Abstract：The aim of this study is to construct a DNA fingerprinting database of 21 annual ryegrass (Lolium multiflorum Lam.) varieties using SSR makers, which is necessary to annual ryegrass varieties identification and germplasm management. The 21 annual ryegrass varieties or strains in the experiment, including the most domestic varieties, introduced varieties and six strains which were cultivated by our research group, were all tetraploid. Since annual ryegrass belongs to the typical cross-pollinated species, the sampling strategies were closely related to the authenticity and efficiency of molecular genetic diversity analysis and the DNA fingerprinting database. On the basis of others' and our previous research experience, we took the bulked DNA samples of 20 individual plants for each group in the experiment. Twelve evenly distributed SSR primer pairs with high polymorphisms and good repeatability were successfully screened out from 100 candidates to construct the fingerprinting database. Among the 21 varieties, 12 primer pairs produced 108 polymorphic genotypes, the ratio of polymorphism was as high as 94.15% and 9 genotypes were detected by each SSR primer pair on an average with the range from 5 to 22; the polymorphic information content (PIC) values ranged from 0.744 to 0.934 with the mean of 0.843. The twelve SSR primer pairs with high polymorphism, good repeatability and specification could be used as the core primer to construct the DNA fingerprinting of more annual ryegrass varieties. Different SSR primer pair could distinguish the different number of annual ryegrass species with the range from 5 to 21. The primer 15-08C amplified 22 polymorphic bands and it could distinguish all the 21 varieties at once, while the primer 07-07G amplified 6 polymorphic bands distinguishing only 5 varieties. The limited combination of different primer pairs could greatly improve the primer pairs' identification ability. Nine varieties had specific genotypes by 7 primer pairs, which couid be identified at the soonest by the feature primer. The genetic similarity coefficient of 21 accessions varied from 0.4956 to 0.8571. UPGMA cluster analysis of genetic similarity showed that all the materials were clustered into three groups at the genetic similarity of 0.69. For distinguishing germplasms more simply, through selection of the primer pairs according to its PIC step by step , we found that the primer 15-08C could distinguish all the germplasms in the experiment separately. At last, we constructed the standard mode image of DNA fingerprinting of 21 annual ryegrass varieties by the primer 15-08C. Each germplasms has its own fingerprinting(bandtype), which provids a basis of the cultivars assessment, germplasms protection and cross breeding of annual ryegrass varieties.
