DUS Testing And Construction Of DNA Fringerprints In Stylosanthes

Stylosanthes is an excellent tropical forage which plays an important role for livestock development in the south of China. Since the Stylosanthes was introducted to China, the genetic background of Stylosanthes has become very complexed with the constant cultivation, natural selection, which make it difficult to the resource collection, identification and classification of species, the application and protection of new varieties of Stylosanthes. In this study, through the collection of information in the existing resources of Stylosanthes, the shape of Stylosanthes was observed and statistic to establish morphology-based databases in accordance with DUS testing; in addition, the polymorphic of core primers between different species were screened by SSR markers, the DNA fingerprinting databases of Stylosanthes was constructed to provide a reference for the collection, identification and protection of species, breeding of new varieties. The main results are as follows:1. A total of192species of Stylosanthes and their basic information were collected, through the Guidelines for the conduct of tests for distinctness, uniformity and stability-Stylosanthes, the morphology-based databases of Stylosanthes was established by testing192varieties on morphological traits.2. The genetic diversity analysis and clustering analysis of DUS testing traits was through the software of POPGEN, the results showed that:there were130allelic variation of192Stylosanthes species in24morphological traits, an average of5.417for each trait alleles (1～9); effective variance was3.750(2.000～6.268), Nei’s diversity index was0.644(0.000～0.887), Shannon’s information index was1.002diversity (0.000～1.389). Using the NTSYS-pc and Structure software to analysis, the genetic similarity coefficient was between0.550and0.985, the similarity coefficient was greater than0.695, it can be divided into4categories of Stylosanthes.3. Based on the database of DUS testing by the morphology to genetic diversity analysis and clustering analysis, the guidelines for the conduct of tests for distinctness, uniformity and stability-Sty losanthes were revised, and completed the standard varieties.4. According to the result of the transcriptome sequencing, developed2002EST-SSRs markers by MISA and Primer3software. The DNA extraction method of Stylosanthes was improved and optimized SSR-PCR systems by orthogonal design, also improved the gel electrophoresis of polyacrylamide, established a simple and efficient SSR-PCR markers, in addition, a simple method of transcriptome sequencing was introduced in development of more SSR markers in Stylosanthes. Seven different kinds of Stylosanthes with123pairs of SSR markers among eight varieties of Stylosanthes were screened by optimized SSR-PCR systems, the polymorphic of25SSR primers were higher, which proved that SSR primers has a good versatility in all species of Stylosanthes.5. While marking the25pairs of polymorphic primers in the192varieties of Stylosanthes, DNA marker fingerprint database of Stylosanthes was constructed with the characteristic band, take the POPGEN and NTSYS-pc to analyze the DNA fingerprinting diversity and take UPGMA to cluster analysis.25SSR markers produced200alleles in14varieties of Stylosanthes, the valid variance was5.1412(2.5505-9.4251), Nei’s diversity index was1.3459(0.7015-2.8200), Shannon’s diversity index was1.1201information of diversity0.5747-2.2414, all of this showed the rich resources of Stylosanthes species. The results of cluster analysis is that:the genetic similarity coefficients ranged between0.600and0.995, the similarity coefficient is greater than0.700, it can produce4groups.6. Compared DUS testing data with SSR markers in genetic distance by mantel test in TFPGA software, we found the correlation coefficient of r=0.7627, and they have certain relevance.Using SSR markers detection system to test distinctness, uniformity and stability of Stylosanthes, and established the evaluation standard.The study not only established the morphological foundation database by the DUS testing, and revised the guidelines for the conduct of tests for distinctness, uniformity and stability-Stylosanthes, but also constructed DNA fingerprinting database by using SSR markers, as well as applied SSR markers to DUS testing. All of these will provide important basis on the breeding and improvement of the varieties in Stylosanthes.