A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea: Genome analysis, contig assembly, and gene cloning

Heng Zhu, Sangdun Choi, Andrea K. Johnston, Rod A Wing, Ralph A. Dean

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ~26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.

Original languageEnglish (US)
Pages (from-to)337-347
Number of pages11
JournalFungal Genetics and Biology
Volume21
Issue number3
DOIs
StatePublished - Jun 1997
Externally publishedYes

Fingerprint

Magnaporthe
Bacterial Artificial Chromosomes
Organism Cloning
Fungi
Clone Cells
Genome
Genes
Libraries
Nucleic Acid Hybridization
Retroelements
Chromosomes, Human, Pair 2
Membranes
Oryza
DNA
Nylons
DNA Probes
Gene Library
Morphogenesis
Adenylyl Cyclases
Restriction Fragment Length Polymorphisms

Keywords

  • Bacterial artificial chromosome (BAC)
  • Chromosome walk
  • Contig
  • Genome analysis
  • Magnaporthe grisea
  • Physical mapping

ASJC Scopus subject areas

  • Genetics
  • Microbiology

Cite this

A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea : Genome analysis, contig assembly, and gene cloning. / Zhu, Heng; Choi, Sangdun; Johnston, Andrea K.; Wing, Rod A; Dean, Ralph A.

In: Fungal Genetics and Biology, Vol. 21, No. 3, 06.1997, p. 337-347.

Research output: Contribution to journalArticle

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abstract = "Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ~26{\%} of the clones contain single-copy DNA. Approximately 35{\%} of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.",
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