Isolation and Characterization of the Resistance Gene Sequences with NBS-LRR Domains in Citrus

Citrus bacterial canker disease, caused by Xanthomonas axonopodis pv. Citri (Xac), is one of the most severe diseases in citrus species and cultivars and one of quarantine pests in many countries. Amplifying the resistance gene analogs (RGAs) with the degenerate primers designed according to the conserved domains of the known resistance genes is an effective approach to identify disease resistant genes in plants. For searching citrus RGAs against Xac, in this study, a specific fragment of 500bp was amplified and cloned from each genomic DNA of six citrus cultivars which presented diverse resistance to Xac with degenerate primers designed on the conserved domains of some NBS-LRR resistant genes, and those RGAs were then characterized. The fragments amplified were classified to different types based on restriction enzyme digestion analysis, and totally, nine different sequences of RGAs, belonging to the non-TIR-NBS-LRR type in terms of their putative amino acid sequences, were obtained. Among those nine RGAs, RGAs 1-4, RGAs 4-1, RGAs 6-1 were similar to Citrus grandis x Poncirus trifoliate 18P32 gene (AY130787.1 - AY130789.1）, and the others hit the Poncirus trifoliata resistance protein-like protein (RGA 19) gene (AAU89644.1, AAU89653.1 and AAU89647.1). Eight RGAs with complete ORF all contained the motif P-loop, kin-2, “PLAL” as well as motif NBS-A, B and C. The RGAs cloned showed homology of 31.5%~39.1% to 2C-1gene, 27.1%~39.9% to Xa1 gene, 27.6%~28.0% to RPS5 gene and 30.6% ~90.1% to the 4 reported RGAs (AAU89653, AAU89644, XM002321779, and AY130787), respectively. Southern analysis indicated that among 5 citrus varieties, Ju Yuan, Chen Orange, San Ye, Shen Orange and Sweet Orange showed 2 copies, 3 copies, 1 copy, 3 copies and 2 copies of RGAs, respectively.