Abstract:In this study, the samples of Tan sheep, small tail Han sheep and Tan Han hybrid sheep were researched by macrogenomics to discuss the intraspecific polymorphism of sheep breeds which were easy to be confused. The whole genome of mixed samples was sequenced by high throughput sequencing, and the genetic diversity of sheep population was evaluated and analyzed. The SSR site of the splicing sequence was found by MISA. SSR sequence analysis and then designed as a fluorescent marker primer through PRIMER3 Input (version 2.3.7). Finally, the genetic diversity of Tan sheep, small tail Han sheep and Tan Han hybrid sheep was analyzed by polymorphism SSR markers. The sequencing results showed that the sequence information of the whole genome of the mixed sample with the total size of 6.1 GB was obtained, and the average fragment length of the sequence was 150 bp. It was found that compound SSR repeats existed, and the ratio of 3 / 2 / 1, 2 / 1 and 1 / 3 of the number of units was higher, and the total number of the three patterns accounted for 87.35% of the total number of complex repeats. 46 polymorphic markers were screened from 100 pairs of SSR primers, with a polymorphic rate of 46 and an average PIC value of 0.358. The samples of three species of Sheep genus and three mixed mutton species of sheep genus were grouped into one group, respectively, and showed monophyletic properties. The results are valuable for the study of sheep genome engineering, the development of molecular markers among sheep species and the identification and protection of germplasm resources.