Abstract:

Chromosome 6B of wheat (Triticum aestivum L.) in mitotic metaphase spreads was micro-dissected with Nd:YAG laser microbeam into four segments and then each segment was collected by glass needles. The DNAs of the isolated chromosomal segments were separately amplified by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The presence of region-specific DNA from each of four segments was verified by Southern hybridization. The second round PCR products from four segments of chromosome 6B were cloned into a pGEM T-vector to create four chromosome region-specific libraries, named R1, R2, R3 and R4, which included 2.1×10⁵; 2.74×10⁵; 2.45×10⁵ and 2.93×10⁵ recombinant clones, respectively. A total of 150 randomly selected clones from each library were characterized by mini plasmid DNA preparation and enzyme restriction. Results showed that the size of inserts ranged from 300 to 1 800 bp with an average of 820 to 870 bp, of which 43%-48% were low/unique copy and 42%-47% were medium/high copy sequences. A set of microsatellite sequences located on chromosome 6B and other chromosomes of wheat were used for the verification of PCR products from micro-dissected chromosomal segments. The results reported here should facilitate the molecular genetics analysis of different fragments from single chromosomes of a plant.

Key words: wheat, chromosome microdissection, LA-PCR, chromosome region-specific DNA library