Construction and characterization of chromosome 1B specific DNA library of wheat

Abstract

Chromosome 1B was microdissected and collected from chromosome spreads of wheat (Triticum aestivum L. ‘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 using DIG-labeled genomic DNA of wheat. The results initially showed the DNA was from 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 results confirmed that the 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 insert size ranged between 0.5 and 2.0 kb, with an average of 1 kb. Randomly selected 288 clones were characterized with dot blot hybridization, of which 57.2% were medium/high copy clones and 42.8% low/single copy clones. The application of this technique to establish high-density molecular maps for chromosome 1B is discussed. Key words: Wheat, chromosome microdissection, chromosome-specific library