| Aleurites plant of Vernicia montana is specific industry oil plant in China, it has a long history of cultivation, high content of oil and wide applications, it is the one of most ideal biodiesel raw material plants at present in our country. Vernicia montana research at home and abroad mainly concentrated in physiological character, nursery stock and resource exploitation, etc; the research in the field of molecular marker and genetic breeding is little. This thesis use RAPD molecular marker as the method of markers experiments for Vernicia montana of 11 provenances and 33 individuals, and do the research of genetic structure difference and genetic relationship variation patterns, which provides theory basis for genetic structure variation patterns, genetic relationship division, germplasm resources protection and thoroughbred seed selection of different Vernicia Montana provenances.Comparing CTAB method and plant genomic DNA extracted kit method, this thesis select plant genomic DNA extracted kit method as the best way of extracting Vernicia montana's total DNA because of this method can extract good DNA quality about high purity, less impurity content, less protein and phenol, less RNA pollution, moderate consistence, integrated DNA bands, more DNA content, clear and bright DNA bands and less trailing DNA bands.Doing gradient optimization experiment of dNTP, Tag enzyme, Primer, template DNA and Mg2+and annealing temperature, the best RAPD-PCR procedures of 94℃anticipate degeneration for 3min, 94℃degeneration for 1min, 36.4℃annealing for 1min, 72℃extensions for 1.5 min, circulating 44 times, again after 72℃extension for 5min. The best Polymerase chain reaction (PCR) system for total 25ul of dNTP (2.5mmol/l)3.0ul, Tag enzyme(2.5U/μl)0.4ul, prime(r5μmol/l)1.5 ul, DNA (10ng/μl) 2.0 ul, Mg2+(25 mmol/l)3.0 ul, 10×PCR buffer2.5ul, sterilization ultra-pure water12.6 ul. Screening random primer of 105, and screened 14 specific primer for RAPD amplification of 11 provenances and 33 samples of Vernicia Montana. And obtained 130 DNA bands, in which polymorphism DNA bands is 119, polymorphism percentage is 91.54%, and every primer amplificated out 8.5 bands. These results indicate that different Vernicia montana provenances and samples have more genetic differences. 11 provenances'Nei 's observation alleles number is 1.8923 and 1.8769, Nei' s effective alleles number is 1.5248 and 1.4896, Nei 's genetic diversity H = 0.3088 and 0.2949, Shannon' s information index I = 0.4639 and 0.4472, the four important index of measuring genetic diversity all shows high level of genetic diversity between 11 provenances. Zhenghe(H=0.2631, I=0.3892), Tancheng(H=0.2669, I=0.3887), Shufang(H=0.2617, I=0.3819), Jiguan(H=0.2533,I=0.3751)and Xiaohu(H=0.2218, I=0.3316 ), these five provenances'Nei 's genetic diversity index and Shannon' s information index is higher and close, they constitute the first level of genetic diversity. Shaxian (H= 0.1679, I= 0.2452), Youxi (H=0.1287, I=0.1886) and Putian (H=0.1226, I=0.1797), these three provenances'Nei 's genetic diversity index and Shannon' s information index successively reduce, these three provenances'geographical position is more decentralized, their distance is farther, hybridization and communication is less, germplasm preservation is good and pure degree is high. Huangkeng(H=0.1045, I = 0.1530), Xushi(H = 0.0898, I = 0.1289) and Zhangdun(H=0.0748,I=0.1079)these three provenances'Nei 's genetic diversity index and Shannon' s information index is lower and close, the genetic diversity of them is not high.Between 11 provenances of Vernicia Montana, the farthest genetic relationship is Jiguan provenance and Zhangdun provenance, their genetic distances is 0.3693; the father genetic relationship is Jiguan provenance and Huangkeng provenance, Zhangdun provenance and Zhenghe provenance, Xiaohu provenance, their genetic distance is 0.3271, 0.3286 and 0.3174. The closest genetic relationship is YouXi provenance and Xushi provenance, their genetic distance is 0.0510; the closer genetic relationship is Huangkeng provenance and Xushi provenance, their genetic distances is 0.0603.The similarity coefficient range is 0.9503 to 0.6912 between 11 provenances of Vernicia Montana, the highest similarity is Youxi provenance and Xushi provenance; the lowest similarity is Jiguan and Zhangdun provenance, these index indicate that between each provenance, they communicate with each other very frequently, their polymorphism is abundant, their genetic range is wide, and the complexity of the geographical environment will also affect the exchange and variation between provenances. The similarity coefficient between every provenance is basically under 1, but the gap is not big, the maximum similarity coefficient is 0.9503, the minimum similarity coefficient is 0.6912, the gap between them is 0.2591; each individual of provenances can polymerize together, most individuals have gathered in distance 0.27, these index show that most of the individuals'genetic background is similar and the impact is not big by geographical environment. Using popgen software to cluster the genetic relationship of different provenances of Vernicia Montana, 11 provenances are obviously divided into two big clustering and four small clustering. Putian provenance obviously becomes a single, this indicated that Putian provenance is far away from other provenances, their communication is less, pure degree is high; the other two provenances of Sanming cluster together with Xushi provenance which is closer with each other, this indicated that their communication is more because of their geographic distance is close, their similarity is higher and genetic relationship is closer. Because of landform is complicated, Huangkeng provenance and Zhangdun provenance in Jianyang city communicate with the outside world is less, the differences between other provenances in Jianyang city is larger. Tancheng, Zhenghe, Shufang, Xiaohu and Jiguan provenance cluster into one large clustering, this indicated that the geographical position and the genetic relationship is close between these 5 provenances. Using the Dps software to cluster the genetic clustering of 33 individuals of Vernicia Montana, 33 individuals of 11 provenances are divided into four parts, this basically as same as the result of provenance clustering figure, present that provenance inward and between provenance, 33 individuals are all polymerizing, the polymerization degree is high for closer geographic distance, vice is low, the polymerization degree is also high in geographical border area, the degree of distinction is also more meticulous. |
