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 and Yasunari Ogihara*
Dr. Yasunari Ogihara, Kihara Institute for Biological Research, Yokohama
City University Y. ogihara@yokohama-cu.ac.jp
The Plant Journal (2000) 23(5), 687-695
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.