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RESEARCH ARTICLE Open Access Genomic scan of selective sweeps in thin and fat tail sheep breeds for identifying of candidate regions associated with fat deposition Mohammad Hossein Moradi1*, Ardeshir Nejati-Javaremi1, Mohammad Moradi-Shahrbabak1, Ken G Dodds2 and John C McEwan2 Abstract Background: Identification of genomic regions that have been targets of selection for phenotypic traits is one of the most important and challenging areas of research in animal genetics. However, currently there are relatively few genomic regions identified that have been subject to positive selection. In this study, a genome-wide scan using ~50,000 Single Nucleotide Polymorphisms (SNPs) was performed in an attempt to identify genomic regions associated with fat deposition in fat-tail breeds. This trait and its modification are very important in those countries grazing these breeds. Results: Two independent experiments using either Iranian or Ovine HapMap genotyping data contrasted thin and fat tail breeds. Population differentiation using FST in Iranian thin and fat tail breeds revealed seven genomic regions. Almost all of these regions overlapped with QTLs that had previously been identified as affecting fat and carcass yield traits in beef and dairy cattle. Study of selection sweep signatures using FST in thin and fat tail breeds sampled from the Ovine HapMap project confirmed three of these regions located on Chromosomes 5, 7 and X. We found increased homozygosity in these regions in favour of fat tail breeds on chromosome 5 and X and in favour of thin tail breeds on chromosome 7. Conclusions: In this study, we were able to identify three novel regions associated with fat deposition in thin and fat tail sheep breeds. Two of these were associated with an increase of homozygosity in the fat tail breeds which would be consistent with selection for mutations affecting fat tail size several thousand years after domestication. Background The domestication of livestock represents a crucial step in human history. The rise of civilizations could not happen without domestication of plants and animals. Sheep (Ovis aries) is the first grazing animal known to have been domesticated [1]. Multiple mitochondrial lineages suggest that domestication occurred several times, as in other livestock species such as cattle, goat and pig [2]. Recognition of the origin of domestication is difficult by the fact that the first domestic animals were no different from their wild counterparts [3]. In spite of this, the archaeozoological evidence suggests that the domestication of sheep occurred during the Neolithic revolution approximately 9000 years ago [4] in a region in northern Iraq and nearby regions in Iran [5]. Since domestication, sheep have established in a wide geographical range due to their adaptability to poor nutrition diets, tolerance to extreme climatic conditions and their manageable size [6]. Fat tail breeds are an important class of sheep breeds that are first documented as being present 5000 years ago. The earliest depiction of a fat tail sheep is on an Uruk III stone vessel of 3000 BC and fat and thin tail sheep appear together on a mosaic standard from Ur dated around 2400 BC [3,7]. The fact that the fat tail breeds are now prevalent in the Fertile Crescent, where sheep were originally domesticated, while thin tail sheep breeds are predominant in peripheral areas [7] and that the wild ancestor of sheep is thin tail suggest that the * Correspondence: Hoseinmoradi@ut.ac.ir 1Department of Animal Science-Excellent centre for improving sheep carcass quality and quantity, University of Tehran, PO Box 3158711167-4111, Karaj, Iran Full list of author information is available at the end of the article Moradi et al. BMC Genetics 2012, 13:10 http://www.biomedcentral.com/1471-2156/13/10 © 2012 Moradi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 许瑞霞 高亮 n. 驯养；教化 first domesticated sheep were thin tail and fat tail was developed later. The evidence shows that sheep were being farmed throughout Europe 5000 years ago [8]. The fat tail is considered as an adaptive response of animals to a hazardous environment and is a valuable energy reserve for the animal during migration and win- ter. Until recently it had additional value to the herder because it was used to preserve cooked meat for longer periods of time and also as an energy reserve during times of drought and famine. Therefore the climatic var- iation as well as the associated requirements of humans led to artificial selection for higher fat tail weight across generations [9-11]. Nowadays in intensive and semi- intensive systems most of the advantages of a large fat tail have reduced their importance and therefore, a decrease in fat tail size is often desirable for producers and consumers. Fat deposition requires more energy than the deposition of lean