Genetic Diversity By Molecular Markers And Evaluation Of Super Germplasm Resources On Dactylis Glomerate L.

Dactylis glomerate ssp. glomerata L. (common orchardgrass) is one of the most important member of the Gramineous genus Dactylis, as a world-famous cool-season forage propagated widely. There is abundant native orchardgrass germplasm in temperate climates zone with great potential of utilization.Twenty accessions of orchardgrass, among which eighteen are selected from the former research, were collected and tested in germplasm appraisal at morphological, productivity and ecological adaptability by a comparison with two representative cultivars D. glomerata L. cv. Baoxing and D. glomerata L. cv. Amba. On the other hand, the levels and patterns of genetic diversity were evaluated among 45 collections of orchardgrass obtained from seven geographically and environmentally distinct countries and regions by RAPD, ISSR, SRAP and SSR molecular markers. The main results as follows:1. The orchardgrass germplasm produces rich morphological diversity. Among the twelve observed properties, morphological variances most significant occur in the length and width with flag leaf and inflorescence, internode length and 1000-seed weight. Moderately significant variances were tested in leaf length and width. But no significant variances occur in features of plant height and internode diameter. The significant correlation was tested among morphological characters of orchardgrass, such as the length and width with leaf and flag leaf. These morphological properties showed us the value in use of accessions. Three main clusters generated by dendrogram with morphological characters revealed the difference of procreating growth. The cluster analysis suggested the high universality of distributing and ecological adaptability in orchardgrass germplasm, too.2. Twenty accessions were clustered as pre-matured type and slow-growing type by the observation of phonological stage. D. glomerata L. cv. Baoxing and D. glomerata L. cv. Gulin belong to the former while D. glomerata L. cv. Chuandong belongs to the latter. Accession 02123 showed the longest generation time (247d) among the twenty accessions, accession YA02-116 rank the second one (244d), and both of them would be bred as slow-growing type. Accessions 01821 belong to the pre-matured type, which showed the shortest generation time (216d). All the tested overseas accession could vegetate well with good ecological adaptability. And most of them belong to the slow-growing type except the accession 01821. All the growth period of orchardgrass was consisted in slow-growth stage, high-velocity-growth stage and fast-growth stage according to the growth velocity analysis. But the rule of growth was different among the different plant apparatus or different growth types.3. The yield characterizes of the twenty accessions was significantly different. The hay yield of accession YA02-116 was the highest among all the accessions, amounting to 32342.10kg/hm~2 in hay. Which increase by 24.17% and 40.34% than that of D. glomerata L. cv. Baoxing and D. glomerata L. cv. Amba, respectively. The hay yield of accession 02123(32342.10kg/hm~2) was the higher. Which increase by 16.79% and 31.99% than that of two controlling accessions, respectively. On the other hand, D. glomerata L. cv. Amba is popular forage in China as a registered cultivar, but the yield characterize is poor in the area of southwest in Sichuan province, and which is not the good choice for forage production in southwest of Sichuan province. The research suggested that leaf-stem ratio of most accessions belong to the range of 1.5-2.0, the leaf-stem ratio of accessionYA01-103, YA01-104 and YA02-116 was lower than 1.5, and accession 02123 was lower with leaf-stem ratio, too. The survival rates in summer of most accessions were lower than 60% except accession YA02-116(60%) and 02123(63%). and some oversea accessions were difficult to survival in summer with lower survival rates, such as accesion YA02-117(39%) and 01472(32%). The correlation analysis suggested the accessions can be more productive in fresh grass production with bigger-leaf and more tillers, and the hay yield were better by accessions with long-small leaf, big internode and high plant.4. The genetic diversity of 45 accessions were evaluated by RAPD marker, Twenty primers were used and 120 bands were produced, of which 97(80.83%) were polymorphic, and each primer generated 4.8 polymorphic bands. The coefficient range of genetic similarity (GS) was 0.6154-0.9254 with 0.7984 on average, which showed rich genetic diversity of orchardgrass germplasm. 45 accessions could be divided into 5 main groups by cluster and principal component analysis on the genetic characteristics. In most cases, accessions from the same continent were classified into the same group, indicating the geographical distribution of genetic diversity of orchardgrass. The more efficient measures must be taken for the collection and protection of orchardgrass germplasm according to the rich genetic diversity. The research suggested RAPD was a useful marker system for primary or ordinary genetic research.5. The genetic diversity among 45 orchardgrass accessions was tested by ISSR. A total of 117 bands were amplified by twelve ISSR primers, among which 100 (84.04%) bands were found to be polymorphic, the percentage polymorphic band was higher than other species by ISSR marker. Each primer generated 8.42 polymorphic bands. The ISSR-based genetic similarity values among 45 accessions ranged from 0.6116 to 0.9231 with 0.8151 on average. Abundant genetic diversity among orchardgrass accessions could be revealed, especially for Chinese and American accessions. Analysis of cluster and principal component showed that all the 45 accessions could be distinguished by ISSR markers and divided into 5 groups, indicating the better geographical distribution rule of genetic diversity than RAPD. The result showed that ISSR marker fit not only the genetic diversity research but also the cultivar identification according to exact and simple operation for any species.6. The present study is the first report of evaluating the levels and patterns of genetic diversity among 45 collections of orchardgrass by SRAP, a new molecular marker with few applications in forage and grass. Twenty-one primer combinations were used and 438 bands were produced in SRAP, of which 363(82.08%) were polymorphic, the percentage polymorphic band was higher than other species by SRAP marker. The coefficient range of genetic similarity was 0.6248-0.966 with 0.7958 on average, which showed rich genetic diversity of orchardgrass germplasm, too. 45 accessions could be divided into 4 main groups bycluster and principal component analysis, which not only revealed the geographical distribution of accessions more clearly than RAPD and ISSR but also reflected the difference of karyotype. The result suggested that the genetic diversity of orchardgrass were correlative with karyotype and geographical distribution of accessions. In addition, SRAP marker can test much genetic variance of orchardgrass like AFLP, which could be applied to more genetic research.7. Using the primers pairs and PCR amplified program of Triticum aestivum L., The genetic diversity among 45 orchardgrass accessions was analyzed by means of comparing with T. aestivum L. cv. Chinese Spring. 80% of primer pairs for selecting could amplify band from orchardgrass genetic DNA. A total of 295 bands were amplified by twenty SSR primer pairs, among which 187 (61.15%) bands were found to be polymorphic. The SSR-based genetic similarity values among 45 bermudagrass accessions ranged from 0.7848 to 0.9513. Analysis of cluster and principal component analysis showed that all the accessions could be distinguished by SSR markers and divided into 6 groups. The dendrogram based on genetic similarity of SSR revealed the geographical distribution of accessions. The cluster analysis can suggest not only the ecological adaptability and productive performance but also the narrow genetic basic of Chinese orchardgrass cultivars. The results also showed that it is feasible of applying T. aestivum L. primer pairs to the testing of genetic variance in orchardgrass. Furthermore, our research showed us that the genetic diversity for plant without designed primers could be revealed by using SSR primer pairs of some other species with near genetic relationship.Overall, rich diversity can be observed in different dimensions in morphology, production and DNA formation. The genetic variance was correlated with wide geographic distribution and karyotype. The further selection of orchardgrass germplasm suggested the better ecological adaptability and productive performance of some accessions with higher application potential, such as accession YA02-116 and 02123. Both of two accessions could be bred as slow-growing cultivars. It is efficient to breed more excellent orchardgrass with wide ecological adaptability and genetic basic by means of super quality germplasm.