Molecular Markers Linked To Resistance Genes Ty-1、Ty-3a And Mi And Screening For Germplasm Resources In Tomato

Tomato yellow leaf curl disease(TYLCD)and tomato root-knot nematode disease are two kinds high degree of harm in the production of tomato,spreading a wide range.The experimental materials of this study were the tomato plants with resistant homozygous genotype(Ty-1/Ty-1,Ty-3a/Ty-3a),resistant heterozygous genotype(Ty-1/ty-1,Ty-3a/ty-3a)and the susceptible homozygous genotypes(ty-1/ty-1,ty-3a/ty-3a)of TYLCD and the resistant homozygous genotype(Mi/Mi),resistant heterozygous genotype(Mi/mi)and the susceptible homozygous genotypes(mi/mi)of root knot nematode disease.And the appropriate specific primers were designed with reference to the published literature.The stabilized molecular markers of Ty-1,Ty-3a and Mi were obtained separately,and then we established two duplex PCR systems on the basis of the establishment.One of the two duplex PCR systems could evaluate Ty-1 and Ty-3a,the other could evaluate Ty-1 and Mi.The molecular marker of Ty-1 gene: the specific segment of length 398 bp was found in each of the experimental materials after the amplified of the PCR reaction with the primer of TY-1F/TY-1R.After digested with Taq I enzyme,only one segment of length 398 bp was found in the susceptible homozygous genotypes,two segments of length 303 bp and 95 bp were found in the resistant homozygous genotype,three segments of length 398 bp,303 bp and 95 bp were found in the resistant heterozygous genotype.The sequences of primers are TY-1F: 5’-TAA TCC GTC GTT ACC TCT CCT T-3’,TY-1R: 5’-CGG ATG ACT TCA ATA GCA ATG A-3’.The molecular markers of Ty-3a gene: the specific segment of length 689 bp was found in each of the experimental materials after the amplified of the PCR reaction with the primer of TY-3a F/TY-3a R.After digested with Taq I enzyme,only one segment of length 500 bp was found in the susceptible homozygous genotypes,two segments of length 350 bp and 150 bp were found in the resistant homozygous genotype,three segments of length 500 bp,350 bp and 150 bp were found in the resistant heterozygous genotype.The sequences of primers are TY-3a F: 5’-GCA AAC AAG TTC CCC CAC TA-3’,TY-3a R: 5’-CGC AGA ACC AGC TAC TTT CC-3’.The molecular markers of Mi gene: the specific segment of length 750 bp was found in each of the experimental materials after the amplified of the PCR reaction with the primer of MIF/MIR.After digested with Taq I enzyme,only one segment length 750 bp was found in the susceptible homozygous genotypes,two segments length 570 bp and 180 bp were found in the resistant homozygous genotype,three segments length 750 bp,570 bp and 180 bp were found in the resistant heterozygous genotype.The sequences of primers are MIF: 5’-TCG GAG CCT TGG TCT GAA TT-3’,MIR: 5’-GCC AGA GAT GAT TCG TGA GA-3’.On the basis of the three single gene molecular markers above,the primers TY-1 and TY-3a were added to a PCR system simultaneously,the primers TY-1 and MI were added to a PCR system simultaneously,and improved the experimental conditions repeatedly such as concentration of template,Primer’s ratio and annealing temperature and so on,at last we established a double PCR system which could detected dual resistance gene Ty-1 and Ty-3a simultaneously,another double PCR system which could detected dual resistance gene Ty-1 and Mi simultaneously.We used the obtained double PCR reaction system to identify the breeding materials with unknown genotype,we got 3 kinds of resistant homozygous genotype breeding materials which contained Ty-1 and Ty-3a,as well as we got 7 kinds of resistant homozygous genotype breeding materials which contained Ty-1 and Mi.The double PCR technique provided in this experiment not only provid an effective method for the breeding of disease resistance gene,but also laid a foundation for further cultivating new tomato varieties with compound disease resistance,it was great significant to improve the resistance of tomato cultivars and the sustainable development of tomato production.