| In this study, 125 samples of the 25 natural Korean pine (Pinus koraiensis) populations which distributed in Liao ning, Ji lin and Hei longjiang provinces of China were used for Molecular phylogeography research. Through the DNA extraction, purification, RFLP-PCR, restriction endonuclease digestion, PCR product sequencing and statistical analysis of the sequencing results, and ultimately got the chloroplast DNA non-coding region of the psbA-trnH sequence variation. The establishment of haplotype phylogenetic trees, combined with the geographical distribution of various groups to explore the geography phylogenetic pattern of natural Korean pine in Northeast China, initial guessed when Quaternary Ice Age came where was the refuge of natural Pinus koraiensis for the Ice Age in China, as well as spread ways after the Ice Age.Natural Korean pine cpDNA of the psbA-trnH sequences of non-coding region was amplified, 115 DNA sequences were obtained after direct sequencing from PCR products, the actual length between 611bp to 614bp, showed length polymorphism. A, T, C, G ratio of the four nucleotides were 27.5% -28.0% (average 27.7%), 32.1% -32.4% (average 32.3%), 19.7% -20.0% (average 19.9%), 19.9% -20.4% (average 20.2%), A + T content (60%) slightly higher than the G + C content (40%), this character appears in many non-coding region.After aligement and analysis by ClustalX ver1.81 software, the result showed that 16 variation sites existed in a total of 115 DNA sequence, there have been 17 kinds of mutation types, and delimited 18 cpDNA haplotypes.From the existence of haplotypes in the populations, we can see that there was a wide range of variation within the population of natural Korean pine in China; From the distribution of the haplotypes during populations, we can see that HA haplotype was the ancestor, on behalf of the parts with same sources of various groups, the majority of haplotypes existed only in a particular population, a small number of the existence of haplotype-sharing phenomenon showed that geographical differentiation was existed between haplotypes. Haplotype combinated with the geographical distribution, we can see that there were certain populations shared haplotypes HB, HC in Hei longjiang, Ji lin and Liao ning region, But the haplotype sharing HD and HE only existed in Ji lin, which indicated that there was some gene flow between populations in the three provinces, especially in the internal population of Ji lin Province, there was a short distance of gene flow, no spreading to the greater..Construction of natural Korean pine haplotype phylogenetic trees by Molecular evolutionary genetic analysis software MEGA 4.0, cpDNA haplotypes from the NJ and MP phylogenetic trees can be seen, all haplotypes constituted two major single-source groups. Contribution of two methods showed consistency in the classification. NJ tree based on haplotype clustering and geographic distribution of population can be seen that at the end of lineage tree haplotypes are located in two places in Ji lin and Hei longjiang. According to haplotype clustering and geographical distribution of the populations can prove once again, there is a certain degree of gene flow in Liao ning, Ji lin and Hei longjiang populations, Liao ning-Ji lin, Jilin-Hei longjiang were the major pattern.Through analysis of the distribution of all haplotypes in the northeastern region of China, synthesized haplotype-sharing and haplotype-specific distribution, we can speculate that natural Korean pine in China have been differentiated as early as the Quaternary Ice Age occurred, the Ji lin and Hei longjiang regions may be the refuge for natural Korean pine in our country when Quaternary Ice Age arrived. Bottleneck effect caused a random fixed of different haplotypes in the reguges. when the glaciers receded, Korean pine in these two areas spreaded to around, at the same time, Korean pine in Ji lin spreaded to the north. The specific proliferation way within the provinces remains to be further studied.
|
PDF Full Text Request