Phylogeography Of Bretschneidera Sinensi Hemsl

Bretschneidera sinensi has been listed as the National First-Grade protected plants in China. It is the important scientific research material for phylogeny, paleogeography and paleoclimate. The phylogeography and genetic diversity of B. sinensi have been researched using non-coding sequence of chloroplast DNA markers. The main results are as follows:(1) Establishment and optimization of cpDNA PCR reaction system for B. sinensi. The optimum cpDNA PCR amplification system were:30μL reaction system contained30ng genome DNA,10×pfu PCR buffter,2.9mM Mg2+,120mM dNTP,11μM primers and3units Taq polymerase. The optimum amplification program was, predenaturing at94℃for4min,35cycles of denaturing95℃for50s, annealing for50s,72℃for5min, and10min of final extension. The annealing temperatures were different according to different primers.(2) Screened stable and repeatable primers for chloroplast DNA PCR amplification system of B. sinensis.Using the optimized PCR system,3pairs of cpDNA primers were screened, which were PipetB1411F-PipetD738R、psbA-trnHGUG and trnh-tmF. The annealing temperatures respectively are52℃,59℃and56℃.(3) Analyzed the population genetic structure of B.sinensis populations.A total of10cpDNA haplotypes (Hap1-10) were identified. The Hapl is the most widespread haplotype and existed in every population. DY, MES, NKS and TBS populations have only Hapl. Another9haplotypes have the specificity in each population. The phylogenetic tree of chloroplast haplotyes was constructed by maximum parsimony.10haplotypes were classied into3clades:Hap1was the1st clade, Hap2,7,8were classied the2nd clade, Hap3,4,6,9were the3rd clade, and Hap5,10were classied the4th clade. The genetic differentiation coefficients (Gst) among populations was0.230, Nst was0.314. The analysis of molecular variation (AMOVA) result suggested80.96%of total variation between the populations, and19.04%among population, the fixation index was0.19046. This indicated that the differentiation among populations were relatively high and had obvious phylogeographic structure. The neutral test suggested that the population of B.sinensis experienced population expanding.