- Several genomic analyses have been conducted in sheep breeds from limited geographic origins to identify the genetic factors underlying this trait. - Results: To minimize the bias and distinguish the true candidates, we used an extended data set of 968 sheep representing 18 fat-tailed breeds and 14 thin-tailed breeds from around the world, and integrated two statistical tests to detect selection signatures, including Genetic Fixation Index (F ST ) and difference of derived allele frequency ( Δ DAF). - The results showed that platelet derived growth factor D (PDGFD) exhibited the highest genetic differentiation between fat- and thin-tailed sheep breeds. - Analysis of sequence variation identified that a 6.8-kb region within the first intron of PDGFD is likely the target of positive selection and contains regulatory mutation(s) in fat-tailed sheep. - This study provides insights into the selection of fat-tailed sheep and has important application to animal breeding, as well as obesity-related human diseases.. - Keywords: PDGFD, Fat-tailed sheep, Genomic scan, Fat deposit, Adipogenesis. - One of the main. - It is be- lieved that fat-tailed sheep were selected in response to the steppe and desert conditions in central Asia from 3, 000 BCE (Before Common Era) [3], which is several thousand years after the domestication of its thin-tailed ancestor, and then spread east into north China and west into South Africa.. - This is quite surprising, as all the fat- tailed sheep seem to be of same origin with relatively short history and thus are expected to have the similar genetic basis underlying the trait. - To clarify this, we performed a comprehensive genomic analysis of 18 thin-tailed and 14 fat-tailed sheep breeds from around the world. - We inte- grated two selection tests, including F ST and the difference of derived allele frequency (ΔDAF) (referred to as DAF Fat-- tailed sheep – DAF Thin-tailed sheep. - to identify positively selected genes specifically in fat-tailed sheep. - Additionally, histological and gene expression analysis of sheep tail tissues were carried out to understand the asso- ciation of the expression of the most predominant candi- date gene PDGFD with fat deposition in sheep tail during embryonic development. - These samples contained eight wild Mouflon sheep individuals, 18 thin-tailed sheep breeds and 14 representative fat-tailed sheep breeds from different countries or regions within countries (Fig. - The geographic distributions of the sheep breeds in this report include South Asia, East Asia, Middle East, Europe, Africa, North America, and. - After a series of quality control filters, a total of 45,337 SNPs and 828 individuals from 30 diverse sheep breeds world- wide were retained for further analysis.. - Because the wild ancestor of domestic sheep, the Mouflon sheep, is thin-tailed, modern thin-tailed sheep are expected to keep initial allele state while fat-tailed sheep exhibit derived alleles at the loci related to fat tail phenotype. - Under this scenario, we integrated two statis- tical measures, the F ST and the ΔDAF, to increase the power of identifying genomic loci specifically selected in fat-tailed sheep breeds. - For comparison with previous studies, we separately performed F ST and ΔDAF analysis in three group-pair comparisons, including Middle East fat-tailed sheep (MEF) vs South Asian thin-tailed sheep (SAT), MEF vs European thin-tailed sheep (EUT), and Chinese fat-tailed sheep (CHF) vs SAT. - 0.5) in more than 30% of the thin- tailed (or fat-tailed) sheep breeds, after examining the DAF of these SNPs in additional three American thin-tailed sheep breeds and three African fat-tailed sheep breeds (See Methods) (Additional file 5: Fig. - The remaining eight SNPs are highly diverged between thin- and fat-tailed sheep, and keep ancestral allele in most thin-tailed sheep breeds while derived allele in most fat-tailed sheep breeds worldwide (Fig. - Principle Component Analysis (PCA) and phylogenetic tree analysis using these eight SNPs showed clearly separated clades between thin- and fat-tailed sheep, which is quite