| Most known plant resistance genes have a NBS domain and plant genomes contain many NBS-coding genes (so-called RGA, resistance gene analog). Tobacco (N.tobacum) genome has been partially sequenced. In this study, we first annotated RGAs based on the genome sequences and then determined NBS sequences from both cultivated and wild tobacco populations and developed NBS SSR markers. The main results are as follows:1. Thirty NBS-coding genes were found in the tobacco genome through sequence assembling and gene finding.2. Total 426 NBS-coding genes including motifs of P-loop, Kinase2 and GLPL were determined in 24 cultivars and 5 wild tobacco lines through directly or cloning sequencing of PCR products using the above annotated RGAs as models. Of the 426 genes, 293 come from 11 tobacco RGA sub-families and others from other 19 subfamilies. These genes cover all known types of plant resistance genes.3. Population genetic investigation revealed at least two characteristics of tobacco RGAs. Firstly, the genetic diversity between cultivated and wild tobacco is significant. The densities of 65.3 SNP or 4.2 indel per Kb were observed between them based on four RGAs. Secondly, highly genetic similarity was observed among tobacco cultivars, respectively.4. Pervasive purifying selection characterizes the evolution of tobacco RGAs. In other hands, strong positive selections on several sites of some RGAs were detected.5. Forty-three SSR markers were designed based on the tobacco genomic sequences containing NBS domain as a pilot study of development of RGA related markers. Polymorphism was observed in 8 primer pairs, resulting in a successful rate of 18.6%. As a control, 50% rate was achieved based on non-RGA SSR primer designs. Our results indicate that SSR is a good choice for molecular marker development in tobacco genomes.
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