Reference Report for IND22054028
| Title: | A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance |
| Authors: | Tompkins, J.P., Mahalingam, R., Smith, H., Goicoechea, J.L., Knap, H.T., Wing, R.A. |
| Source: | Plant Mol. Biol. 1999, 41(1):25-32 |
| Abstract: | A soyabean bacterial artificial chromosome (BAC) library was constructed from soyabean plant introduction (PI) 437654. The library contains 73728 clones stored in 192 384-well microtiter plates. Random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones did not contain inserts. Some 90% of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. 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 soyabean cyst nematode (SCN) (Heterodera glycines) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analysed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analysed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse 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 pathogenic attack. |
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