Identification of expressed resistance gene analogs (RGA) and development of RGA-SSR markers in tobacco

Tobacco is an important cash crop and an ideal experimental system for studies of plant-pathogen interactions. Identification of tobacco resistance (R) genes and resistance gene analogs (RGAs) is propitious to elucidate the underlying resistant mechanisms. In recent years, the public tobacco EST (expressed sequence tags) data set, which provides a rich source for identifying expressed RGAs, has enlarged substantially. In this study, 149606 Uni-ESTs were assembled from 412325 tobacco ESTs available in GenBank, scanned with 112 published plant R-genes protein sequences, and 1113 Nicotiana (tobacco) RGAs (NtRGAs) were identified. The majority of them comprised the common R-genes domains, such as NBS-LRR, LRR-PK, LRR, PK and Mlo, while we were unable to identify 109 RGAs using published domains of R-genes. Upon sequence alignment, 1079 NtRGAs were allocated on 712 loci within the Nicotiana benthamiana genome. A total of 78 simple sequence repeats (SSRs) were identified from 72 NtRGAs, and out of 64 newly designed primer pairs, 54 primer pairs generated clear bands upon PCR amplification using tobacco genomic DNA. Only nine primer pairs displayed polymorphism in 24 varieties of tobacco, with 2-4 alleles per locus (2.56 alleles on average), while 41 primer pairs were able to detect polymorphisms in six wild species of genus Nicotiana, with 2-4 alleles per locus (2.61 alleles on average). nema