The PCR (Polymerase Chain Reaction) Amplification And The Exploitation Of SCAR Molecular Mark Of Single Chromosome From Cunninghamia Lanceolata (Lamb.) Hook

The microdissection and microcloning of single chromosome of Cunninghamia lanceolata were carried on. In this study by means of chromosome microdissection, microcloning and random amplified polymorphic DNA (RAPD) locating molecular markers on chromosome and screening specific DNA fragments of single chromosome were investigated. The main results were highlighted in the following:1.Single chromosome microdissection and DOP-PCRChromosome microdissection can be finished by glass needle and glass capillary. The virtues of glass needle were as follows: It was nicety and directional to get single interest chromosome or specific chromosome segments by glass needle. But the process was difficult and it was easy contaminated by cytoplasmic DNA. The virtues using glass capillary were as follows: The process was easy. It can avoid loss of microdissected chromosome. And it was not easy to be contaminated by cytoplasmic DNA. But it was impossible to get specific chromosome segments and it could be contaminated by non-target chromosome. DOP-PCR amplification was carried out on isolated chromosome. Two rounds amplification were performed for each sample. The range of DOP-PCR products was between 200bp to 1000bp, with predominant fragments at 300bp~600bp. The Southern hybridization showed that the PCR products were derived from the Cunninghamia lanceolata DNA.2.Study on polymorphism among different single chromosome of Cunninghamia lanceolata by RAPDCunninghamia lanceolata has a symmetric karyotype comprising 11 pairs of metacentric and submetacentric chromosomes, one pair of which has satellite. After a satellite chromosome and other two different non-satellite chromosomes were isolated, DOP-PCR amplification was carried out on isolated chromosomes. Then the DOP-PCR products from three different single chromosomes were tested with RAPD. The results indicated: the polymorphism among different single microdissected Cunninghamia lanceolata chromosome was evident. This research provides a new method to identify microdissected Cunninghamia lanceolata chromosome on molecular level.3.Screening satellite chromosome specific fragments of Cunninghamia lanceolataA pair of satellite chromosomes and 20 other non-satellite chromosomes in one cell was isolated. The two kinds of chromosomes were amplified by DOP-PCR. And the DOP-PCR products were analyzed by RAPD-PCR using pairwise combinations of primers. There were fourspecific fragments between 500bp~250bp which were belong to satellite chromosomes in the RAPD profile with pairwise primers OPB07 and OPBIO. Another specific about 900bp fragment of satellite chromosomes was obtained in the amplified products with pairwise primers OPB07 and OPB18. And a specific about 250bp fragment was also belonging to satellite chromosomes, which was get in the RAPD profile using pairwise primers OPD07 and OPD05. It was got two specific homologous (about 99%) 907bp fragments after the approximate 900bp specific RAPD fragment was cloned and sequenced. It was successful to convert the specific 907bp RAPD marker to SCAR marker and locate it on satellite chromosomes. This research will make it possible to identify the satellite chromosome on molecular level and correspond a genetic linkage group of Cunninghamia lanceolata to a specific chromosome.