WPGP/WHISP Research on Major Gene Resistance (MGR)
Positioning of MGR on a genetic map
MGR was mapped in several sugar pine families (Devey et al, 1995; Harkins et al, 1998). Conifer genomes are large and hundreds of genes could reside in the map interval that contains MGR.
Cloning by homology
In the mid-nineties, progress had been made in cloning and characterizing genes from a wide range of plant taxa that confer resistance to a variety of pathogens and pests, including viruses, bacteria, fungi, and nematodes. Surprisingly, most of these genes share significant similarities in sequence homology and structural motifs. Conserved regions present in plant resistance genes allow similar sequences to be isolated from other species using homologous primers and the polymerase chain reaction (PCR) (Kanazin et al, 1996; Leister et al, 1996; Yu et al, 1996; Lagudah et al, 1996; Rivkin et al, 1996a & b). Our research employed a similar approach toward cloning the R gene mediating resistance to WPBR. By using degenerate primers from the NBS domain in lettuce, we amplified sequences in sugar pine that shared sequence identity with many of the NBS-LRR class resistance genes catalogued in GenBank. The PCR products were used to probe a cDNA library constructed from needle tissue of sugar pine seedlings. A full-length cDNA was obtained that demonstrated high predicted amino acid sequence similarity to the CC-NBS-LRR subclass of NBS-LRR resistance proteins in GenBank. The resistance gene analog (RGA) sequence was given the designation, Pl_RGC-CC_ 1 (DQ017061) and characterized as 2926 bp in length that translates into a protein of 777 amino acids. Resequencing of Pl_RGC-CC_1 in megagametophyte DNAs from three sugar pine trees known to be heterozygous for MGR revealed no variation throughout the entire length of the cDNA. Therefore, no association could be found between observed SNPs and the MGR. A full description of Pl_RGC-CC_ 1 and details of resequencing can be found in Jermstad et al. (2006).
RAPD to SCAR conversion
Two closely flanking RAPD markers (OP_G16_950 and BC_432_1110) were recently converted to sequence characterized amplified regions (SCAR) markers. The appropriate RAPD band was excised from agarose gel, gel-purified, cloned into a plasmid vector, and DNA sequenced. Conversion to sequence-based markers will allow them to be mapped readily in other conifer species for comparative studies and also may be used as probes for BAC library screening.