Microdissection of chromosome 7B of common wheat and cloning of low-copy specific DNA sequences

The 7B chromosome of common wheat was microdissected from pollen mother cel ls of the 7B monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase I treatments, the isolated chromosomes w ere subjected to 1-3 rounds of DOP-PCR amplification, which produced contin uous DNA fragments ranging from 150 to 700 bp. Genomic Southern hybridizati on confirmed that the PCR products were originated from the wheat genome. C loning of portion ( > 200 bp) of the 3rd round DOP-PCR products (50 mu L) c ould generate about 20 000 recombinant clones. Characterization of 50 rando mly chosen clones indicated that 21 clones produced discrete PCR products w ith the size of 240-600 bp. Dot-blot hybridization showed that among the 21 clones, 11 (similar to 55%) were of low-copy nature while 10 (similar to 4 5%) were repetitive. Southern hybridization with the complete set of the CS "nullisomic-tetrasomic (NT)" lines demonstrated that all the 6 low-copy cl ones were specific to either chromosome 7B or the 7th homoeologous group, w hereas the 3 arbitrarily chosen repetitive clones were non-specific, disper se sequences.