PAG-IV Plant Genome IV Conference

Town & Country Conference Center, San Diego, CA, January, 1995.

P13
Construction and Characterization of a Bacterial Artificial Chromosome Library for Gossypium hirsutum L.

SANGDUN CHOI, Michael S. Zwick, Robert E. Hanson, Sung-Sick Woo, David M. Stelly, H.J. Price and Rod A. Wing
Dept. of Soil and Crop Sciences, Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2123, USA

We have constructed a cotton BAC (bacterial artificial chromosome) library using high molecular weight DNA prepared from agarose microbead-embedded leaf nuclei from cotton MAR line CD3PIHP45H-2-89 (Gossypium hirsutum L.). The library contains 22,240 clones with an average insert size of 100 kb. Cotton DNA was cloned in the pBeloBAC11 vector using HindIII and BamHI sites as the cloning sites. Assuming a haploid genome size of 2,118 Mb, the BAC library contains 1.05 haploid genome equivalents with a 65% probability of isolating a specific genomic region. The library was screened with ten cotton cDNA probes and six probes hybridized to at least one BAC clone. FISH (fluorescent in situ hybridization) analysis was used to screen 12 large BACs for chimerism and physical location. Direct sequencing of BAC insert ends was performed to generate sequence-tagged sites suitable for mapping and chromosome walking. The BAC library will be a valuable resource for isolation of genomic clones, map-based cloning, and development of an integrated genetic map that encompasses recombination maps and physical maps.

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