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

J. P. Tomkins, R. Mahalingam, H. Smith, J. L. Goicoechea, H. T. Knap, R. A. Wing

Research output: Contribution to journalReview article

57 Scopus citations

Abstract

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73728 clones 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, 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 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 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 suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI 437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed 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.

Original languageEnglish (US)
Pages (from-to)25-32
Number of pages8
JournalPlant Molecular Biology
Volume41
Issue number1
DOIs
StatePublished - Sep 1 1999
Externally publishedYes

Keywords

  • BAC fingerprinting
  • BAC library
  • Cyst nematode
  • Soybean
  • cDNA subtraction

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

Fingerprint Dive into the research topics of 'A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance'. Together they form a unique fingerprint.

  • Cite this