Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/6/2005
Publication Date: 12/1/2005
Citation: Ling, P., Chen, X. 2005. Construction of a hexaploid wheat (triticum aestivum l.) bacterial artificial chromosome library and identification of candidate bac clones containing stripe rust resistance gene markers. Genome 48:1028-1036. Interpretive Summary: A genomic library containing large DNA insertions, such as bacterial artificial chromosomal (BAC) libraries, is essential for efficient cloning of genes of importance. When we initiated a project to clone wheat genes for resistance to stripe rust, a devastating disease in the US and many other countries of the world, BAC libraries were not available for common (hexaploid) wheat. This paper presents the results of construction of a BAC library using a wheat genetic stock possessing the Yr5 gene for resistance to all races of the wheat stripe rust pathogen identified so far in the United States. We demonstrated that the high quality of common wheat BAC library is useful for cloning the Yr5 gene through screening the library using direct molecular markers for the gene. A method for efficient screening BAC libraries also was developed. The BAC library should be useful in cloning not only Yr5 and other stripe rust resistance genes, but also genes controlling other important traits of wheat. The BAC library should also be useful in basic and applied research on wheat genome and manipulation.
Technical Abstract: A hexaploid wheat (Triticum astivum L.) bacterial artificial chromosome (BAC) library was constructed for the positional cloning of the wheat strip rust resistance gene Yr5. The Yr5 near-isogenic line were used to isolate the high molecular weight DNA as intact nuclei. The HindIII partial restricted large DNA inserts were cloned into pECBAC1 and pIndigoBAC-5 vectors. The wheat BAC library consists of 410,000 clones arrayed in 1,100 micro-titer plates of 384 wells each. Random sampling of 300 BAC clones indicated an average insert size of 140kb with the size range from 25kb to 365kb, and less than 5% of the clones did not contain inserts. Ninety percent of the clones in the library have an insert size greater than 100kb. Based on estimated hexaploid wheat genome size of 15,966 Mb, the BAC library was estimated to have a total coverage of 3.58 X wheat genome equivalents, giving approximately 96% probability of identifying a clone representing any given wheat DNA sequence. Twelve BAC clones containing Yr5 locus specific marker sequences were successfully selected by PCR screening three-dimensional BAC pools. The Yr5 locus co-segregating marker STS7/8 with high homology to many known plant resistance genes was used in the BAC pool PCR screening. The methodology for screening and isolation of candidate clones and the possible physical representation of those BAC clones related to Yr5 locus region were discussed. The T. aestivum BAC library constructed described here is a valuable genomic resource for positional cloning of Yr5 and other target genes of interest in the hexaploid wheat system.