A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clon es stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, an d 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 kb. Base d on a genome size of 1115 Mb, library coverage is 9 haploid genome equival ents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low ( 1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppressio n subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 4 37654 were hybridized to the BAC library filters. The 488 BACs identified f rom positive signals were fingerprinted and analyzed using FPC software (ve rsion 4.0) resulting in 85 different contigs. Contigs were grouped and anal yzed in three categories: (1) contigs of BAC clones which hybridized to for ward subtracted cDNAs, (2) contigs of BAC clones which hybridized to revers e subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a p athogenic attack.