distinct from the results obtained based on genome-wide variants showing geo- graphic clustering (Additional file 6: Fig. - To better investigate these selected genes, we extracted their genome sequence variants of 16 thin-tailed and 13 fat-tailed individual sheep with diverse geographic origins from NextGen project (Additional file 7: Table S5). - most divergent locus between fat- and thin-tailed sheep (Fig. - Haplotype comparison analysis using all the SNPs (n = 122) within this region re- vealed a consistent differentiation between sheep breeds with different tail types (Fig. - 0.8) between thin- and fat-tailed sheep and. - 1 Identification of positively selected loci in fat-tailed sheep. - a The geographic distribution of the sheep breeds used in this study, each of which is represented by a dot on the world map. - MEF: Middle East fat-tailed sheep from Middle East. - SAT: South Asian thin-tailed sheep. - EUT: European thin-tailed sheep. - CHF: Chinese fat-tailed sheep. - d The derived allele frequency (DAF) of the 16 positively selected loci identified in all three group-pair comparisons in all the studied sheep breeds. - large derived allele frequency in fat-tailed sheep (>. - We also examined the DAF distribution of the top candidate SNPs that were proposed in previous studies and the results showed that the majority of these SNPs are not consistently diverged between thin- and fat-tailed sheep populations (Additional file 10: Fig. - To gain insight into tail fat deposition in fat-tailed sheep breeds, tail tissues from a fat-tailed Chinese sheep breed, namely Tan sheep, at four different embryonic time points, including embryonic day 60 (E60), E70, E80 and E90, were collected. - a The distribution of the absolute difference of allele frequency Δ AF of sequence variation within the 24 identified genes with selection signatures between 13 fat-tailed sheep individuals and 16 thin-tailed sheep individuals from different regions. - The bottom panel shows the distribution of the absolute difference of allele frequency Δ AF of sequence variation within PDGFD gene. - b Haplotype compassion between thin- tailed and fat-tailed sheep obtained based on all the variations within the highly differentiated 6.8-kb genomic interval (Chr bp). - c Derived allele frequency of the 13 top candidate SNPs in additional fat-tailed sheep (Chinese Tan sheep) and thin-tailed sheep (Chinese Tibetan sheep) obtained by Sequenom MassARRAY. - a Morphological changes of tail tissues from fat-tailed sheep at embryonic day 60 (E60), E70, E80 and E90. - b Hematoxylin and Eosin (HE) staining for tail tissues of fat-tailed sheep at E60, E70, E80 and E90. - c Oil Red O staining for tail tissues of fat-tailed sheep at E60, E70, E80 and E90. - 4a), confirming the morphologic observation and histological results that cells in tail tissues of fat-tailed sheep undergo adipogenesis from E60/70 to E80/90.. - a Expression of several marker genes involved in adipogenesis in tail tissues of fat-tailed sheep at different stages of embryonic development revealed by RNA-seq. - PDGFD expression in tail tissues of fat-tailed sheep at different stages of embryonic development revealed by b RNA-seq and c qPCR. - g PDGFD expression is higher in fat tissues of thin-tailed and fat-tailed sheep at E70 and h adult stage revealed by qPCR. - Further qRT-PCR analysis showed that PDGFD expres- sion is greater in tail tissues of fat-tailed sheep than that in thin-tailed sheep at both E70 and adult stages (Fig. - To accurately map the candidate gene(s) underlying the fat tail phenotype of sheep, herein we comprehensively an- alyzed genomic variation data from a large cohort of sheep breeds with different tail types from around the world and integrated two different selection tests to detect genomic regions with signals of selection. - We demonstrated that PDGFD gene locus, with known involvement in adipogen- esis [12, 13], exhibited the highest genetic differentiation between fat- and thin-tailed sheep and further found that the potential causal mutations are located within regula- tory region. - Compared to several