Plant & Animal Genome V Conference
Town & Country Hotel, San Diego, CA, January 12-16, 1997.
PAG-V: W75 - CONSTRUCTION AND SCREENING OF A SOYBEAN BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY AND IDENTIFICATION OF CLONES LINKED TO A MAJOR SOYBEAN CYST NEMATODE (SCN) RESISTANCE LOCUS.
W75
CONSTRUCTION AND SCREENING OF A SOYBEAN BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY AND IDENTIFICATION OF CLONES LINKED TO A MAJOR SOYBEAN CYST NEMATODE (SCN) RESISTANCE LOCUS.
DANESH, DARIUSH, Silvia Penuela, Vergel C. Concibido, Heather Nordstrom, Nevin Dale Young
Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, U.S.A
A BAC library consisting of 30,720 clones with an average insert size of 120 kb has been constructed from soybean nuclear DNA. The library was constructed primarily, using the EcoRI site of pECSBAC4 plus a small number in the HindIII site of pBeloBAC11. Several variables were tested and optimized. The library, which consists of approximately three genome equivalents, was screened with four RFLP markers linked to a major SCN resistance locus on linkage group G. Multi-probe hybridization of high density filters representing 1,536 BAC clones each, uncovered eight positive clones ranging in size from 80 to 220 kb and an average insert size of 125 kb. Comparative restriction digestion analyses of the parental soybean DNA and the positive BACs showed that four of the clones map near the major SCN resistance locus representing 0.4 Mb of the soybean genome in this region. Two BAC clones are positive for B053, which is the RFLP marker closest to the SCN gene. These overlapping clones appear to contain three copies of B053 in a region of 180 kb. Another positive BAC clone, 110 kb in size, hybridizes to a second RFLP locus, Bng 122. Southern blot analysis, using whole genomic DNA as probe, indicate that these BACs are mostly low copy DNA, which is unusual in the highly repetitive soybean genome. The characterization of a fourth 120 kb positive BAC clone is in progress. Pulsed field gel electrophoresis analyses to estimate the physical-to-genetic relationship in the SCN region have indicated that there are 120-150 kb per cM. Therefore, further characterization of positive BACs by end-cloning, sub-cloning and contig construction will provide more information to physically bridge closely linked markers in the region and establish a framework for positional cloning of SCN in soybean.