tissue, animal fat has lost much of its market demand and monetary value and sheep producers have easy access to other forms of aux- iliary feeding [12]. These breeds are commonly found in a wide range of countries in Asia especially the Middle East and North Africa [7]. To date, several investigations into the inheritance of fat tails have been undertaken [10,11,13-18], nevertheless the genes affecting fat deposi- tion in fat tail breeds are still unknown. The study of genes underlying phenotypic variation can be performed in two different ways, firstly from phenotype to genome, which is performed by LD based association mapping or by targeting particular candidate genes identified based on homology to known genes, and secondly from genome to phenotype, which involves the statistical evaluation of genomic data to identify likely targets of past selection using selective sweep ana- lysis [19,20]. The elimination of standing variation in regions linked to a recently fixed beneficial mutation is known as a “selective sweep” and has recently been the focus of much theoretical and empirical attention [21-23]. In contrast to natural populations, domesticated spe- cies provide an exciting opportunity to understand how artificial selection promotes rapid phenotypic evolution [22]. With an hypothesis that different selection pres- sures operated in thin and fat tail breeds over the his- tory of time and somehow the selection acts on a variant that is advantageous only in one breed, it is expected that the frequency of that variant may differ across populations to a greater extent than predicted for variants evolving neutrally in all populations [21]. Identi- fying of these genome regions, which have been subject to such selective sweeps could reveal the mutations which are responsible for the fat deposition in these breeds. The examination of variation in SNP allele fre- quencies between populations, which can be quantified by the statistic FST, is a promising strategy for detecting signatures of selection [21,24]. To date a relatively small number of examples have successfully identified genomic regions subject to posi- tive selection in different domestic animals [20,25-38]. The constraint to identifying selection signatures in sheep has been the limited density of markers. This lim- itation has recently been reduced with the availability of tens of thousands of single nucleotide polymorphisms (SNPs) using the Ovine SNP50k BeadChip (http://www. sheephapmap.org.) In this research two independent resources have been investigated to identify regions associated with fat deposition in these breeds. The first data set was comprised of one thin and one fat tail Ira- nian sheep breed while the second was comprised of several other fat and thin tail breeds selected from the Ovine HapMap project. Sheep production constitutes the most important component of the Iranian livestock industry with a total of approximately 50 million head. Twenty seven breeds and ecotypes have been documented in Iran, the major- ity derived in situ. The Lori-Bakhtiari sheep breed is one of the most common indigenous breeds. It is well adapted to the hilly and mountainous Bakhtiari region stretched out to southern Zagros Mountains (Figure 1), with a population of more than 1.7 million. The animals are kept mostly in villages under semi-intensive systems. Relative to other Iranian fat tail breeds the Lori-Bakh- tiari is a large breed, having the largest fat tail by girth and weight [39]. Sheep breeds vary in tail length and this breed, perhaps surprisingly, has a rather short tail length. Zel is the only thin-tail Iranian breed and it is present largely on the northern slopes of the Elburz mountain range near the Caspian Sea [40] representing around 3% of Iranian sheep. The aim of this study was to find selective sweeps between the Iranian thin and fat tail breeds using dense SNP markers and to compare with those from similarly divergent breeds extracted from the Ovine HapMap dataset (http://www.sheephapmap.org.) Our work pro- vides the first genome wide characterization of selective sweeps in thin and fat tail breeds. Each animal was gen- otyped with approximately 50,000 SNPs and a variety of selection sweep tests were utilized. Results Genotyping and data mining A total of 94 animals consisting of 47 samples per breed were genotyped on the Illumina OvineSNP50K Beadchip assay in the Zel-Lori Bakhtiari data set. One animal in each breed had greater than 5% missing data and these were excluded from further analysis. The second data set used in this study was SNP genotyping data from similarly divergent breeds in the Ovine HapMap project Moradi et al. BMC Genetics 2012, 13:10 http://www.biomedcentral.com/1471-2156/13/10 Page 2 of 15 (Table 1). After data cleaning (see methods) a total of 45,611 and 48,053 SNPs passed the filtering criteria in the Zel-Lori Bakhtiari and HapMap