Abstract: 【Objective】In order to obtain a high density genetic map of tetraploid orchardgrass previously established, we used EST-SSR and genomics-SSR markers to enhance the density of the genetic map. These results will be beneficial and helpful to orchardgrass selection and QTL analysis，especially QTL analysis of recessive genes.【Method】Based on the proposed test-hybridization strategy, an F1 population of 214 individuals derived from the cross between two Chinese orchardgrass cultivars–Kaimo (tall height plant, more tillers, broad leaves and early-maturing) and 01436 (dwarf, less tillers, narrower leaves and late-maturing) was used for map construction. 574 pairs of EST-SSR markers and 150 pairs of Genomic-SSR markers were selected as the screening primers. Five of the 214 progenies were randomly selected and amplified together with their parents. Amplified fragments were separated on 8% denatured polyacrylamide gels. The primers which could amplify clear bands and the presence of separated polymorphic were used for population and parental DNA amplification. The amplified results were statistically analyzed according to the marker type. According to the presence or absence of bands (with band count 1, or count 0), the amplification products of the DNA were statistically analyzed. According to theχ2 test, the marker which separation ratio was in accordance with 1﹕1 (Aaaa × aaaa or aaaa × Aaaa) and 3﹕1 (Aaaa × Aaaa) for genetic linkage map construction by using HighMap software.【Result】Finally, 31 pairs of EST-SSR primers and 17 pairs of Genomic-SSR primers were used for population and parental DNA amplification, The primer polymorphism percentage were 5.4%, 11.3% and 6.6%.A total of 169 clear bands were obtained, and 89 were used to construct the genetic linkage map of orchardgrass. There were 79 markers with Aaaa × aaaa or aaaa × Aaaa segregation types, 10 with Aaaa × Aaaa and the remaining 80 with distorted markers. A high-density linkage map of orchardgrass was constructed using 2,551 markers, which were distributed on seven linkage groups spanning 758.4 cM. The encrypted map including 4187 SNP markers, 84 SSR markers, the number of markers in the LGs from166 to709, with average 364. LG1 contains the largest maker with 709, while the LG7 was the least with 166. The sizes of the individual LGs ranged from 60.28 to 147.09 cM, with average inter-marker distances ranging 0.19—0.76 cM. The average distance between adjacent distance markers was reduced from 0.37 cM to 0.3 cM. Due to the change of marker density, the position of the markers distribution on each linkage group also changed greatly.  【Conclusion】 A high-density genetic linkage map of tetraploid Dactylis glomerata was reconstructed using 2551 markers, which were distributed on seven linkage groups spanning 758.4 cM. The new map added a number of SSR markers, which total length increased by 42.63 cM and average distance between adjacent distance markers was reduced from 0.37 cM to 0.3 cM.

Key words: orchardgrass, SSR, genetic linkage map