Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R |
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Authors: | Yihua Zhou Zanmin Hu Benyuan Dang Huai Wang Xiangdong Deng Lanlan Wang Zhenghua Chen |
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Institution: | (1) Institute of Genetics, Chinese Academy of Sciences, Beijing 100101, P.R. China, CN |
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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.
Received: 5 December 1998; in revised form: 15 April 1999 / Accepted: 29 April 1999 |
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