Construction of poplar (Populus tremuld) chromosome 1 - Specific DNA library by using a microdissection technique

摘要

A method for single-chromosome microdissection and microcloning was established in forest plants using poplar (Populus tremula) as a model. By use of meristernatic cell division in root tip and the wall degradation hypotonic method, well-spread poplar metaphase chromosome spreads showing low contamination were quickly prepared and fitted for chromosome microdissection. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with a fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by the Sau3A linker adaptor-mediated PCR technique, by which 200- to 3000-bp smear DNA fragments were obtained. Southern hybridization results showed that the PCR products from the single poplar chromosome were homogeneous with poplar genomic DNA, indicating that DNA from the single chromosome has been Successfully amplified. Next, the second-round PCR products from the single chromosome I were cloned into T-easy vectors to generate a DNA library of the chromosome 1. About 3 x 10(5) recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230-2200 bp, with an average of 800 bp. Therefore, this research suggests that microdissection and microcloning of single small chromosomes in forest plants is feasible.