existing papers that have studied sheep breeds from limited geographical areas and reported quite different candidates associated with fat tail phenotype [6–11], this study analyzed the largest co- hort of samples until so far, to the best of our know- ledge, originating from around the world. - For instance, three SNPs, OAR ORA and s27419.1, at the BMP2 locus, exhibited relatively higher DAF in fat-tailed sheep than that in thin-tailed (Additional file 10: Fig. - A possible reason for this is the low abundance of de- rived allele in some fat-tailed sheep populations, such as Afshari, LocalAwassi and EthiopianMenz (Additional file 10:. - Therefore, BMP2 has lower priority than the PDGFD locus as the candidate for fat-tailed phenotype.. - Furthermore, a recent study based on whole-genome re- sequencing data also found that PDGFD is the most highly differentiated loci between fat-tail sheep populations from China as well as Middle East, and thin-tailed Tibetan sheep, followed by BMP2 as the second top highly diver- gent gene [8]. - Our morphological and histological ana- lysis suggests that tail tissues undergo fat deposition from E60/E70 to E80/E90 in fat-tailed sheep during embryonic development (Fig. - Despite a continuous decline of expression during develop- ment, we found that PDGFD sustains higher expression in tail tissues of fat-tailed sheep than that of thin-tailed sheep from embryonic (E70) to adult stage (Fig. - Supporting this, a previous study reported that the expression of PDGF D is significantly upregulated in tail adipose tissue from fat- tailed Hulun Buir sheep as compared to thin-tailed Tibetan sheep [17]. - We discovered a list of mutations that are located within the first intron of PDGFD and narrowed down the candi- date casual mutations to 13 SNPs which display high fre- quency in fat-tail sheep while low abundance in thin-tailed sheep (Fig. - Since in- tronic mutations mainly affect the transcriptional efficiency of genes by creating or disrupting binding sites for tran- scriptional factors [22, 23], it is likely that the elevated expression of PDGFD in fat-tailed sheep is attributed to some/one of these mutations. - Future mutagenesis studies are warranted to determine the effect of mutated allele of these candidate SNPs identified in fat-tailed sheep on PDGFD expression to pinpoint the true casual mutation(s).. - Genome-wide SNP data of 968 individual sheep from five South Asian thin-tailed sheep breeds (i.e., Tibetan, Changthangi, Deccani, IndianGarole and Garut sheep), six European thin-tailed sheep breeds (i.e., Churra, Leccese, Comisana, Altamurana, MacarthurMerino and MilkLa- caune sheep), three American thin-tailed sheep breeds (i.e., BarbadosBlackBelly, MoradaNova and SantaInes sheep), six Middle East fat-tailed sheep breeds (i.e., Afshari, LocalA- wassi, Karakas, Norduz, Moghani and CyprusFatTail sheep), four African fat-tailed sheep breeds (i.e., Ethiopian- Menz, NamaquaAfrikaner, RedMaasai and RonderibAfrika- ner) and eight Mouflon sheep individuals (wild sheep) were downloaded from Ovine HapMap project (http://www.. - SNP data of four Chinese fat-tailed sheep breeds (i.e., Hu, Tong, Large tail Han and Lop sheep) were obtained from a previous study [9]. - SNP data of four Nepalese thin-tailed sheep breeds (i.e., Bhyanglung, Baruwal, Lampuchhre and Kage sheep) were generated in our lab [25]. - The detailed information of these sheep breeds was provided in Additional file 1: Table S1. - A pruned set of 32,450 SNPs were used to investigate the genetic relationship among these sheep breeds from. - After PCA analysis, a total of 45,337 SNPs for 828 individuals from 30 diverse sheep breeds were retained for downstream analyses (Additional file 1: Table S1).. - Genomic screen for positively selected genes in fat-tailed sheep. - Three previous studies compared the genomic variations between Middle East/European thin-tailed versus (v.s.) Middle East fat-tailed sheep [10], European thin-tailed v.s. - Middle East fat-tailed sheep [11], and Chinese thin- versus fat-tailed sheep [9], respectively. - Therefore, we used sheep