CONSTRUCTION AND CHARACTERIZATION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY FOR SOYBEAN PI437654

Clemson University, Genomics Institute, Room 100, Jordan Hall, Clemson, SC 29634 USA

We have constructed a soybean BAC library using the strain PI 437654. The library contains 73,728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kbp with a range of 20 to 325 kbp, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kbp. Of the clones larger than 100 kbp, 92% are greater than 120 kbp. Based on a genome size of 1,115 Mb, library coverage is 9 haploid genome equivalents. High-density 23 x 23 cm colony filters were made using the Genetix Q-Bot allowing the entire library to be represented on a set of four filters (18,432 clones per filter). Screening the BAC library filters with cpDNA probes showed that contamination of the genomic library with cpDNA clones was low (1.85%). Library screening was also performed using three RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulting in an average of 18 hits per probe (range = 7 to 30). Pools of cDNAs obtained from suppression subtractive hybridizations of SCN infected and noninfected roots of PI 437654 were hybridized to the BAC library filters. The 629 BACs identified from filter hits were fingerprinted and analyzed using FPC software resulting in 85 different contigs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.