Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus

Numerous disease resistance gene-like DNA sequences were cloned from an int ergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenera te primers designed from conserved NBS (nucleotide-binding site) motifs fou nd in a number of plant resistance genes. Most of the cloned genomic sequen ces could be translated into polypeptides without stop codons, and the sequ ences contained the characteristic motifs found in the NBS-LRR class of pla nt disease resistance genes. Pairwise comparisons of these polypeptide sequ ences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1-RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage o f amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mappi ng of these sequences and to assess their potential Linkage relationship wi th disease resistance genes in Poncirus, we developed CAPS markers by desig ning specific primers based on the cloned DNA sequences and subsequently id entifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and o ne fragment (Pt8a) was associated with the major gene responsible for the c itrus nematode resistance (Tyr1); both genes are from Poncirus and of impor tance to citrus survival and production. These polymorphic fragments were l ocated on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences am plified with the NBS-derived degenerate primers are valuable sources for de veloping markers in disease resistance-gene tagging, mapping, and cloning.