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GrainGenes Reference Report: TPJ-24-687

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Reference	TPJ-24-687
Title	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
Journal	The Plant Journal
Year	2000
Pages	687-695
Author	Liu YG [ Show all 6 ]
Abstract	Summary: Three large-inert 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
Keyword	[ Hide all but 1 of 46 ] agrobacterium agrobacterium tumefaciens artificial chromosome chinese spring chromosome clone common wheat culture development dna libraries escherichia coli gene transfer genetic transformation genome genomic dna genomic library grass genomes haploid haploidy hexaploid hexaploid wheat isolate library maintenance marker method multigene family pcr polymerase chain reaction pooled pcr positional cloning regulatory sequences screening sequence single single-copy spring sts sts marker system tac target transfer triticum triticum aestivum vector

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