Development Of SSR Primers And Genetic Diversity Study On Dayaoshania Cotinifolia

Dayaoshania cotinifolia W.T.Wang,a new kind of species belonging to the family Gesneriaceae ,was firstly found in 1983 by Mr.Wang wencai in Guangxi Dayaoshan natural reserve, and was the monospecific genus uniqe to China. The plants in the family Gesneriaceae are undergoing sharp differention, this means that some of the genus and species are weakly adapting to its surrounding environment, and even hard to compete with other commensal plants. As the more original species in Gesneriaceae, it has great value not only in scientific research but also in economic field. But due to the greatly limited amounts of resource, the paticular habitat, the narrow range, and the impact from human activites, Dayaoshania cotinifolia are losing its population size and distribution area. As a result of its condition, the species are listed as the I–level national key protected wild plants in The List of National Key Protected Wild Plants (The First) promulgated by State Council in 1999,and are reffered to an critically endangered species on the China Species Red List(First Volume).In general, we must firstly know the genetic structure and genetic diversity of species when we make the conservation strategy. If not, any expected results through protection measures can not be achieved. Molecular markers are useful tools in the Genetic Diversity study owning to the fact that it can reflect the genetic differention of a species on the DNA level. Simple Sequence Repeats (SSR) is widely used in the study of genetic diversity because of its incomparable traits: such as high species specifity, high polymorphism, codominance and neutral marker. In this paper, we selected 10 pairs of SSR primers which were developed by our own to study the sixty-one individuals in the only two population. The main results are as follows: (1)We had structured a genomic library containing lots of SSR sequences successfully. After cloning and solid culturing, we checked the positive clones by PCR amplification using M13F and M13R primers and finally, eighty one positive clones were selected and sequenced on ABI 3730 DNA analyze, of which 64(about 79%) contained SSRs. PCR primers were designed for 38 sequences using program PRIMER 5.0. These primers were tested for polymorphism in 10 Dayaoshania cotinifolia W.T.Wang individuals which came from the only 2 different populations. The Products were checked with 2.0% agarose gels. 15 pairs of primers showed the single and clear band. Finally, 10 of themwere selected, and the forward or reverse primer was labeled with one of the fluorescent dye (FAM or HEX) for polymorphism detection.(2)We e analyzed the total 61 individuals in the only two population using the computer program package Arlequin ver. 3.1, and finding that there were two monomorphic loci which contained only one allele . While in the rest eight polymorphic loci, the number of alleles per locus ranged from 2 to 12, with an average of 4.5 alleles. The heterozygosity analyzing results were as follows: on the species level, the observed heterozygosity value ranged from 0.01639 to 1,on average 0.50830; the expected heterozygosity value ranged from 0.01639 to 0.65831,with an average 0.41556. There were 5 loci which deviated from Hardy-Weinberg equilibrium significantly(P<0.01), JT14, JT26, JT29, JT17 and JT34.And the locus JT21 deviated from Hardy-Weinberg equilibrium notablely(P<0.05). We also detected the linkage disequilibrium between the allele pairs, of which 4 pairs were notable, P<0.05, they were JT14 and JT29, JT3 and JT29, JT14 and JT34, JT34 and JT17. The remaining 3 pairs were significantly(P<0.01),they were JT29 and JT34,JT14 and JT34,JT17 and JT34.(3) We also detected the bottleneck effect with the software bottleneck and our results showed that: under the different models, there were some loci which the observed heterozygosity value was higher than the expected heterozygosity value notablely or significantly, but the allele frequency distribution was normal“L”shape, this implied the species had not experienced the bottleneck effect. The most probable reason was that the loci number we selected were too small, or the bottleneck effect had happened not long ago, its results had not changed the allele frequency obviously, so we could not detect this effect.