关键词: 欧洲山杨, 染色体, 显微分离, 特异性文库, 荧光原位杂交

Abstract: This study was performed to establish a method for single chromosome microdissection and microcloning in forest plants using poplar ( Populus tremula) as a model. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with the fine glass needle controlled by a micromanipulator. The dissected chromosome was amp lified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 200 bp to 3 000 bp smear DNA fragments were obtained. Southern hybridization result showed the PCR products from the single poplar chromosome were homogeneous with the poplar genomic DNA, indicating thatDNA from the single chromosome has been successfully amplified. Then, he second round PCR productswere used as a complex probe mixture for fluorescent in situ hybridization ( FISH) on the metaphasespreads of poplar. Hybridization signals were observed, mainly, along the entire chromosome 1, at the same time, signalswere also present on telomeric and centromeric regions of other chromosomes. The second round
PCR p roducts from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. App roximately 3 ×10⁵ recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230 bp to 2 200 bp with an average of 800 bp. This library will facilitate specific probe screening, molecular map construction, gene tagging and gene cloning on this chromosome.

Key words: Populus tremula, Chromosome, Microdissection, Chromosome specific DNA library, Fluorescent in situ hybridization