Detection,pyramiding Of Five Disease Resistance Genes In Tomato And Construction Of Universal Primer Multiplex PCR System

Tomato is a quite popular horticultural crop in our country and is one of the vegetables with the highest economic benefits.Due to the special environment of facility cultivation and the influence of continuous cropping obstacles,tomato diseases occur more frequently,which affects seriously the quality and yield of tomato.Conventional insecticides are not ideal for controlling insect pests and diseases,which may make them become resistant to drug and cause serious pollution to the ecological environment.Therefore,it is a highly effective way to solve this problem by breeding tomato varieties with resistance gene against multiple diseases.This study using the Functional Marker-assisted Selection and the Gene Polymerization Technology,237 tomato varieties were screened with the functional markers of resistant genes Tm-2~a,Cf-9,I-2,Ph-3,and Mi-1.Multi-resistant germplasms were selected to pyramid the five resistant genes by crossing.At the same time,we construct the universal primer multiplex PCR system to improve the detection efficiency and save the detection cost of tomato diseaseresistance genes,also to accelerate the process of disease resistance breeding.Then,multiplex PCR techniques were employed to test the existence of these resistant genes in F1 generation.The main results were as following:(1)The gene sequences of Cf-9 was used to develop functional marker,which was verified to be useful with resistant/susceptible germplasms.(2)Functional markers of Tm-2~a,Cf-9,I-2,Ph-3,and Mi-1 were used to screen 237 tomato germplasms.Every germplasm was found to have at least one resistant gene.Among the 237germplasm,107 only had one resistant gene,70 had two resistant genes,23 had three resistant genes,and only seven had four resistant genes.They were 11CT387-2M,11CHT164-2, 11CT150,11CT271,11CT730,11CT764 and 11CT774.(3)the seven germplasm with four resistant genes were selected to test the fruit weight,fruit shape index,skin thickness,flesh thickness and fruit dehiscence.The small fruit combination 11CT387-2M×11CT150 and big fruit combination 11CT271×11CT774 were selected to cross to pyramid the five resistant genes.(4)Six universal primers UP1～UP6 were used to chimerize with the 5’ends of the specific primers I-2 to form UP1-I-2～UP6-I-2 to test the specificity and susceptibility.UP6 was found to be suitable for constructing the universal primer multiplex PCR system.(5)Ph-3 functional marker and the multiplex PCR system of the other four resistant genes were used to test five F1 individual of the two cross combinations.Four F1 individual were found pyramid the five resistant genes and could be used in further breeding in the future.