data sets respec- tively and were included in the final analyses. The over- all average distances between 2 adjacent SNPs were relatively consistent among the chromosomes, being about 60 kb in the Zel-Lori Bakhtiari and 58 kb in the HapMap data sets. In all cases the locations used were obtained from OAR true chromosomes (ver.1.0, as at 5/ 2008) from CSIRO [41]. The principal component ana- lysis (PCA) in Zel-Lori Bakhtiari data set, using the individual SNPs as the data, resulted in the first two principal components (PC1 and PC2) explaining 18.6% and 2.9% of the variance respectively. We found that PC1 separated out the two breeds from each other while one animal in each breed was separated from all other animals for PC2 (Figure 2). These two animals were excluded from further analysis. Finally, 45 animals (36 females and 9 males) per breed passed the data cleaning steps and 95% and 95.1% of all remaining SNPs were in Hardy-Weinberg equilibrium at the 5% level in Zel and Lori-Bakhtiari breeds respectively. For the SNPs Figure 1 Traditional distributions of the two Iranian breeds used in this study are shown in (a) with breed examples of Lori Bakhtiari (b) and Zel (c). Moradi et al. BMC Genetics 2012, 13:10 http://www.biomedcentral.com/1471-2156/13/10 Page 3 of 15 analyzed in this study, the average MAF over all samples was 0.29 (SD = 0.13) in the Zel-Lori Bakhtiari data set and 0.30 (SD = 0.12) in the HapMap data set. Genomic distribution of FST in Zel-Lori Bakhtiari data set The plot of windowed FST against location is shown in Figure 3. The average of differentiation between Zel and Lori Bakhtiari breeds was 0.024 (SD = 0.035). As shown in this figure, in several instances outlier SNPs tended to cluster to similar regions. Specifically, we found evi- dence of selection in seven regions with windowed FST value > 0.20 on chromosomes 2 (between 55,861-56,300 kb), 2 (between 73,631-73,784 kb), 3 (between 146,615- 146,676 kb), 5 (between 47,149-47,263 kb), 7 (between 30,512-30,585 kb), 7 (between 46,642-46,843 kb) and X (between 58,621-61,452 kb). The average FST for autosomal and X-linked SNPs was significantly different (0.024 and 0.035, respectively; t test, t = 6.2, P < 10-10). A higher average FST for X-chro- mosome SNPs could occur because of its smaller effec- tive population size compared with that of the autosomes. Another genetic distance measurement including unbiased estimates of FST as described by Weir and Cockerham [42] was also calculated. Because the results were highly correlated (r = 0.995) with the above results, so have not been presented. Study of Bovine genes and published QTLs in regions showing evidence of selection Seven regions showing the largest signals of selection in Zel-Lori Bakhtiari data set were chosen for further ana- lysis. As the current annotation of the sheep genome is not as comprehensive as cattle, the regions of interest in O. aries were compared to the corresponding areas in B. taurus. Dot plots for corresponding areas of Ovine and Bovine genomes showed strong co-linear relationships between the two considered sequences in all regions and rearrangements were not observed (Additional file 1: Figure S1). A summary of orthologous area in both species and published bovine genes and QTLs is presented in table 2. Orthologus genes in the bovine genome were identi- fied using the BLAT genome search with UCSC Gen- ome Browser [43]. Online databases of published QTL in beef and dairy cattle, show that the regions identified here were pre- viously been found to be in regions harboring QTL affecting fat and also carcass yield traits (Table 2). For example, the regions on chromosomes 2 overlapped with QTLs previously suggested as being related with fat depth, and also chromosomes 2 (second region) and 5 with fat thickness and both regions on chromosomes 7 with milk fat yield. Table 1 Origin and sample size of different breeds included in this study Data set Breed Code1 Tail status Sampling area Sample size Zel-Lori Bakhtiari data set Zel ZEL Thin tail Iran 47 Lori-Bakhtiari LOR Fat tail Iran 47 Ovine HapMap data set Deccani DEC Thin tail India 23 Scottish Black Face SBF Thin tail United Kingdom 56 Bunder Oberland Sheep BOS Thin tail Switzerland 24 Gulf Coast Native GCN Thin tail North America 94 Karakas KAR Fat tail Turkey 18 Norduz NDZ Fat tail Turkey 20 Afshari AFS Fat tail Iran 37 1 Code is the 3-letter code for each breed. Figure 2 Animals clustered on the basis of principal components analysis using individual genotypes. Zel and Lori Bakhtiari breeds are shown by green and blue circles respectively. Moradi et al. BMC Genetics 2012, 13:10 http://www.biomedcentral.com/1471-2156/13/10 Page 4 of 15 Genomic distribution of FST in Ovine HapMap data set and comparison with Zel-Lori Bakhtiari data set results An independent resource comprised of similarly diver- gent breeds for thin and fat tail breeds