Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R

Chromosome 1R was microdissected and collected from mitotic metaphase sprea ds of rye (Secale cereale L.) by using glass needles. The isolated chromoso mes were amplified in vitro by Sau3A linker adaptor-mediated polymerase cha in reaction (PCR). After amplification, the presence of rye-specific DNA wa s verified by Southern hybridization. The second-round PCR products from fi ve 1R chromosomes were cloned into a plasmid vector to create a chromosome- specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evalu ated ranged in size from 300-1800 bp with an average size of 950 bp, of whi ch approximately 42% were medium/high copy and 58% were low/unique copy clo nes. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that m any chromosome 1R-specific sequences were present in the library.