Submitted to: Canadian Journal of Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/11/2004
Publication Date: 4/6/2005
Citation: Zhang, F.Y., Yin, W.B., Shi, R., Hu, Y.K., Yan, Y.M., Chen, Y.H., Zhou, Y.H., Hu, J., Wang, R., Hu, Z.M. 2005. Construction and characterization of chromosome 1b specific dna library of wheat. Canadian Journal of Plant Science 85:309-316 Interpretive Summary: Mapping of cloned molecular markers from the whole genome of wheat is time-consuming and costly. The availability of chromosome micro-dissection technique circumvents the long procedure. By isolating the 1B chromosome of wheat first and then establishing a library of PCR products specific to this chromosome, we created a library of 248,000 recombinant clones that cover single-, low-, medium- and high-copy DNA sequences. These clones will be useful in constructing high density molecular map for 1B chromosome of wheat.
Technical Abstract: Chromosome 1B was microdissected and collected from chromosome spreads of wheat (Triticum aestivum L. cv. 'Jing 411'), using a glass needle. DNA of the isolated chromosome was amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The second-round PCR products were verified by Southern hybridization with DIG-labeled genomic DNA of wheat. The result initially showed they were from the DNA of wheat genome. A pair of SSR primers specific to chromosome 1B was used to verify the origin of the PCR products from the isolated chromosome. The result confirmed that PCR products originated from chromosome 1B. The second round of PCR products from chromosome 1B were cloned into plasmid pUCm-T vectors to create a chromosome specific library, which included approximately 248,000 recombinant clones. Characterization of 100 randomly selected clones of the library showed that the inert size ranged between 0.5 and 2.0 kb, with an average of 1000bp. Randomly selected 288 clones were characterized with dot hybridization blotting, of which 57.2% were medium/high copy clones and 42.8% low/single copy clones. The application of this technique for establishing high-density molecular maps for chromosome 1B was discussed.