| Paeonia ludlowii, a species only distributed in Tibet of China, belongs to the section Moutan DC. of the genus Paeoniaceae. This plant had extremely high economic, ornamental and breeding values. Sequence Related Amplified Polymorphism (SRAP) analysis was applied to study the genetic diversity of79samples from five natural populations. Through the improved CTAB method, genomic DNA was extract. And then according to the results of phenotypic datas, cluster analysis and principal coordinate analysis(PCA) were did.The purpose for all the research was to provide theoretic foundations for the conservation and utilizition.The results were as follows:1.The L16(45) othogonal design was used to optimize PCR system in Paeonia ludlowii with five factors(template DNA, dNTPs, Mg2+,Taq DNA polymerase and primer). The result showed that the total volume of SRAP optimized PCR system was20μL, of which template DNA30ng, Mg2+3.0mmol/L, dNTPs0.4mmol/L, primer0.5μmol/L, Taq DNA polymerase2.0U,10×PCR buffer2.0μL, sterile double-distilled water6.4μL, and the mixtures were covered with15μL paraffin oil.2.Primer pairs were selected and polymorphic loci were obtained. Two materials of Paeonia ludlowii different in morphological characters was used to selecting sixteen pairs of high polymorphism SRAP primer combinations. These primers selected were used to evaluate genetic diversity of79samples from five natural populations. A total of396valid loci were obtained, of which357were exhibited polymorphism.3. Various genetic parameters with SRAP analysis were as follows:the percentage of polymorphic loci(Ppl) and the mean diversity estimated with Shannon’s index of phenotypic diversity(Hsp) at species level were90.15%and0.252, respectively. At population level, Ppl was31.82%and Shannon’s index(Ho) ranged from0.068to0.343, with an average value(Hpop) of0.131. Among the five population, population C was especially more diverse(Opl=82.32%, Ho=0.343). Those results indicated that there was a relatively high level of intraspecific genetic diversity. Analysis of molecular variance(AMOVA) demonstrated that a high degree of genetic differentiation occurred among the populations (ΦST=0.416, P<0.001). The proportion of genetic variation among populations was41.6%, while the proportion within population was58.4%. A similar analysis result was exhibited by the genetic differential index(GST) of0.431and the proportion of diversity among populations with Shannon’s index [(HSP-HPoP)/HSP]of0.482. Mantel test showed that there was no significant correlation between geographical distance and genetic distance (r=0.5975, P=0.9055) among populations. A dendrogram conducted by UPGMA method using NTSYSPc(2.1) demonstrated that the range of genetic similarity coefficient was0.47～0.99. The most of the individuals came from the same population had close genetic relationship, such as population of B, D, E, but individuals from population C could not be clustered together completely. The UPGMA results were also supported by the principal coordinate analysis of SRAP phenotypic data.4.The results showed that it was suitable for SRAP technique to be used to study the genetic diversity of Paeonia ludlowii. A strategy for the conservation and utilizition based on genetic diversity was proposed. It was quite necessary to take effective measures for the extreme endangering plant. Such as in-situ protection combined with ex-situ conservation, the individuals with smaller gene frequency returned to nature throngh expanding propagation based on tissue culture and seedling breeding, developing directive breeding and so on. |
PDF Full Text Request