Cloning Of Cold-related Resistance Genes And Molecular Marker Development In Wheat

Wheat(Triticum aestivum L.) is the most widely cultivated food crop followed by rice and maize, serving as a staple food for about 40 % of the world's population. In the limited arable land, higher yield breeding is the fundamental way for enhancing wheat total production level in the world. Low temperature is one of the limit factors for wheat growth, development and distribution. In China, frozen injury almost occur in all wheat growing areas from north to south. In the event of cold injury, it is difficult to control. The improvement of wheat cold resistance is relying mainly on variety itself. Therefore, the primary target of breeders is to enhance the cold resistance of wheat. Plant cold hardiness is a complicated quantitative trait. Although researchers have unearthed a large number of cold resistance gene and function loci using modern molecular biology techniques there were few be used in the molecular breeding production practice. This study is mainly to develop molecular markers by candidate gene SNP sites and explore the superior allelic variation. The main results are summarised as following.1. On the basis of the c DNA sequence, the 5? upstream regulatory region of Wcs19 gene was isolated by Genome Walking. Sequence analysis revealed the upstream regulation shared not only the typical TATA-box and CAAT-box but also contain the CRT/DRE elements which were conservative in Cor genes. By assemble the sequences of 5? upstream regulatory region and coding region, we obtain a 1920 bp Wcs19 sequence. And then the Wcs19 was mapped on the chromosome 2A in wheat by CS nullisomic-tetrasomic lines. Fourteen SNPs were found between different cold resistance wheat varieties. However, these SNPs have no relevance with cold resistance.2. Based on the wheat genome database, the Wcor15 were isolated from A, B and D genomes in wheat, and Wcor15 were mapped on the homologous group 2 in wheat by CS nullisomic-tetrasomic lines. We also uncovered that WCOR15-2A, WCOR15-2B and WCOR15-2D may belong to the chloroplast-targeted LEA3 protein. The Wcor15-2A, Wcor15-2B and Wcor15-2D sequences were highly-conserved among different cold resistance varieties, and the conservative property is Wcor15-2A=Wcor15-2D>Wcor15-2B. In this study, we also cloned and sequenced the Wcor15-2A, Wcor15-2B and Wcor15-2D genes from 23 diploid relative species, 10 tetraploid species and 106 hexaploid wheat species and analyzed their molecular evolution to reveal the origin of the A, B and D genome in Triticum aestivum. Comparative analyses with their sequences in diploid, tetraploid and hexaploid wheat suggestting that T. urartu might be the direct donor of the Wcor15-2A locus in tetraploid and hexaploid wheat. Ae. speltoides, in turn, might be the direct donor of the Wcor15-2B locus in tetraploid and hexaploid wheat. At the same time, present results supported that ssp. strangulata a typical subspecies of Ae. tauschii might be the direct donor of the Wcor15-2D locus in common wheat. Based on the sequences of all the Wcor15 genes, during the first polyploidization, A and D genome kept invariable, however, B genome got mutation.3. Based on the wheat genome database, the Wcor14 were isolated from A, B and D genomes in wheat, and Wcor14 were mapped on the homologous group 2 in wheat by CS nullisomic-tetrasomic lines. Sequence analysis revealed the upstream regulation of Wcor14 shared the CRT/DRE element which was conservative in Cor genes. The consistency of the three homologous Wcor14 genes is >83.6% and > 97.9% in DNA and AA level respectively. The Wcor14-2A and Wcor14-2D sequences were highly-conserved among different cold resistance varieties. By compareing the Wcor14-2B sequences from different cold resistance varieties, there were 15 polymorphic sites with 14 SNPs and 1 In Del. The 15 polymorphic site formed a typical haplotype in the Wcor14-2B. Two alleles of Wcor14-2B were designated as Hap14 a and Hap14 b accordingly. According to the 6bp In Del in the first exon, we developed a molecular marker 14AF/14 AR to distinguish the Wcor14-2B alleles. Because Hap14 a had a significantly positive effect on cold resistance, it was considered a potentially superior allele.4. Based on the Os BC1 and Zm BK2 sequences and wheat genome database, the TaCOBL were isolated from A, B and D genomes in wheat, and TaCOBL were mapped on the homologous group 5 in wheat by CS nullisomic-tetrasomic lines. TaCOBL-5B had two copies on the chromosome 5B. TaCOBL-5A and TaCOBL-5D putatively encoded a polypeptide with 449 amino acid residues. However, TaCOBL-5B putatively encoded a polypeptide with 450 amino acid residues. Cluster analysis showed that TaCOBL, Bc1 and Bk2 were belonging to the same clade, the homology of their amino acid residues with COBRA family conservative domain structure was 86.4%. So, we inferred that TaCOBL shared similar function with Bc1 and Bk2 which associated with the synthesis of cellulose. The TaCOBL-5A and TaCOBL-5D sequences were highly-conserved in among different cold resistance varieties. By comparing the TaCOBL-5B sequences from different cold resistance varieties, there were two alleles designated as Hap5B-a and Hap5B-b. According to the 4bp In Del in the 5? upstream regulatory region, we developed a molecular marker 5BF/5BR to distinguish the TaCOBL-5B alleles. Because Hap5B-a had a significantly positive effect on cold resistance, it was considered a potentially superior allele.