Abstract: 【Objective】Annual ryegrass is a world-cultivated forage, but frequent exchange of breeding material causes intervarietal genetic differences become smaller due to the cross-pollination characteristics, therefore, cultivar identification using traditional agronomic traits is becoming increasingly difficult. Efficient, rapid identification of cultivar plays an important role in breeding, and also provides a scientific basis for DUS testing and intellectual property protection.【Method】In this study, the cultivar identification among 6 backbone varieties of annual ryegrass was conducted by using 20 pairs of SSR primers from 165 for 3 bulk of DNA samples each attracted from 30 random individual plants equally based on strong stability, high polymorphism and co-dominant characteristics of SSR markers, and the specific bands of bulked samples appeared at least twice could be recorded in amplification profile. And the combination of 30 individuals and bulked samples using SSR markers, and the comparation of amplification profiles were used to identify 6 varieties of annual ryegrass.【Result】Comparative analysis of 6 varieties of annual ryegrass using SSR markers between individuals and bulked samples by using 3 primer pairs (13-07A, 12-10F and 15-08C) showed that the amplification bands of bulked samples were more obvious and more bands than individuals, but also some diffuse bands. And the frequency bands more than 40% in individuals appeared the same bands in bulked samples. The results of identification of 6 varieties of annual ryegrass using 3 bulked samples showed that, 20 primer pairs amplified 143 legible bands, comprising 127 polymorphic bands, and the polymorphism ratio reached 88.81%, and the amplified brands to each pair of primers were 5 to 11, and the average amplified brand, polymorphic bands, and polymorphism information content (PIC) were 7.15, 6.35 and 0.571, respectively. Although some primers polymorphic information content is low, high stability, i.e. three groups had almost same amplification results. The genetic similarity coefficient of 6 varieties varied from 0.4755 to 0.7552, the largest genetic similarity coefficient was between Abundant and Aderenalin, and the smallest genetic similarity coefficient was between Changjiang No.2 and Tetragold. The 6 varieties of annual ryegrass were divided into three groups on the average genetic similarity coefficient of 0.615, one included Angus1, Abundant, Aderenalin and Double Barrel, one was ChangjiangNo.2, and one was Tetragold. Nine of 20 SSR primer pairs could directly identify 6 varieties of ryegrass, however, the other primer pairs could only identify 3 or 4 varieties, the identification ability could be improved by combining with different primer pairs.【Conclusion】For cross-pollinated plants, polymorphism is not the only criterion to measure the effectiveness of primers, the stability of primers is also an important indicator. By comparative analysis of the 2 methods, bulked samples using SSR markers is better than the method of individuals. The screened 20 pairs of SSR primers could clearly distinguish 6 varieties of ryegrass, indicating that the use of SSR markers is feasible and efficient for cultivar identification.

Key words: annual ryegrass (Lolium multiflorum Lam.); cultivar identification, sampling methods, simple sequence repeats markers, DUS testing