Different Environment Conditions Was Anemone Obtusiloba ISSR Analysis Of Genetic Diversity

Anemone obtusiloba is a perennial herbaceous plants within the family Anemone L. ofRanunculus subfamilies in Ranunculaceae. There are1-3independent stalks with a singleflower on the top. The flower usually is small (1-2cm), and the color is yellow, light yellowor white. At present, the research of A. obtusiloba mainly focus on its medical value,sexual and reproductive allocation, and so on. This research is combined with a specificresearch conditions, using ISSR molecular markers technology to study the geneticdiversity level and genetic differentiation degree in A. obtusiloba, the results of the studyare as follows:1. The study of the genetic diversity of A. obtusiloba by ISSR technique, from primerscreening, amplification conditions, the testing method and other aspects of optimization,establishes the most appropriate response system for A. obtusiloba ISSR: the20μlpolymerase chain reaction (PCR) system, contain DNA template40ng, Mg2+1.2mmol/L,10x PCR Buffer2μl, dNTPs0.3mmol/L, primer0.3mmol/L and6U Taq DNApolymerases. ISSR-PCR reaction procedure:94℃the degeneration5min;94℃degeneration30s, primer annealing45s,72℃extension90s, circulation35times;72℃extensions7min,4℃preservation. Under the optimized conditions, good repeated, stableand high polymorphic ISSR bands can be got.2.13polymorphic ISSR primers are screened from40ISSR primers. And701bandsare amplified from220individuals of11groups in4areas using the13primers, amongwhich392brands are polymorphic. It means that polymorphic sites’ percentage is55.92%,each primer amplified a number of bands for39-73, on average every primer amplified53.92bands, expansion bands are between300bps-1200bps. The polymorphism within A.obtusiloba is between27.8%and54.1%, with an average of42.4%. Average effectiveNumbers of alleles of each site is1.1673. Nei’s levels of genetic diversity in the populationis between0.0578and0.1699, on average of0.1135, and in species level is0.2497;Shannon information index level i the population is from0.1000to0.2632, with anaverage of0.1830, in species level is0.3719.3. Through the analysis of Popgene1.32software, A. obtusiloba of all groups of genediversity (Ht) is0.2551, gene diversity within populations (Hs) is0.1173, and within species is0.1378, genetic differentiation coefficient (Gst) is0.5010, estimate value of geneflow between populations (Nm) averages0.5243for each generation groups. Amongpopulations under different environmental conditions Genetic differentiation coefficientrises, however, gene flow reduces as the altitude increases. AMOVA analysis revealed:variation in percentage between species is38%, variation in percentage between thepopulations is62%. That is38%genetic variation is within populations,62%variationoccurs between species. The differences between populations and between species are verysignificant (P<0.001).4. By NTSYS software, the change’s range of similarity coefficient withinpopulations of A. obtusiloba is from0.569to0.917, among all groups genetic similaritycoefficient is0.7027on average. Genetic distance between populations (GD) ranges from0.0862to0.5642, on average of0.3656. Under the same condition, the similarity of A.obtusiloba changes small, however, under different environmental conditions, A.obtusiloba has a greater variation in similarity. To the genetic similarity coefficient0.73asa benchmark,11materials can be devided into three classes, the first one is A. obtusilobasfrom Hezuo, those from Luqu and Awancang are for the second class, and the last class isfrom Azi.5. SPSS19.0software are Used for A. obtusilobas correlation between environmentalfactors and genetic diversity index analysis. Studies have shown that A. obtusilobaspopulation genetic diversity is positively related to altitude, moisture content and effectivephosphorus, and has certain negative correlation with longitude, latitude and rainfall, witha significant negative correlation of rainfall（P<0.05）.