Isolation of a full-length CC-NBS-LRR resistance gene analog candidate from sugar pine showing low nucleotide diversity

The nucleotide-binding-site and leucine-rich-repeat (NBS-LRR) class of R proteins is abundant and widely distributed in plants. By using degenerate primers designed on 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 polymerase chain reaction 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 coiled coil (CC)-NBS-LRR subclass of NBS-LRR resistance proteins in GenBank. Sequence analyses of this gene in megagametophytes from two sugar pine trees segregating for the hypersensitive response to white pine blister rust revealed zero nucleotide variation. Moreover, there was no variation found in 24 unrelated sugar pine trees except for three single-nucleotide polymorphisms located in the 3' untranslated region. Compared to other genes sequenced in Pinaceae, such a low level of sequence variation in unrelated individuals is unusual. Although, numerous studies have reported that plant R genes are under diversifying selection for specificity to evolving pathogens, the resistance gene analog discussed here appears to be under intense purifying selection.