Microdissection of a single chromosome and construction of the microclone library from soybean

A single chromosome was microdissected from the metaphase chromosomes of so ybean by using a glass needle. After two rounds of Sau3A linker-adaptor-med iated PCR, smear DNA fragments ranged from 0.3-2.5 kb were obtained. Southe rn hybridization showed that the PCR products from the dissected chromosome were homologous with the soybean genomic DNA. In situ hybridization of the chromosome PCR products to the mitotic metaphase spreads showed that the P CR products were hybridized to the soybean, but the chromosomal signals wer e not restricted to the microdissected chromosome. The second-round PCR pro ducts from the microdissected chromosome were cloned into plasmid vector to generate a chromosome-specific microclone library, which included approxim ately 200,000 recombinant clones. 178 randomly selected clones were estimat ed to range in size from 0.3 similar to1.8 kb, with an average size of 830 bp, of which approximately 44% were high/medium copy clones, while 56% were low/unique copy clones.