Genetic Diversity Study Of Rareand Precious Species Taihanggia Rupestris

Taihangia Rupestris Yu et Li was firstly found in 1974, and then formally described in 1980 as the only species of the Taihanggia belonging to the family Rosaceae. T. Rupestris is an perennial herb endemic to China. T. Rupestris is the most primitive diploid species (2n = l4) in the tribe Dryadeae belong to the family Rosaceae according to its morphology, anatomy of carpel, chromosome counting, microscopic and submicroscopic structure of pollen, so it is an ancient relic species. T. Rupestris, with the characterization of evolvement from bisexual flower to unisexual flower, is at an important systematic position in the tribe Dryadeae, and study on it could throw a new light on evolution of the tribe Dryadeae. However, due to its small distribution area, unique habitats and rare plants, the destruction range of Taihangia Rupestris becomes smaller. It is endangered specie and listed as protected plants in China.In general, we must know the genetic structure and genetic diversity of species before we make the conservation strategy. If not, any protection measures can not achieve the expected results. Molecular markers are useful tools in the 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 ten populations. The main results are as follows:(1)We filtered and optimized the DNA extraction method of Taihangia Rupestris. The total DNA was isolated from the leaf of Taihanggia rupestris YU et LI 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,and A260/ A280 was 1.8 or so. With these leaf DNA serving as template, PCR at two loci of mitochondrial genomes was effective and special highly.(2) We selected 12 ISSR primers from 50 ISSR primers for the Amplification of 220 samples. Amplification of the ISSR fragments in 220 individuals sampled with 12 primers generated 184 clearly identifiable bands, with a mean of 15.33 bands per primer. In all, there were 148 polymorphic bands, the percentage of polymorphic band (PPB) was 47.01 % on average and 80.43% at the species level. The Nei's gene diversity (h) was estimated to be 0.1865 on average at the population level, 0.2479 at the species level, while Shannon indices (I) were 0.2726 and 0.3785 respectively. The number of alleles was estimated to be 1.8043 at the species level and the average effective number of alleles per locus was estimated to be 1.4126.(3)Pairwise Nei's unbiased measures of genetic identity of the ten populations was high as 0.9233 on average, QPG and XXT are the closest with 0.9728, whereas DPB and SSM are the most distant populations with a identity coefficient of 0.8706. Genetic distance was detected to be 0.034～0.144 between populations, QPG and XXT are the Least, whereas DPB and SSM are the largest.(4)The UPGMA analysis clustered 10 populations into two groups. GroupⅠcontained populations DPB, YDS, LHP, QPG and XXT. GroupⅡincluded populations HHS,JG,SSM,NY,DLG. Principal coordinate analysis (PCA) shows: Samples from the southern population were founded in the lower and right side of the diagram, samples from the northern populations in the upper and left side, Principal component analysis supports the results of UPGMA clustering.(5)Mantel test was performed using GenAlEx 6.1.The mantel test showed that there was significant positve correlation between geographical distance and genetic distance (r = 0.611, P<0.001), indicating the role of geographic isolation in shaping the present population genetic structure of Taihanggia Rupestris.(6)Using POPGENE 1.32 to estimate the level of genetic differentiation among the different populations. The result shows that the total genetic diversity of ten populations(Ht) was estimated to be 0.2465 and genetic diversity within populations(Hs) was detected to be 0.1865. Genetic differentiation coefficient between Population(Gst) was 0.2435 and gene flow(Nm) among populations was estimated to be 1.5537.Analysis of molecular variance revealed that 73.51% of the total genetic variability occurred within populations, 26.49 % was found among populations, in which only 9.24% 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.