Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/1997
Publication Date: N/A
Citation: N/A Interpretive Summary: A limiting factor in isolating genes from major crop plants is the difficulty in isolating genetic markers near the gene controlling the trait of interest. This is due to the large size of the genome (amount of DNA per cell) in most crop plants. To aid in obtaining DNA markers near specific genes, a technique called chromosome microdisection, cutting out specific chromosome regions, was developed for a type of specialized chromosome in the immature corn tassel called pachytene chromosomes. As these chromosomes are more elongated than most of plant cell chromosomes, they permit the dissection of more specific chromosome regions than previously possible. After microdissection, the DNA in the isolated chromosome fragment is converted to a library of individual pieces. This technique should speed the efforts to isolate genes of agronomic interest in corn.
Technical Abstract: A limiting factor in isolating genes from major crop plants is the difficulty in isolating genetic markers near the gene controlling the trait of interest. This is due to the large size of the genome (amount) of DNA pr cell in most plants. For the first time, pachytene chromosomes were used for microdissection, amplification, and cloning using the short arm of maize chromosome 6. For this purpose, methods for the preparation of unstained pachytene chromosomes were developed. In two separate experiments, 10 and 12 segments of the satellite region were isolated utilizing glass needles, and DNA was amplified by adaptor-polymerase chain reaction (PCR). After amplification, maize specific DNA was verified by genomic Southern hybridizations. Chromosome in situ suppression-hybridization confirmed that the adaptor PCR-products originated from the microdissected 6S-region. The adaptor PCR-products were cloned into a plasmid vector and the chromosome region-specific libraries were characterized. This technique should speed the efforts to isolate genes of agronomic interest in maize.