breeds from South Asia, Middle East and Europe to identify the positively selected genes in fat- tailed sheep. - Three group-pair comparisons between thin- and fat-tailed sheep were considered, including MEF v.s. - Two statistics, including the F ST and Δ DAF were applied to evaluate the genetic differentiation of each SNP between thin- and fat-tailed sheep populations. - Δ DAF was cal- culated as the derived allele frequency in the fat-tailed sheep minus the DAF in thin-tailed sheep (DAF Fat-tailed sheep. - DAF Thin-tailed sheep ) using R version 3.3.3 (https://www.r- project.org. - For each group-pair comparison, F ST and Δ DAF at each SNP marker was calculated between each thin-tailed breed against each fat-tailed breed and averaged across breed-pairs to produce an overall value for each SNP. - The top 1% SNPs with large overall F ST or Δ DAF value were considered as the significant SNPs in each test and the significant SNPs overlapped in both tests were con- sidered as positively selected SNPs in fat-tailed sheep. - Fi- nally, positively selected SNPs that were identified in all three group-pair comparisons were considered as the loci under positive selection in fat-tailed sheep and genes within 150 kb of these loci were retrieved from Ensembl BioMart database (http://useast.ensembl.org/biomart/martview/625 a397ecca62a421509935f099cdaa7). - Because the high aver- age value of F ST or Δ DAF may have resulted from the ex- tremely large values in several specific breed-pairs due to population structure, we additionally examined the DAF of the candidate SNPs in three thin-tailed sheep breeds from Americas and three fat-tailed sheep breeds from Africa and further filtered the promising candidate list by removing SNPs with DAF >. - 0.5 in more than five thin-tailed sheep breeds or DAF <. - 0.5 in more than four fat-tailed sheep breeds (>. - 30% of total thin- or fat-tailed sheep breeds).. - Genomic variations stored in Variant Call Format (VCF) file for 16 thin-tailed sheep individuals and 13 fat-tailed sheep individuals from 17 different breeds from around the world were downloaded from NextGen of Ensembl Projects (http://projects.ensembl.org/nextgen/) (Additional file 7:. - PLINK [26] was used to perform the SNP quality control (removing SNPs with call rate ≤ 90% or MAF ≤0.05) and to calculate the allele frequency of each SNP in thin- and fat-tailed sheep group. - The absolute ΔAF) of each SNP between the fat- and thin-tailed sheep group were calculated using R.. - 0.8) and derived allele frequency larger than 0.8 in fat-tailed sheep were selected for fur- ther validation in this expanded cohort. - Positively selected SNPs identified in comparison of Middle East fat-tailed sheep vs South Asian thin-tailed sheep.. - Positively selected SNPs identified in comparison of Middle East fat-tailed sheep vs European thin-tailed sheep.. - Positively selected SNPs identified in comparison of Chinese fat-tailed sheep vs South Asian thin-tailed sheep.. - The distribution of derived allele frequency (DAF) of the 16 candidate SNPs in each breed. - Phylogenetic analysis of the studied sheep breeds. - Replication of the top candidate SNPs proposed by three previous studies in all sheep breeds used in this study.. - (A) Derived allele frequency (DAF) of the top candidate SNPs previously proposed. - Expression of the top candidate genes in tail tissues of fat-tailed sheep during embryonic development revealed by RNA-seq. - MEF: Middle East fat-tailed sheep. - EUT: European thin-tailed sheep;. - This study followed the recommendations of the “ Regulations for the Management of Affairs Concerning Experimental Animals ” (Ministry of Science and Technology, China, revised in June 2004). - A genome wide survey of SNP variation reveals the genetic structure of sheep breeds. - Genomic scan of selective sweeps in thin and fat tail sheep breeds for identifying of candidate regions associated with fat deposition. - Genome-wide analysis of the world's sheep breeds reveals high levels of historic mixture and strong recent selection
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