Studies On Laser Micro-dissection Of Chromosome In Populus

Technique of chromosome micro-dissection and-clone is a bridge connecting classic genetics and cytogenetics, being playing important roles in many fields of biological research, such as genetics, phylogenesis and evolution. Techniques of chromosome preparation, Laser micro-dissection and collection of chromosomes for Leica LMD 6000 laser micro-dissection system, amplification and AFLP marker of the isolated chromosomes were studied by using pollen mother cells (PMCs) of a Populus species as materials. Some results as follows,1. Chromosome preparation for laser micro-dissection system was solved, which indicated that laser micro-dissection of PMCs meiotic chromosomes in Populus was feasible. Chromosome preparation for laser micro-dissection is difficult, because the chromosomes must spread on the PEN or POL membrane. We tested many chromosome preparation methods, and high-quality meiotic chromosome preparations were obtained by applying heat-shocks of high temperatures, chromosomes spread evenly to be fit for chromosomes laser micro-dissection.2. PMC meiotic chromosomes of P. canadensis Moench were successfully isolated by the laser micro-dissection system (Leica LMD 6000, Germany), which provided a new way for genetic studies of the genus Populus. The effectiveness of laser micro-dissection is high with a laser beam of power 80-110 at a speed of 2-5 by trial and error. And we successfully separated 1-4 bivalents connected with nucleoli in Pollen mother cells at diakinesis of Populus.3. Molecular biology of laser micro-dissectioned chromosome in Populus was studied. We found that the AFLP amplification products of laser micro-dissectioned chromosomes showed significant polymorphism by digesting with Sau3A I, LA-PCR amplification and AFLP marker; then the specific AFLP fragment was separated, purificated, cloned and sequenced, however the result showed homology very low with Populus trichocarpa by the BLAST program at the Genbank database. More studies are necessary to explain the reason.