PCR cloning of partial nbs sequences from grape (Vitis aestivalis Michx)

Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R genes. Here, we report a 4-week undergraduate laboratory exercise that mimics the research environment to PCR-clone partial nbs sequences using degenerate primers corresponding to the phosphate-binding loop (P-loop) and GLPL motifs within the NBS domain of potential R gene products from the North American grape, Vitis aestivalis Michx. Students were able to complete the laboratory procedures successfully and obtained four different clones, among which three are new. Through the laboratory exercise, students learned a variety of important molecular techniques including genomic DNA isolation, DNA quantification, PCR, agarose gel electrophoresis, DNA extraction from agarose gel, ligation, bacterial transformation, and plasmid DNA isolation and purification. They also used currently available web-based bioinformatic programs for sequence analysis. The laboratory exercise provides students the hands-on experience on PCR cloning and shows them how it is done in a research environment. The clones obtained may be further tested for their potential use as markers to differentiate resistant cultivars from the susceptible ones, a useful tool in breeding programs.