Cloning Of NBS-LRR Type RGAs From Zizania Latifolia (Griseb.) And Their Genetic And Epigenetic Changes In The Introgression Lines

More than 50 plant disease resistance genes have been cloned and sequenced in the past ten years. Sequence comparision of cloned resistance genes from a wide range of plant taxa has revealed significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. Proteins encoded by resistance genes usually contain leucine-rich repeats (LRR) and nucleotide binding site (NBS). In this study, a pair of degenerative oligonuleotide primers designed from conserved sequences from coding regions of two disease resistance genes, N (tabacco) and RPS2 (Arabidopsis), were used to amplify related sequences from a wild rice species (Zizania latifolia Griseb.). The products of polymerase chain reaction from Z. latifolia genomic DNA using these primers were cloned into the pGEM-T vector, and eight distinct RGAs (resistance gene analogs) were obtained.The eight RGAs were sequenced and analysed with bioinformatic software Bioedit and Clustal X and then classified into seven groups. Based on their structural characteristics, the eight RGAs were deduced to belong to the non-TIR-NBS-LRR subfamily. Furthermore, based on the near complete rice genome sequence information, the eight RGAs were located in four different rice chromosomes.Southern blot analysis was performed with each of the eight RGAs as probes to the genomic DNA of Z. latifolia, the parent rice cultivar (Matsumae) and the introgression lines, whose genomic DNA was digested by the enzymes TaqI, MspI and HpaII respectively. The results showed that, compared with the rice parental line, the introgression lines showedchanges at the both the primary sequence level (variation in nucleiotides or indels) and DNA methylation patterns (including hepermethylation and demethylation). These results indicate that, on the one hand, the possible reasons why the parent rice was susceptible to diseases while the introgression lines were highly resistant to them; and on the other hand, thought-provoking ideas on R gene evolution from the aspect of epigenetics. Meanwhile, this study is helpful to a better understanding of the introgression lines of rice and wild rice.