Genome scan of selective sweeps
To determine genetic modifications that occurred under different temperatures, we analyzed selective sweeps between the two cattle groups: northern (MG and YB) and southern (YN and HN). Selective sweep analyses were preformed over the whole genomes based on the distribution of F ST values, with the top 5% calculated in 50-kb windows with 25-kb steps using a Fisher’s exact test to determine the frequency spectrum in southern versus northern cattle. A total of 269 candidate genes were found to be with strong selective sweep signals in Chinese cattle (Fig. 2a , Additional file 1: Fig. S3and Table S3 ). The top enriched KEGG pathway of the candidate genes was RNA degradation (P = 5.5×10-3)(Additional file 1: Table S4 ), which mainly acts on gene expression and regulation and plays an important role in cellular mechanisms [31]. Several candidate genes (e.g., AOC3 and FASN ) were involved in pathways related to the activation of thermogenic metabolism, such as tyrosine metabolism (P = 5.3×10-2) and fatty acid biosynthesis (P = 5.8×10-2)(Additional file 1: Table S5 ). This is expected as cold exposure could increase tyrosine hydroxylase, leading to the increased secretion of norepinephrine and enhanced non-shivering thermogenesis in BAT [32]. In addition, AOC3 is related to diabetes and membrane-bound WAT [33-34]. FASN plays a role in the de novobiosynthesis of fatty acids, and the down-regulation of FASN can induce browning in WAT [35]. Many fatty acids exert a positive effect on thermogenesis by activating BAT [36-38]. We also found many candidate genes (e.g., APOA5, ACOX3 and ALDOB ) involved in fatty acid, fructose and mannose metabolism and associated with signalling pathways, such as the PPAR signalling pathway (Additional file 1: Table S5 ). APOA5 treatments can increase gene expression levels of UCP1 in adipocytes, and fat accumulation can cause ACOX3 to increase in blood [39]. ALDOB is involved in insulin biosynthesis and secretion, as well as insulin receptor signalling [40]. It is well-known that insulin pathways and fat metabolism are inseparable and could affect the development of BAT, leading to obesity and insulin resistance, for example [41-42]. Consistently, enrichment analysis of GO terms revealed that several candidate genes (LOC100337001, PRDM16, SH3PXD2B and ZFPM1 ) are related to fat cell differentiation (GO:0045598), brown fat differentiation (GO:0050873) and white fat cell differentiation (GO:0050872) (Additional file 1: Fig. S4 , Table S6 andS7 ).
Among those genes with signals of selective sweep, PRDM16 is of most interest because it was reported to increase thermogenesis by promoting the expression of the key gene UCP1 (Additional file 1: Fig. S5 ). In addition, though the F ST (0.26) andθ π (0.2×10-3) values ofPRDM16 were similar to that of three other significant genes (LOC100337001, SH3PXD2B and ZFPM1 ), the lowestP -value was observed for PRDM16 (P = 3.8×10-8 (Additional file 1: Table S3 andS8 ). In addition, Tajima’s D analysis also supported the idea that PRDM16 was under selection (D= -0.34) (Fig. 2b, Additional file 1: Table S8 ). The PRDM16genotypes found in northern and southern cattle were well-distinguished from each other and consistent with the phylogenetic tree created using the SNPs of this gene (Fig. 2c ). We discovered five non-synonymous SNVs of which one (c.2336 T > C, p.L779P) was found at a higher level (93%) in southern cattle than northern cattle (Additional file 1: Fig. S6 and Table S8 ).
We then compared the PRDM16 protein sequences to other species whose BAT had been found and reported (Fig. 2d ,Additional file 1: Fig. S7 ) and it is interesting to find that the substitution that occurred at position Leu779 of the PRDM16 gene in northern cattle is the same as that in species that have complete BAT function (e.g., mouse, rat and hamster)(Fig. 2d ). In rodents, BAT is intact and persists throughout their lifetimes, and its thermogenesis activity is complete [43-44]. However, in many large mammals, BAT is incomplete or even non-existent. For example, the BAT is incomplete in human adults and sheep in which it exists only during infancy and degenerates as they grow [45-46]. Conversely, the substitution in southern cattle, which is proline, is the same as that in species that have an incomplete or null BAT function (sheep, pig, whale, horse, platypus, elephant, sirenian, marsupial, human and rabbit (Fig. 2d ). Thus, we hypothesized that this substitution of residue 779 in thePRDM16 gene is probably related to the BAT function, and this locus is likely to play a role in cold tolerance.