Development of EST-SSR Primers and Application in Analysis of Genetic Realtionships in Tree Peony

Tree peonies are famous ornamental flowers all over the world because its flower is large, bright-colored and strong fragrance. They are not only have high ornamental value, but also a high medicine value. There are so many cultivarsand the germplasm resources are very abundant. The conservation and use of evaluation is an important prerequisite for use. The genetic relationship at the molecular level research has made great progress. However, in revealing the genetic relationships of interspecific species and species groups, and origin of cultivated peonies did not be consistent. Therefore, Development of tree peony EST-SSR primer that would aid in characterizing population genetic structure of populations and it can be employed in genetic diversity analysis and molecular marker-assisted breeding.Microsatellites mining through the public EST sequences has been widely used in the study of plant genomics and more recently applied to animal systems. EST-SSR is from the analysis of EST sequences to explore the repeating unit and design the primers of containing the location at both sides. Therefore, it reflects the gene encoding part of the gene, some of the important traits can be directly identified.Based on the existing ESTs of tree peony in the NCBI database, ESR-SSR marker was developed. 29 pairs of tree peony EST-SSR primers were selected to investigate genetic relationships of 66 tree peony varieties. The main results are as follows:1. Firstly, 2 204 ESTs of tree peony (Paeonia suffruticosa Andrews.) were downloaded from the database of NCBI and some redundant sequences were removed and 1 658 non-redundant ESTs were obtained. Subsequently, MISA, a web tool for SSR hunting, was used to analyze the SSR motifs in the EST sequences. The results showed that a total of 1 111 SSRs were detected which distributed in 901 ESTs, with the frequency for the non-redundant ESTs being 67.00%, the average distribution distance of the EST-SSRs were about 1 004 bp. Among the different SSRs motifs, mononucleotide repeats were most abundant, followed by dinucleotide and trinucleotide repeats. Distribution of hexanucleotide repeat and tetranucleotide repeat were very few, and there is no existance of pentanucleotide A/T, AG/CT and AAG/CCT are the most frequent motifs, accounting for 98.19%, 83.78% and 35.71% in mononucleotide, dinucleotide and trinucleotide repeats respectively.2. A part of EST sequence was randomly selected from the 901 available sequences, and 29 pairs of primers were designed by using Premier 3.0 software according to primer designed principle.3. Twenty-nine pairs of primers were selected on agarose gel electrophoresis (T=0.80%) in“Luoyanghong”according to the standard of bands polymorphism and stability. The 13 pairs of primers amplified 68 alleles in all, with an average of 4.5 alleles per primer. The polymorphism number ranged form 2-7, and the average polymorphism rate was 85.30%.4. The 13 pairs of primers were used to amplificated on the 55 cultivars of tree peony and showed on Polyacrylamide gel electrophoresis (T=8.00%). Based on the SSR results, the similarity coefficient of the 55 cultivars distributed in the range of 0.53-0.95, it showed that the gene differentiation was remarkable among tree peony cultivars. According to the constructed UPGMA cluster, the 55 cultivars could be classified into two groups at 0.53, 11 central plains species and 3 French species are classified in the first group, two French species“jinge”and“jindi”together first of all, the rest were in the second group. It could be classified into two sub-groups at 0.64, 8 South species together in the first sub-group, the second sub-group could be classified into four groups at 0.68, Most of the Central Plains cultivars, Northwest varieties, Japan varieties, the United States cultivars together, respectively, seven Japanese and five American varieties together, it showed a close genetic relationship, but there are also some of the same origin of the phenomenon is not together.which was approximately in accordance with their diversity of cultural area, and provided a scientific basis for selection of parents for hybridization of tree peony.