title: Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R.
abstract: Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 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 evaluated ranged in size from 300-1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library.
authors.affiliation: Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, P.R. China.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: Z
authors.initials: Z
authors.lastname: Hu
authors.forename: B
authors.initials: B
authors.lastname: Dang
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: X
authors.initials: X
authors.lastname: Deng
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D002875:Chromosomes; D003001:Cloning, Molecular; D017931:DNA Primers; D017403:In Situ Hybridization; D016133:Polymerase Chain Reaction; D012434:Secale / Q000235:genetics*
chemical_list: D017931:DNA Primers