Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifolia
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O R I GI N A L P A P E R
Z. Deng á S. Huang á P. Ling á C. Yu á Q. Tao C. Chen á M. K. Wendell á H.-B. Zhang F. G. Gmitter Jr
Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.) Received: 8 November 2000 / Accepted: 23 February 2001 / Published online: 25 April 2001 Ó Springer-Verlag 2001
Abstract A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identi®ed and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Eorts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be con®rmed through genetic complemenCommunicated by R. Hagemann Z. Deng á S. Huang á P. Ling á C. Yu á C. Chen M. K. Wendell á F. G. Gmitter Jr (&) University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA E-mail: [email protected]¯.edu Tel.: +1-863-9561151 Fax: +1-863-9564631 Q. Tao á H.-B. Zhang Texas A & M University, Department of Soil and Crop Sciences, Crop Biotechnology Center, College Station, TX 77843, USA
tation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial. Keywords Citrus tristeza virus (CTV) á Resistance gene á Genetic mapping á BAC contig development á Chromosome landing
Introduction Map-based cloning (MBC) strategies have been used successfully to isolate disease resistance (R) genes in several annual or biennial plant species. Examples include Pto (Martin et al. 1993) and Cf-2 (Dixon et al. 1996) in tomato, RPS2 (Bent et al. 1994; Mindrinos et al. 1994) and RPM1 (Grant et al. 1995) in Arabidopsis, Hs1 pro-1 in sugar beet (Cai et al. 1997), and Mlo in barley (Buschges et al. 1997). These R genes mediate resistance to diverse pathogens; their cloning has signi®cantly enhanced our understanding of
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