Molecular Markers Linked To Resistance Genes Sw-5 Of Tomato Spot Wilt Virus And Screening For Germplasm Resources

Tomato spotted wilt virus disease is a kind of key disease,which is widespread and harmful in the world.The experimental materials in this study were the tomato varieties with disease-resistant homozygous(Sw-5/Sw-5),resistant heterozygous(Sw-5/sw-5)and susceptible homozygous(sw-5/sw-5).Reference to relevant published literature,according to the gene sequence of Sw-5,specific primers were designed with software of Primer 5.0.The target fragment was obtained by PCR amplification,and we gained the SCAR marker of “SW-5-2” closely linked to disease resistance gene Sw-5.The marker can distinguish homozygous and heterozygous,and it is co-dominance SCAR marker.SCAR marker “SW-5-2”: Throughing the PCR amplification with specific primer SW-5-2F/SW-5-2R,disease resistant homozygous materials appeared the specific segment of 574 bp,resistant heterozygous materials appeared the specific segments of 464 bp and 574 bp,and susceptible homozygous materials appear the specific segments of 464 bp.Primer sequences are SW-5-2 F: 5’-AAT TAG GTT CTT GAA GCC CAT CT-3’;SW-5-2 R: 5’-TTC CGC ATC AGC CAA TAG TGT-3’.Nine kinds of tomato materials from different genetic backgrounds and sources were used to prove the effectiveness of the marker “SW-5-2”.It was proved that the SCAR marker was clearly and easily recognizable,which was a reliable co-dominant marker.“SW-5-2” SCAR marker was used in the assisted selection of the 63 breeding materials of unknown gene-types.Finaly we gained 6 disease-resistant homozygous materials and 9 resistant heterozygous materials.On the basis of the marker “SW-5-2”,combined molecular markers of single disease resistance gene Ty-1 and I-2 respectively,the experimental conditions such as concentration of substrate,primers’ ratio and annealing temperature and so on were optimized repeatedly.As a result,the double PCR system for resistance genes Sw-5 and Ty-1 was established,and the double PCR system for resistance genes Sw-5 and I-2 was established respectively.The double PCR system for genes both Sw-5 and Ty-1: template DNA concentration was 1.5μL,primer’s ratio of SW-5-2 and TY-1 was 1μL:1μL,and annealing temperature was 57℃.The double PCR system for genes both Sw-5 and I-2: template DNA concentration was 1.5μL,primer’s ratio of SW-5-2 and I-2 was 0.5μL:1μL,and annealing temperature was 53℃.With the breeding materials of unknown gene-types,the two double PCR systems were used in the assisted selection.Resultly,we gained 2 disease resistant homozygous materials for genes Sw-5 and Ty-1,1 disease resistant homozygous materials for genes Sw-5 and I-2.The molecular marker “SW-5-2”,double PCR system for genes Sw-5 and Ty-1 and double PCR system for genes Sw-5 and I-2 gained in this study,not only provide an effective method for the breeding of disease resistance,but also lay a foundation for the further developing new tomato varieties with compound resistance genes,which is very important to the sustainable development of tomato production.