Development of SCAR marker linked to a root-knot nematode resistant gene in peanut
Root-knot disease caused by Meloidogyne spp. is the most important nematode disease of peanut. Even though many management strategies have been applied to control this disease on peanut, resistance is the most recommendable. Marker-assisted selection has been used as a useful tool for screening of resistant individuals in segregating populations. However, it requires many laborious steps. Thus, there is a need for PCR - based markers, which are more practical, rapid, and efficient.
In this study, we tried to develop a SCAR marker linked to root-knot nematode resistance locus in peanut based on the RFLP marker R2430E. The entire sequence of R2430E was 2217 bp and contained one putative open reading frame (ORF) of 713 nucleotides. Thirteen primers including 5 forward and 8 reverse primers were synthesized to sequence the entireR2430E. Based on the results of BLAST searches, R2430E appeared to encode an AAA ATPase containing von Willebrand factor type A (VWA) domain from Magnetococcus sp. MC-1 (106 bits). To determine if there is a portion of the R2430E that hybridizes only to a band co-segregating with the resistance locus, we generated 4 probes spanning different parts of the gene. Southern analysis using these probes revealed identical banding patterns for each probe. Therefore, we concluded that there is very limited if any sequence polymorphism between different alleles detected by the R2430E probe. Additionally, this conclusion is supported by the experiment in which we tested 25 primer pairs derived from the R2430E using genomic DNA from both resistance and susceptible genotypes. In this experiment, all primer pairs amplified identical PCR fragments, suggesting again that there is little or no sequence divergence between putative alleles as differentiated by southern blotting. To identify possible single nucleotide polymorphisms (SNPs) between polymorphic R2430E RFLP bands, we cloned several fragments that span the entire R2430E transcribed sequence. Surprisingly, no SNPs were identified in the transcribed region of this gene. We propose that polymorphism detected by this RFLP marker is outside of the R2430E.