Population Genomics Reveal That The Relationship Between The Thermogenesis Related Gene PRDM16 Mutation And Cold Tolerance In Domestic Cattle

Temperature is one of the most important environmental factors that drive evolutionary changes in diverse organisms.Mammals are endotherms;they require a constant body temperature to ensure optimal biological activity.This leads to strong selection pressure on the heat production system,including shivering and non-shivering thermogenesis.In China,due to the large latitude difference between the South and north,the annual average temperature is greatly different.Therefore,there is a vast difference in the annual average temperature of the habitats among mammals and the domestication,including cattle.In summer,southern cattle are exposed to extreme heat environment compared to northern cattle,and in winter,northern cattle are exposed to extreme cold environment compared to southern cattle.These living conditions just drive the characteristics that resistance of northern cattle can tolerant extremely cold environment and southern cattle can tolerant extremely hot environment.Compared with the research on heat tolerance of cattle,there are few studies on the regulation mechanism of cold tolerance of cattle.1.We sequenced genomes of 28 cattle,including 14 cold-tolerant cattle lineages（annual average temperature of habitat:2–6℃）and 14 cold-intolerant cattle lineages（annual average temperature of habitat:20–25℃）.The Neighbor-joining trees,PCA,LD and population structure analysis were clearly indicated that cattle samples could be classified into northern and southern groups and PSMC analysis showed two bottlenecks and two expansions,with population peaks at～50 and～700 kilo years ago（Kya）and population bottlenecks at～30 and 400 Kya,respectively.2.Selective sweeps analyses were performed over whole genomes based on the distribution of population-differentiation statistic(F_ST)values.First,we identified highly differentiated regions using F_ST,and then determined the top 5%calculated in50 kb windows in 25 kb steps.Then,final candidate genes were determined and ranked using Fisher’s exact test（q<0.01）.A total of 197 candidate genes had strong selective sweep signals and Among genes with signals of selective sweep,two candidate genes（PRDM16 and CPT2）were involved in thermogenesis;PRDM16 was of the most interest as it is known to increase thermogenesis by promoting the expression of the key gene UCP1.PRDM16 had the lowest P-value(Fisher’s exact test P=3.8×10^-11)and the highest F_ST（0.52）among genes related to thermogenesis.3.The PRDM16 genotypes found in northern and southern cattle were well-distinguished and consistent with the phylogenetic tree created using the SNPs of this gene.We discovered five nonsynonymous single nucleotide variants（SNVs）,of which one（c.2336 T>C,p.L779P）was found at a higher level（93%）in southern cattle than northern cattle.we compared the PRDM16 protein sequences to other species,and found that the substitution that occurred at position Leu₇₇₉of the PRDM16 gene in northern cattle is the same as that in species that have complete BAT function（e.g.,mouse,rat,and hamster）.Conversely,the substitution in southern cattle,which is proline,was the same as that in species with an incomplete or null BAT function（sheep,pig,whale,horse,platypus,elephant,sirenian,marsupial,human and rabbit）.Moreover,we also explored the genetic pattern of these substitutions（c.2336 T>C,p.L779P）across cattle genomes worldwide,and found that cattle in cold regions have a high frequency of the c.2336 C>T mutation,consistent with the pattern in China.4.In biochemical experiment,despite the similar differentiation efficiency between the two ectopic PRDM16-overexpressing groups,the m RNA expression levels of four BAT-selective genes（UCP1,C/EBPb,PGC1-a,and CIDEA）were significantly lower in the PRDM16 MU（c.2336 T>C,L779P mutation of PRDM16）group than in the PRDM16 group.Moreover,overexpression of PRDM16 increased UCP1expression to a much greater degree than PRMD16 MU.In general,on the one hand,well-functioning PRDM16 in northern cattle is required to resist extreme cold,and on the other,the functional inactivation of PRDM16 impairs beige adipocyte formation,which is beneficial for the environmental adaptability of southern cattle.These results could improve understanding of adaptive genetic variations in cattle and other livestock species living in regions different temperatures.