Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Yao Guang Liu, Kiyotaka Nagaki, Masako Fujita, Kanako Kawaura, Masahiko Uozumi, Yasunari Ogihara

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.

Original languageEnglish
Pages (from-to)687-695
Number of pages9
JournalPlant Journal
Volume23
Issue number5
DOIs
Publication statusPublished - Oct 2 2000
Externally publishedYes

Keywords

  • Binary P1 phage
  • Gene screening
  • Genomic library
  • Hexaploid wheat
  • Pooled PCR
  • TAC

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

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