Establishment Of Multiplex PCR For Simultaneous Detection Of Resistance Genes Mi?Tm-2~2 And Cf-9

In this study,the tomato plants with resistant homozygous genotype(Mi/Mi),resistant heterozygous genotype(Mi/mi)and the susceptible homozygous genotype(mi/mi)of tomato root knot nematode disease and the resistant homozygous genotype(Tm-2~2/Tm-2~2),resistant heterozygous genotype(Tm-2~2/tm-2~2)and the susceptible homozygous genotype(tm-2~2/tm-2~2)of tomato mosaic virus as well as the resistant genotype(Cf-9/-),and the susceptible genotype(cf-9/cf-9)of tomato leaf mould are used as experimental materials.The specific primers are designed referring to the published literature,performing PCR amplification and enzyme digestion reaction,the single gene molecular markers of Mi?Tm-2~2 and Cf-9 are established respectively,and based on those the triplex PCR system which can simultaneously identify the above three genes is established and it is preliminary application in breeding for disease resistance.(1)The molecular marker of gene Mi the use of specific primer for PCR amplification and enzyme digestion reaction,susceptible homozygous genotype materials without enzyme site,still for 750 bp specific segment;the resistant homozygous genotype materials appear two specific segments 570 bp and 180 bp;the resistant heterozygous genotype materials appear three specific segments 750 bp,570 bp and 180 bp.The sequences of the primer are CAPS1F:5'-TCG GAG CCT TGG TCT GAA TT-3';CAPS1R:5'-GCC AGA GAT GAT TCG TGA GA-3.(2)The molecular marker of gene Tm-2~2 the use of specific primer for PCR amplification and enzyme digestion reaction,the resistant homozygous genotype materials without enzyme site,still for 950 bp specific segment;the susceptible homozygous genotype materials appear two specific segments 500 bp and 280 bp;the resistant heterozygous genotype materials appear three specific segments 950 bp,500 bp and 280bp.The sequences of the primer are CAPS2F:5'-CAC CTT TCC CTC TCC AA-3';CAPS2R:5'-CAC CTT TCC CCT AAA GC-3'.(3)The molecular marker of gene Cf-9 the use of specific primer for PCR amplification,the resistant genotype materials appear 2.7 kb specific segment;the susceptible genotype materials do not appear specific segment;after enzyme digestion reaction,the resistant genotype materials appear three specific segments 1170 bp,460 bp and 390 bp.The sequences of the primer are CAPS3F:5'-AGT GCA GAA ATG GAT TGT GTA-3';CAPS3R:5'-CGG AAA TCT TTG AAT CCT GGA-3'.Based on the single gene molecular markers above,the three primers CAPS1?CAPS2and CAPS3 are mixed in the PCR system at the same time,and improve the experimental conditionsrepeatedlysuchasconcentration of template,primer'sratio and annealing temperature and so on,so that the triplex PCR system is finally established which can assay the three resistant genes Mi?Tm-2~2 and Cf-9 simultaneously.Using the triplex PCR reaction to assay the unknown genotype of breeding materials and F2 segregating population of new tomato variety“201”,among those breeding materials,there is a tomato material which can aggregate three kinds of resistant homozygous genotypes Mi/Mi?Tm-2~2/Tm-2~2?Cf-9/-,it is CT-60,there are five tomato materials can aggregate two kinds of resistant homozygous genotypes;ten plants at the same time aggregation of two kinds of resistant homozygous genotypes tomato materials are obtained from the F2 segregating population.The triplex PCR system laid a foundation for assay breeding materials and assistant breeding,and the resistant tomatoes selected in this way can be applied in the future breeding.