Bulking strategies for genetic diversity analysis of annual ryegrass based on SSR markers

Since Annual ryegrass (Lolium multiflorum) belongs to the typical crosspollinated species, the sampling strategies are closely related to the authenticity and the efficiency of molecular genetic diversity analysis. Two Annual ryegrass varieties, Changjiang No.2 and Tetragold were selected and their bulked DNA samples obtained from 5, 10, 15, 20, 25 and 30 individual plants respectively. The result of genetic diversity analysis based on simple sequence repeat（SSR）markers showed that there were no differences in the PIC index among seven sampling methods, PIC reached the highest when bulked plant sample were 20. The number of average allele and the alleles, types of per SSR loc increased along with the plant number in the bulked plant samples increasing, which kept relatively steady when bulked plant samples surpassed 20 (including 20) plants, meanwhile the SSR banding patterns were relatively uniform. The bulked samples 20 were able to detect the most high frequency (10%) alleles with excellent reproducibility, if disregard the undetection of rare alleles (10%). It could be concluded that 20 individual plants was the suitable sampling size in genetic diversity analysis of annual ryegrass based on SSR markers.