Cloning And Analysis Of Resistance Gene Analogs (RGAs) In Wild And Cultivated Tomatoes

With high commodity features, cultivated tomatoes (Solarium lycopersicum) are more susceptible to pathogen infection than wild tomatoes. Wild tomatoes are the main source for resistance gene. In this study,3 wild tomato accessions and 2 cultivated accessions were selected:LA3858 (S.peruvianum), LA1777 (S. Habrochaites), LA716 (S.pennellii), Zhongshu6 and A113 (S.lycopersicum). Degenerate primers were designed to amplify resistance gene analogs (RGAs) in these materials by PCR. The results were as follows:1. Fifteen-one NBS-encoding RGAs with uninterrupted ORF were finally obtained. Their amino acid sequences all contain obvious conserved motifs such as P-loop、Kinase2a、Kinase3a and HD.2. These RGAs could be clustered as 7 group based on multiple alignment and phylogeny tree construction. There were only 3 RGAs belonging to TIR-NBS-LRR while the other RGAs were all non-TIR-NBS-LRR RGAs.3. RGAs from LA3858 were aligned with several nematode resistance genes and RGAs from LA1777 were aligned with a few late blight resistance genes. Results show that most of RGAs from LA3858 clustered tightly around Mi-1.2 and Hero whereas RGAs from LA 1777 also showed certain similarity with late blight resistance genes.4. Only a few RGAs from LA716 showed similarity with plant resistance genes. And there were much less RGAs from cultivated tomatoes clustered with resistance genes than wild tomatoes.5. Based on former fine mapping results of Mi-3 gene and recent development on tomato genome sequencing project, we confined the Mi-3 to an area of about 35kb.6. The sequence was put into FGENESH program to predict genes. After analyzing the domains of predicted genes and considering their potential functions, only four genes were selected as Mi-3 candidate genes.7. The candidate genes were amplified by PCR and over-expression vectors are being constructed.