standard hexaploid triticale cultivars. This study shows that this tetraploid triticale genetic stock can be an interesting pre-breeding germplasm for triticale improvement or can be developed as a new alternative crop.
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Project description:Develpment of a diversity panel for tetraploid wheat which include wild emmer wheat from across its distribution ranges, domesticated emmer wheat, durum landraces and durum varieties.
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Project description:Tetraploid wheat mitochondrion sequencing
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Project description:Durum and bread wheat are well adapted to the Mediterranean Basin. Twenty-three genotypes of each species were grown to evaluate the intra- and inter-genetic diversity based on omega (ω), gamma (γ) and alpha (α)-gliadin profiles. To achieve this purpose, the endosperm storage proteins (both gliadins and glutenins) were extracted from wheat grains and electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results of SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) revealed nine polymorphic loci out of 16 loci with durum wheat genotypes and nine polymorphic loci out of 18 loci with bead wheat genotypes. The polymorphisms revealed by the SDS-PAGE were 56% and 50% in durum and bread wheat genotypes, respectively. Using the cluster analysis, the durum wheat genotypes were clustered into five groups, while the bread wheat genotypes were grouped into six clusters using un-weighed pair group mean analyses based on ω, γ, and α-gliadins profiles. The 46 durum and bread wheat genotypes were grouped into seven clusters based on the combined ω, γ, and α-gliadins profiles revealed by the SDS-PAGE. The in silico analysis determined the intra-genetic diversity between bread and durum wheat based on the sequences of ω, γ, and α-gliadins. The alignment of ω-gliadin revealed the highest polymorphism (52.1%) between bread and durum wheat, meanwhile, the alignment of γ and α-gliadins revealed very low polymorphism 6.6% and 15.4%, respectively. According to computational studies, all gliadins contain a lot of glutamine and proline residues. The analysis revealed that the bread wheat possessed ω and γ -gliadins with a lower content of proline and a higher content of glutamine than durum wheat. In contrast, durum wheat possessed α-gliadin with a lower content of proline and a higher content of glutamine than bread wheat. In conclusion, the SDS-PAGE, in silico and computational analyses are effective tools to determine the intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on ω, γ, and α-gliadins profiles.
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