| Tree peony(Paeonia suffruticosa Andr.) belonged to the family Paeoniaceae is one of the most well-known traditional flowers. It originates in China and has been cultivated for more than 1600 years. As a special cultural symbol of peace and happiness, prosperity and development above that of many other common plants, the tree peony is respected as the ‘King of Flowers’ in China and plays an irreplaceable role in ornamental and medicinal uses. Until now, there are more than 2000 cultivars with various flower colors all over the world, and these cultivars can be divided into ten flower color categories including red, pink, white, blue, purple, purplish red, green, yellow, black and double color. CDDP is a novel method for generating plant DNA markers, which is developed on the basis of data mining for short conserved amino acid sequences in proteins, and designing PCR primers based on the corresponding DNA sequences. So, it is the point that analysis of genetic relationships in tree peony on the level of flower color groups.The main research results were as follows: 1. Based on the results of orthogonal experimental design, the best 20 mL system was established and optimized: 2× Es Taq MasterMix(dyed) 10 mL, 10 pmol/mL primer 1.0 mL, 30 ng/mL DNA template 2 mL and ddH2 O 7 mL. Further researches on its stability and repeatability resulted that the system could be used for the genetic relationships analysis of tree peony and so on. 2. In the present study, 14 primers targeted to flavonoid biosynthetic genes were designed exploiting conserved DNA regions. A set of 16 CDDP primers were used for genetic variability and diversity analysis among 64 tree peony accessions. Only 13 primers that exhibited distinct and reproducible band patterns were selected for further analysis. 3. 13 primers generated a total of 152 fragments with a mean of 11.69, ranging from 7(primer 8) to 17(primer 3) per primer. Of 152 bands, 149 bands(98.03%) were polymorphic. 6–17 polymorphic bands were amplified by each primer, with an average of 11.46 polymorphic bands per primer. Detected polymorphism per primer among the tested accessions ranged from 85.71%(Primer 11) to 100.00%, with an average of 98.03%. These results indicated that certain DNA polymorphismcould be detected among tree peony germplasm using CDDP markers. In addition, the mean value of He was 0.2974 and that of I was 0.4548. All these calculated indices confirm that there is abundant genetic diversity in tree peony. 4. None of CDDP polymorphic primers could discriminate all tree peony alone. The minimum number of primers required to distinguish all 64 samples was 4(such as Primer 3, 9, 5 and 6). Primer 3 and primer 8 could distinguish a maximum of 55 samples and a minimum of 6 samples, with Rp values of 9.34 and 2.97, respectively. There was a significant positive correlation betweenthe Rp of each primer and the number of tree peony samples identified(y= 7.20x- 8.44, r = 0.96, **P < 0.01). 5. To understand the genetic relationships among the various cultivars and species used in this study, a dendrogram was constructed based on SM coefficient. It grouped the 64 genotypes into 5 major clusters at a similarity index value of 0.69. Cluster I comprised of all the 47 Zhongyuan Group cultivars. Cluster II included 7 accessions, which were foreign cultivars. Cluster III contained 2 accessions, a foreign cultivar ‘Hei Bao’ and a wild species P.potanini. Cluster IV included 2 species, which were P. decomposita and P. rockii. Other 6 species were classified into cluster V. Basically, 64 accessions clustered based on flower color. Most of the same flower color cultivars(yellow, pink, purplish red) were clustered together, suggesting varieties of the same color had relatively close relationship. |
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