Homology-based Cloning Of Genes Related With Powdery Mildew Resistance And Grain Protein Content From Wheat Relatives

Wheat is one of the most widely distributed and cultivated crops around the world as well as in China. Therefore, breeding for increased resistance and improved quality is of great significance for wheat production. Common or bread wheat is an allohexaploid species (Triticum aestivum L., AABBDD,2n= 6x= 42), containing three different but related sub-genomes. The genetic background of common wheat is complex as it emerged via two hybridization concomitant with genome doubling. The relatives of wheat species, being riched in genetic diversity, are important resources for wheat genetic improvement. Thus, constant exploitation and utilization of excellent genes in wheat related species can speed up the progress of the modern wheat breeding. In this study, a wheat powdery mildew resistance gene TmPm3 and a series of genes controlling grain protein content (GPC) were cloned from the relatives of wheat using homology-based cloning strategy. The expression, variation and function of these genes were further studied, and the main results were as follows:1. The powdery mildew resistance for thirty-four accessions of einkorn wheat germplasms was tested using Blumeria graminis f. sp. tritici (Bgt) isolates E09. According to the results of resistance identification, fifteen einkorn wheat germplasms showed resistance to wheat powdery mildew. Molecular markers detection for wheat powdery mildew resistance (Pm) Pm3 loci, and Pm3a, b, c, d, e, /genes proved the presence of Pm3 loci in Triticum monococcum L (Accession No.3AA28). Using homology-based cloning strategy, the full length sequence of that candidate Pm3 homologous gene was obtained. The candidate gene was named as TmPm3. Sequence alignment showed that TmPm3 was a new gene, exhibiting 79% similarity with pm3CS. Transient expression of TmPm3 in leaf epidermal cells showed that over-expression of TmPm3 could significantly inhibit the penetration of Bgt conidia spores and the formation of haustoria. Sequence analysis of the Pm3 alleles shed new light on the evolution of the Pm3 genes, providing a better understanding of the molecular basis of disease resistance. This is the first report about the cloning of a functional Pm3 homolog gene from a diploid AA-genome wheat species, demonstrating that homology-based cloning of resistance gene is feasible for the isolation of functional resistance genes from excellent germplasm materials in wheat relatives.2. Haynaldia villosa (2n= 2x= 14, genome VV), carrying powdery mildew resistance gene Pm21 in chromosome 6V, is one of the important relative wild species for wheat genetic improvement. In this study, NAM-V1 gene belong to NAM family was cloned from 6AL/6VS translocation. Phylogenetic tree indicated NAM-V1 might be the orthologous gene of NAM-A1, B1 and D1. Molecular detection of NAM-V1 in Pm21 segregation populations proved that NAM-V1 linked to Pm21, suggesting NAM-V1 also come from chromosome 6V. In addition, a new molecular marker CauNAM-V1 for specifically detection of NAM-V1 gene was developed and could be also used for selecting Pm21 in wheat breeding program. Furthermore, we developed a marker CauNAM-ABD which could amplify and distinguish NAM-A1, NAM-B1, NAM-B2, NAM-D1 and NAM-D2 genes simultaneously in a simple step. CauNAM-ABD allowed us to develop an efficient "one-marker-for-five-genes" procedure for identifying genes and its copy numbers related with grain protein content. To analyze the function of NAM-V1 in 6AL/6VS translation lines, four Pm21 F2 segregation populations W50200, W50175, W50156 and W50176 were constructed. Our data demonstrated that the GPC of the individuals containing with NAM-V1 gene was higher than that containing NAM-A1 gene in all the four segregation populations, suggesting the replacement of NAM-A1 to NAM-V1 gene in 6AL/6VS translocation lines confers to increasing grain protein concentration (GPC) of hexaploid wheat.3. In wheat-Aegilops speltoides translocation 6BS/6SS, Ae. speltoides chromosome 6S short arm carrying Pml2 was translocated into chromosome 6B of common wheat, and confers effective resistance to powdery mildew. Here, a new NAM family gene NAM-S1 was isolated from translocation 6BS/6SS using homology-based cloning strategy. The full-length of NAM-S1 gene is 1,547 bp, containing three exon and two intron, and encoding a protein of 409 amino acids with the molecular weight of 43.6 KDa. According to the sequence alignment results, a molecular marker CauNAM-S1 for specific detection of NAM-S1 gene was developed. A lingage map of NAM-S1 were constructed in Pm12 F2 segregation population which containing 486 individuals. Genetic distance between NAM-S1 and Pm12 was 7.3 cM. Correlation analysis of the phenotype and genotype indicated that NAM-S1 was responsible for the increasing of wheat GPC. This study laid a foundation for using NAM-S1 and Pm12 in wheat breed program.4. Using near-IR (NIR) detector, the GPC of thirty-five accessions of einkorn wheat germplasms were determined. The results demonstrated that the GPC content varies widely among different accessions, and many einkorn wheat germplasms showed high GPC. The GPC of T. monococcum was significantly lower than that of wild species (T. boeoticum), implying that the GPC tends to decline in the process of wheat improvement and domestication. Among them, T. boeoticum 3AA5 represented the highest GPC in all einkorn wheat germplasms. One accession of T. monococcum 3AA22 represented the lowest GPC in all einkorn wheat germplasms. Using homology-based cloning method, twenty-seven NAM-A1 series genes were cloned, including twenty one genes from T. monococcum, four genes from T. boeoticum, and two genes from T. urartu. Sequence alignment showed a "G" insertion in the coding region leading to the frame-shift mutation of NAM-A1 gene in accession 3AA22 of T. monococcum. For this reason, the lower GPC of 3AA22 might be caused by the inactivation of NAM-A1 gene, implying that other NAM-A1 series genes might be functional.