in the Ovine HapMap project were selected to determine whether the regions with large allele differentiation in Zel-Lori Bakh- tiari data set could be confirmed. The pattern of FST across the genome was also calculated in the Ovine HapMap data set (fat and thin tail breeds were pooled) and values were averaged in a sliding window. The windowed FST was then plotted against location in the genome. The windowed FST values, at each common SNP, were correlated in the two data sets with r = 0.413, N = 45,238 and the average of differentiation between thin and fat tail breeds in the Ovine HapMap data set was 0.027 (SD = 0.038). The sliding window average FST revealed good agreement between both data sets for regions on chromosomes 5, 7 (second region) and X (Figure 4). Calculation of empirical p-values for windowed FST values in each data set (Table 3) shows that the 3 regions with the largest differentiation in Zel-Lori Bakhtiari data set are also significant in the Ovine HapMap data set (P < 0.01). On the other hand, combining p-values from independent tests of significance in Zel-Lori Bakhtiari and Ovine Hapmap data sets using Fisher’s combined test revealed the first region on chromosome 2 (P < 0.0001) in addition to these same significant regions. The Weir and Cockerham method of estimating FST [42] was also performed for the ovine HapMap data and a high correlation (r = 0.992) was observed. Previous reports [e.g. [24,52]] also indicated these methods led to similar results; therefore, a strong correlation between these two measures is not surprising. Study of median homozygosity in candidate regions Median run of homozygosity (Figure 5) was increased at the candidate regions on chromosome 5 and X for Lori Bakhtiari (fat tail) and at the candidate region on chromo- some 7 for Zel (thin tail). The largest differences of med- ian homozygosity were located on chromosome X and this was present for a longer distance as well, whereas these statistics were lower on Chromosome 5. A study of med- ian homozygosity in the HapMap data set for thin and fat tail breeds (data not shown) revealed similar results. Estimation of effective population size and haplotype age The average extent of LD in the genome was used to estimate the effective population size at various times in the past. The estimated effective population size show a persistent decline from 2000 down to 20 generations ago, declining from 4900 (both breeds) to 840 in Zel and 532 in Lori Bakhtiari. The graph of effective population sizes suggests a dis- tinctive time point when the breeds separated ~1,100 generations ago (Additional file 2: Figure S2). Assuming an average generation interval in sheep of around 5 years or 5500 before present, this is congruent with the first archaeological evidence for the fat tail sheep breeds (~5,000 years ago). The average effective population size over the period was calculated using reciprocals as described by Fal- coner [53]. Using this average estimated effective popu- lation size and based on the current frequency of sweeps in our regions of interest [54], the age of sweeps under the assumption of selection neutrality and genetic drift has been inferred for chromosomes 5, 7 and X to be approximately 7100, 9600, and 6900 generations before present respectively. Figure 3 Distribution of windowed FST values for Zel versus Lori-Bakhtiari breeds by chromosome. SNP position in the genome is shown on the X-axis, and windowed FST is plotted on the Y-axis. Regions with arrows above had windowed FST value > 0.20 and were later examined for further analysis. Moradi et al. BMC Genetics 2012, 13:10 http://www.biomedcentral.com/1471-2156/13/10 Page 5 of 15 Table 2 Bovine genes and published QTLs in regions showing evidence of selection in Zel vs Lori Bakhtiari data set Chr-Region Location on Ovine genome Location on Bovine genome Gene* RefSeq Number QTL QTL Reference 2-1 2:55859169-56302905 8:62601056-63034390 NPR2 NM_174126.2 Fat Depth [44] SPAG8 NM_001102005.1 Carcass weight [45] HINT2 NM_174340.2 Body weight (birth) [45] TMEM8B NM_001103335.1 2-2 2:73628609-73861345 8:45226663-45415081 — Fat Depth [44] Fat thickness [44] Carcass weight [45] Body weight (birth and mature) [45] 3 3:146615284-146676235 5:33981017-34041987 CACNB3 NM_174509.3 Meat [46] Tenderness DDX23 NM_001102202.1 Hot Carcass Weight [47] Birth Weight [48,49] 5 5:47146900-47332222 7:44936435-45113988 PPP2CA NM_181031.2 Fat thickness [48] SKP1 NM_001034781.1 TCF7 NM_001099186.1 7-1 7:30510065-30587784 10:29363930-29441529 —— Milk fat yield [50] Carcass weight [48] Hot Carcass Weight [48] Body weight (birth) [45] 7-2 7:46639859-46910155 10:45312778-45581699 PTGDR NM_001098034.1 Milk fat yield [50] Hot Carcass Weight [48] Body depth [51] X X:59192476-60151772 X:51751347-52712765 — * Obtained by orthology with the Bovine genome Figure 4 Distribution of windowed FST values for Zel-Lori Bakhtiari a