Plant & Animal Genome IX Conference

Poster: Large Insert Libraries, Gene Isolation, Etc.
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ANALYSIS OF 150 KB OF DNA SEQUENCE SURROUNDING THE CITRUS TRISTEZA VIRUS RESISTANCE GENE (CTV)

¹ Department of Botany and Plant Sciences, University of California, Riverside, CA 92521

² Department of Plant Pathology and Microbiology, Texas A&M University, Weslaco, TX 78596

Citrus tristeza virus (CTV) occurs in most citrus producing areas and causes significant economic losses in many countries. Transfer of resistance genes from other species is one approach to controlling the disease. The Ctv gene occurs in the Citrus relative Poncirus trifoliata and confers resistance or immunity to most CTV isolates. To clone Ctv, we identified molecular markers flanking the gene, prepared a BAC library, and completed a 1.2 Mb chromosome walk between the flanking markers. Fine mapping of the Ctv gene relative to marker-delineated recombination breakpoints within this contig identified a 250 kb region that should contain the resistance gene. Four overlapping BAC clones covering this region were subcloned and sequenced. Two clones that span 150 Kb at the core of this region were sequenced to 6X coverage using a bidirectional sequencing strategy. Several large contigs were assembled and joined into a single 150 kb sequence based on the occurrence of forward and reverse end-sequences of specific subclones in different contigs. Sequence comparisons and database searches of the 150 Kb core revealed several R gene homologs that contain NBS and/or LRR sequence motifs. At least two R gene homologs are closely linked (within 30 Kb) and three others are separated by sequences with high homology to retroelements. About 60% of the total sequence in this region is derived from retroelements, which may play an important role in disease resistance gene evolution.