Genome-wide probing of NBS-LRR encoding genes in red clover (Trifolium pratense L) for the identification of resistance gene analogs in Trifolium alexandrinum L

Abstract

The nucleotide-binding sites (NBS) domain is the highly conserved domain of nucleotide-binding sitesleucine rich repeats (NBS-LRR) class encoded by resistance genes (R genes), and it is used to identify disease resistancerelated genes. This study was based on the genome-wide identification of NBS-LRR encoding genes in Trifolium pratense to recover resistance gene analogs (RGAs) in Trifolium alexandrinum. In T. pratense, 251 protein sequences were identified to have the NBS domain. Of these, 16 NBS proteins were predicted to be localized to the mitochondria, 9 to the chloroplasts, 44 to the secretory pathways, and 182 to subcellular locations other than chloroplasts or mitochondria. The structure pattern of predicted NBS genes of the NBS-LRR group was displayed as single gene architecture having an untranslated region (UTRs) with four CD regions. The conserved sequences of the NBS-LRR group were used for primer designing to recover RGAs from T. alexandrinum. A maximum likelihood method based phylogram was constructed for exploring the phylogenetic relationship amongst NBS genes encoding protein sequences of T. pratense and R genes of Medicago truncatula and Medicago sativa encoding protein sequences having NBS-LRR domain that showed the highly close phylogenetic relationship among them, which supported the highly conserved nature of NBS-LRR class. Hence, we used the conserved NBS-LRR genetic regions to recover the RGAs from the T. alexandrinum genome. Therefore, the DNA of a cultivar of T. alexandrinum was subjected to polymerase chain reaction (PCR) analysis using the primer designed from these regions; and all primers, except RNL3-F/R, RNL5-F/R, and RNL7-F/R, gave the amplification.