Assaying Single Nucleotide Polymorphism Of Fusarium Wilt Resistance Gene1-2in Tomato And Screen Of Germplasm Resources

Because of high yield, nutrient rich, good eating, strong adaptability, tomato become one of the most important crops in the world. But with the enlarged cultivating area and the special environment for cultivation, diseases happened in tomato was more and more severity. Fusarium wilt is one of the most serious diseases in the production. Genetic improvement is the most economical ways to reducing the losses caused by fusarium wilt.In this paper, PCR primers were designed based on the1-2gene. SNPs were discovered by aligning the1-2genes between resistant varieties and susceptible varieties. Allele specific primers and their complementary primers were designed using the SNPs as their3’-end. In addition, we studied the effect of the mismatched bases at the3’-end of the allele-specific primers on PCR. To type these SNPs, the study successfully carried out50groups SNP genotypes with SNPs detected in tomato1-2gene. The main results are as follows:1Designed three nested primers for1-2gene, and compared1-2sequence of resistant varieties and susceptible varieties with the gene in the GenBank. Two SNPs:C/T variation at the site of1793bp and G/A variation at the site of1963bp were detected between resistant varieties and susceptible varieties.2We studied the effect of the mismatched bases at the3’-end of the allele-specific primers on PCR. Introducing proper mismatched based at the first or the second sites of the3’-end of the antiprimer played an important role in inhibiting the combination of allele specific primer and template. The mismatched base pairs C/T is stronger than C/A. Non-specific primer extension was decreased remarkably after introducing a mismatch at the second sites of the3’-end and separated with SNP mutations by a base of the allele specific primers.3Optimizing the reaction system to be annealing temperature58℃, Mg2+2.5mM, and dNTP250μmol·L-1by gradient PCR which had improved the accuracy of the reaction.4Using the SNP markers to screen the tomato germplasms in the laboratory. There are39of50cultivars and lines were resisitant fusarium wilt, and the results were consistent with the artificial inoculation research.