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

Abstract

The 7B chromosome of common wheat was microdissected from pollen mother cells of the 78 monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase I treatments, the isolated chromosomes were subjected to 1–3 rounds of DOP-PCR amplification, which produced continuous DNA fragments ranging from 150 to 700 bp. Genomic Southern hybridization confirmed that the PCR products were originated from the wheat genome. Cloning of portion (>200 bp) of the 3rd round DOP-PCR products (50 μL could generate about 20 000 recombinant clones. Characterization of 50 randomly chosen clones indicated that 21 clones produced discrete PCR products with the sue of 240–600 bp. Dot-blot hybridization showed that among the 21 clones, 11 (∼55%) were of low-copy nature while 10 (∼45%) were repetitive. Southern hybridization with the complete set of the CS “nullisomic-tetrasomic (NT)” lines demonstrated that all the 6 low-copy clones were specific to either chromosome 78 or the 7th homoeologous group, whereas the 3 arbitrarily chosen repetitive clones were non-specific, disperse sequences.