Investigation of variations in NBS motifs in alfalfa (Medicago sativa), M. edgeworthii, and M. ruthenica

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials from Asia, which have a remarkable ability to survive stress , but are genetically distant from cultivated alfalfa (M. sativa L., 2n = 4x = 32). In lieu of hybridization, potentially important genes from the Asian species could be transferred to alfalfa using transgenic techniques. Many plant disease resistance genes (R-genes) belong to the nucleotide binding site-leucine rich repeats (NBS-LRR) superfamily and have several highly conserved NBS region motifs, which can be used to identify them. Thus, genomic DNA of alfalfa and the two Asian species was primed with a degenerate forward P-loop primer paired with degenerate reverse primers designed for GLPLA, RNBS-C, or RNBS-D-non-Toll/Interleukin (RNBS-D-non-TIR) motifs. The P-Loop/GLPLA primer combination yielded the largest number of sequences without stop codons, and alfalfa had the greatest variation within the kinase and RNBS-C domains; however several kinase and RNBS-C sequences were unique to M. edgeworthii or M. ruthenica. Priming with the P-loop/RNBS-C primer pair resulted in some open reading frames, which apparently did not belong to the NBS-LRR superfamily. The only open reading frames for the P-loop/RNBS-D-non-TIR primer combination were for M. ruthenica. These non-TIR-type clones, and the only non-TIR-type clone from the P-loop/GLPLA PCR (alfalfa), had sequences that were markedly different from the TIR-type sequences. The RNBS-C region may be useful in identifying non-TIRtype R-gene analogs in future studies. Screening for unique R-gene analogs in genetically distant Medicago species may be a very effective way of isolating potential new R-genes for transfer to alfalfa. Key words: Alfalfa, disease resistance, kinase, Medicago edgeworthii, Medicago ruthenica, NBS-LRR, R-gene analog