Study On Genetic Diversity Of RARE Opisthopappus Taihangensis (Ling) Shih

ABSTRACT:Opisthopappus Shih was firstly described in1974as the only genera of the Taihanggia belonging to the family Compositae.Optisthopappus taihangensis is an perennial herb endemic to China’ Taihang Mountain.This genera includes Opisthopappus taihangensis (Ling) Shih and Opisthopappus longilobus Shih.Opisthopappus rich in aromatic oil, fragrance lasting after dried,can extract balmy oil,used as a spice it has important ecological,collection and economic value.Due to due to its small distribution area,unique habitats,weak fertility,and grievously aritificial picking, the destruction range of Opisthopappus Shih becomes smaller.It is listed as the precious national preserved plant and the key protected plants of province class.In general,we must know the genetic structure and genetic diversity of species before we make the conservation strategy. Molecular markers are useful tools in the plant Genetic Diversity study. Inter-simple sequence repeat(ISSR)is a molecular marker targeting simple sequence repeats(SSR), by which the length variation of DNA fragment between adjacent microsatellites is detected in the entire genome. Freedom from the necessity of obtaining flanking sequence information, high level of polymorphism and simple technology, ISSR has been widely used for Genetic Diversity studies. Inter-simple sequence repeat(ISSR)markers were employed to estimate the genetic diversity and genetic differentiation of six populations. The main results are as follows:(1)We filtered and optimized the DNA extraction method of Opisthopappus taihangensis.The total DNA was isolated from the leaf of Opisthopappus taihangensis by the conventional CTAB method, improved CTAB method and SDS method.The results showed the extracts by conventional CTAB method were brown and could not dissolve completely,and those by SDS method had a low quality and yield.As for the improved CTAB method,there was a high production rate of the extracts, that was pure with little degradation.With these leaf DNA serving as template,PCR chloroplast genomes was effective and special highly.(2)We selected10ISSR primers from50ISSR primers for the Amplification of116samples. Amplification of the ISSR fragments in116individuals sampled with10primers generated157clearly identifiable bands, with in11～31bands and a mean ofl5.7%bands per primer.In all,there were104polymorphic bands,the percentage of polymorphic band (PPB)was56.23%on average and66.24%at the species level.The Nei’s gene diversity(h)was estimated to be0.1667on average at the population leve1,0.2271at the species level,while Shannon indices(I)were0.2489and0.3619respectively.The number of alleles was estimated to be2.0000at the species level and the average effective number of alleles per locus was estimated to be1.3621.(3)Pairwise Nei’s unbiased measures of genetic identity of the six populations was high as0.9123on average, J-YTS and J-YTD are the highest with0.9349,whereas J-WHA and J-SCKare the most distant populations with a identity coefficient of0.8761.Genetic distance was detected to be0.0720～0.1375between populations, J-YTS and J-YTD are the Least,whereas J-WHA and J-SCK are the largest.(4)The UPGMA analysis clustered6Optisthopappus taihangensis populations into two obvious groups.Group I contained populations J-WHA.Group II included populations J-WHB, J-LHP, J-YTS, J-YTD, J-SCK.(5)Mantel test was performed using IBDWS Version3.23. The mantel test showed that there was unsignificant positve correlation between geographical distance and genetic distance(r=0.2243,P=0.2590), meanwhile there was not significant positive correlation between genetic distance and elevation altitude(r=0.2813,P=0.8650),ease see to that geography factors has not influence in poputations genetic structure.(6)Using POPGENE1.32to estimate the level of genetic differentiation among the different populations.The result shows that the total genetic diversity of six populations(Ht)was estimated to be0.2264and genetic diversity within populations(Hs)was detected to be0.1617.Genetic differentiation coefficient between Population(Gst)was0.2859and gene flow(Nm)among populations was estimated to be1.2489.Analysis of molecular variance revealed that58.78%of the total genetic variability occurred within populations,41.22%was found among populations,in which only6.49%resided between regions,indicating a low level of among-population genetic differentiation.The results of the population structure analysis by AMOVA and Nei genetic diversity is the same.