Study On Genetic Diversity And Molecular ID Establishment Of Tree Peony By CDDP Marker

Tree peony (Paeonia suffruticosa Andr.) is a famous flower in the world for its highornamental and economic value with origin in China. There are many wild species and lots ofcultivars containing abundant gene resources. Study on the genetic diversity and cultivaridentification of tree peony by DNA markers will has an important guiding significance to theeffective protection for tree peony germplasm resources and its germplasm innovation. CDDPis a kind of new method belonging to gene-targeted markers technique. This method tends togenerate candidate function markers which may be a part of or linked with the gene targeted.It is a kind of dominant marker with good polymorphism, easy operation and low cost.Researches on tree peony germplasm genetic diversity by CDDP technique can provide notonly a new way or method for tree peony researches but also an available reference for otherplants to carry out related researches.In this study, optimized CDDP-PCR reaction system for tree peony was established and316genome DNA templates composed of9wild species,8foreign cultivars and299Zhongyuan tree peony cultivars were amplified with18informative and reliable CDDPprimers. Then, the amplification results were used to analyze the genetic diversity, the cultivardistinctness and uniformity of tree peony. At last, core primers were selected to indentifydifferent tree peony germplasm resources and to set up their molecular ID codes. The mainresults were as following:1. After comparing the results of orthogonal experimental design and PCR-Mix reagentapplication, the optimized CDDP-PCR reaction system(20L) were2×Es Taq MasterMix(dyed)10L,10pmol/L primer1.0L,30ng/L DNA template2L and ddH2O7L.Researches on the stability and repeatability of the above reaction system resulted that it couldbe used for genetic diversity analysis and its follow-up studies of tree peony.2. Eighteen out of21CDDP primers with good polymorphism and clear bands wereselected to reveal the differences among genetic diversities of wild species, foreign cultivarsor Zhongyuan cultivars in China. Results showed that the average Nei s gene diversity (H),average Shannon information index (I) and average effective number of alleles of22individuals composed of abroad and Zhongyuan cultivars was0.1950,0.3128and1.3060,respectively. As for299Zhongyuan cultivars, the above indexes were0.1658,1.2569and 0.2695in turn, which were all lower than those of22cultivars. And the values of299Zhongyuan cultivars were higher than those of wild species by20.14%,5.42%and16.63%inturn. These results indicated that the genetic diversity of abroad cultivars was the mostabundant and that of Zhongyuan cultivars was more while that of the wild species was thelowest. In addition, clustering results showed that the22foreign and domestic cultivars wereall clustered with P. spontanea, indicating that tree peony cultivars should originate from P.spontanea, while cultivars from different countries didn t get into a class respectively. As forforeign cultivars, Japan cultivars were in together with Zhongyuan cultivars in China andUSA or France cultivars were clustered together.3. There were10populations with different color. Results of their genetic diversityanalysis showed that average Nei s gene diversity (H), average Shannon information index (I)and average effective number of alleles was0.1451,1.2313and0.2306, respectively.Comprehensive analysis results indicated that the genetic diversity of red or purple populationwas higher while that of multicolor or green population was lower. The genetic distanceamong10populations was close with an average of0.0271and the genetic identity was highwith an average of0.9735. The coefficient of gene differentiation among them was0.1252,showing that there were12.52%genetic variation among populations. The gene flow among10populations was high and its value was3.4939. Results obtained from genetic diversityanalysis and UPGMA dendrogram based on Nei s genetic identity proved the evolutionarytrend of tree peony s flower color at molecular level: pink and red cultivars might be at thecenter of Zhongyuan tree peony resources, then gradually fuchsia, purple, blue, white, yellowor black cultivars were evolved, while green or multicolor cultivars might be degraded.4. After comprehensively analyzing the average, the maximum and the minimum valuesof genetic distances between an arbitrary cultivar and the others cultivars, the criticalthreshold T=0.20was determined as the evaluation criteria of tree peony cultivar distinctness,meaning that when the genetic distance between2cultivars was higher than0.20, they wereregarded as2different individuals. Then the genetic distance among12individuals belongingto the same cultivar were calculated and8cultivars were involved. According to the abovecriteria, tree peony cultivars used in this study had good genetic uniformity.5. Several polymorphism indexes of18CDDP primers showed that Pr2, Pr5, Pr12, Pr19, Pr20or Pr21could be core primers to identify tree peony germplasm resources. Based on theirability to distinguish tree peony gemplasm alone, two or three different primers combination,three primers combination of Pr2+Pr19+Pr20was selected and applied to set up the molecularID of tree peony. In the end, the IDs with18decimal numbers of316tree peony germplasm resources were structured and provided a new method for the realization of digital searchingfor tree peony resources.