Description of the sequencing data
Fecal samples were collected from four different sheep breeds (10 Dorset sheep (DrS), 15 Small Tail Han sheep (STHS), 5 Tibetan sheep (TS) and 10 Dorper sheep (DrS)). We retrieved 1,694,264 raw bases from the sequencing platform, as mentioned above. After quality–filtering (also as described in the methods), 1,359,405 total sequences with an average of 433 bp in length were obtained for the following analysis.
Gut microbiota is associated with sheep breeds
After cleaning the original data, 1,359,405 high-quality available sequences were obtained. According to 97% breeds similarity, 7039, 6887, 4112, and 8257 OTUs were acquired from samples at groups DrS, DsS, TS, and STHS (Table S2), respectively. A total of 26,295 OTUs were detected from all samples, of which 2,448 were core OTUs (Figure 1(A)). The core OTUs accounts for nearly 9.31 % of the entire OTUs. Furthermore, 225,203 OTUs were uniquely detected in DrS and DsS groups, and 168,654 unique OTUs were found in group TS and STHS, respectively. The number of observed OTUs in the TS samples was fewer than that of the other three breeds.
To confirm the quality of our sequencing data, we examined alpha and beta diversities of bacterial fraction of the sheep microbiota. Several alpha diversity indices diverged significantly between the four sheep breeds (Fig.1 (B) and (C)). The Shannon-Wiener index may straightly indicate the heterogeneity of a community according to the OTU counts of breeds and their related abundance [27]. The Shannon-Wiener indicator of groups DsS, STHS, TS and DrS were 9.92, 9.33, 8.09 and 9.06, respectively. Within the groups, Chao1 and Shannon index visually reflected that the abundance and diversity of intestinal microbial population in the TS group were lower than those in groups DsS, STHS and DrS, and the difference among these four groups was significant (P < 0.05; Figure 1 (B) and (C)). The Chao1 index of DsS, STHS, TS and DrS breeds was 3160, 2598, 1585 and 2207 respectively. The ACE indices of these four breeds were 3263, 2646, 1627 and 2267 (Table S3), which was consistent with the Chao1 results, suggesting that the OTU richness of TS samples was lower than other three breeds (Fig.1 (B) and (C)). Collectively, these data pointed towards a more diverse bacterial population in TS compared to others, and also showed that differences in intestinal microbial composition associates sheep breeds.
Comparison of bacterial microbiome diversity among different sheep breeds
The Bray-Curtis distance matrices were measured according to the OTUs abundance of each sample. Based on the distance matrices, the unweighted Unifrac similarity analysis indicated that the similarities among different sheep breeds were significant. The principal coordinates analysis (PCoA) was performed according to the phylogenetic-tree-based Unifrac metric. As shown in Fig. 2 (A), samples were sequestered into three clusters. Scattered points in the principal component denoted different breeds and their relationship between each other. There were significant differences among breeds in relation to microbiome composition (PERMANOVA, p < 0.01). TS were mainly aggregated in cluster B, whereas DsS were mostly converged in cluster A. Moreover, DrS and STHS were more scattered and found between cluster A and cluster B. Both principal components accounted for 28.1% (PC1) and 8.5% (PC2) of the explained variance. Interestingly, two lambs in STHS were clustered separately as shown in Fig. 2 (A), suggesting that there was a general difference in gut microbiome between adult sheep and lambs.
Nonmetric multidimensional scaling (NMDS) was used to further clarify the difference among the bacterial population of all breeds, which was performed using the Bray-Curtis similarity for all samples at OTU level[1]. As a dominant ordination method that could exhibit the non-linear relationship among samples, NMDS has been widely applied in the study of gut microbiome. As shown in Fig. 2 (B), there was distinguishing clustering of TS samples, meanwhile samples from DsS were very close to DrS. However, the samples from STHS were more dispersed (Fig. 2 (B)).
Additionally, we did hierarchical clustering analysis of all samples to exhibit the similarity among samples, which was performed with Unweighted pair-group method with arithmetic means (UPGMA) and the Bray-Curtis similarity. Two primary groups were perceived in this analysis. One cluster contains all TS samples and the other cluster contains all samples from DrS (Fig. 3). Consist with the results above, TS samples were distinctive compared with other breeds. In general, the composition of gut microbiota is greatly influenced by the breed of sheep.
Gut microbial diversities and community composition among different sheep breeds
In order to clarify the diversity of gut bacterial composition in different sheep breeds, we estimated the gut microbiota in different taxonomical levels. The overall bacterial composition of different groups at the phylum level is illustrated in Fig. 4 (A), which shows that Firmicutes was the most predominant phylum in all samples, followed by Bacteroidetes. Higher abundance of phylum Spirochaetes, Proteobacteria and Verrucomicrobia was found in TS than those in other three breeds, but the Deferribacteres was absent in TS (Fig. 4 (A)).
When analyzed on the family level, as shown in Fig. 4 (B), no significant differences were detected among these four groups. Ruminococcaceae and WCHB1-25 were the most abundant families in DsS, DrS and STHS group, whereas Alcaligenaceae, Desulfovibrionaceae and Barnesiellaceae were almost absent. As for TS group, the most abundant families were Spirochaetaceae, S24-7, Prevotellaceae, Barnesiellaceae and Succinivibrionaceae, while BS11 and WCHB1-25 were almost absent in the TS samples (Fig. 4 (B)).
In contrast to the family level, there are significant differences between TS group and the other three groups on the genus level. The main genera in TS group included Treponema, Succinivibrio, 5-7N15 and Prevotella (Table 1), while Bifidobacterium, Sharpea and YRC22 were absent (Fig. 4 (C)). Moreover, in DsS, STHS and DrS group, Treponema remained the predominant population, and Coprococcus and Roseburia were relatively less abundant. However, it is worth mentioning that a large number of microbes in TS samples were relatively abundant, when compared to other three groups.