A selective sweep of >8 Mb on chromosome 26 in the Boxer genome

RESEARCH ARTICLE Open Access A selective sweep of >8 Mb on chromosome 26 in the Boxer genome Javier Quilez 1* , Andrea D Short 2 , Verónica Martínez 1 , Lorna J Kennedy 2 , William Ollier 2 , Armand Sanchez 1 , Laura Altet 1 and Olga Francino 1 Abstract Background: Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome. Results: Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at ~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds. Conclusion: A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds. Background It has been proposed that the majority of modern dog breeds recognised today have resulted from two population bottlenecks in dog evolution [1,2]. During the first genetic bottleneck, pre-domestic breeds diverged from wolves some 15,000 years ago, probably through multiple domestication events. The second bottleneck for most breeds occurred within the last few hundred years, when the breed creation process resulted in the loss of genetic variation due to strong bottleneck events which occurred in parallel with strong artificial selection for behavioural and physical characteristics favoured by humans. The same bottlenecks and artificial selection forces that generated these breed-specific features have, in some instances, provoked undesired health effects. Random fixation of detrimental variants can occur during bottlenecks. Similarly, risk alleles may be in linkage disequilibrium with selected phenotypic variants or these may have pleiotropic effects [3,4]. Several studies have previously aimed to identify genomic regions involved in defined traits and their relationship with disease using association mapping (reviewed in Karlsson and Linblad-Toh [2]). However, phenotypic traits that have been driven to fixation by genetic drift or artificial selection within a dog breed cannot be mapped within that breed with this approach. An alternative in these cases is selection mapping, in which selective * Correspondence: [email protected] 1 Molecular Genetics Veterinary Service (SVGM), Department of Animal and Food Science, Veterinary School, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain Full list of author information is available at the end of the article Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 © 2011 Quilez 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. sweeps (a reduction or elimination of genetic polymorphism in a region owing to strong selection) are searched [2,5-8]. The aim of this work was to identify selective sweeps in the Boxer genome resulting from the breed creation process using high density genome-wide SNP data. These regions are likely to govern phenotypic traits of interest and may be linked to overrepresentation of certain genetic disorders in this breed. Results Detection and replication in Boxer Regions of homozygosity (ROHs) in the Canis familiaris chromosomes (CFA) were identified in 25 Boxers from the United Kingdom (UK) which had been genotyped on microarrays for ~20,000 SNPs (set A, Table 1). Eight ROHs meeting the criteria detailed in Methods were detected (Table 2), representing 22 Mb (~0.9%) of the dog genome. Three of these ROHs, two on CFA 1 and one on CFA 26, showed a remarkable extended low heterozygosity (Figure 1a, Table 2). To confirm these ROHs, a second dataset (set B, Table 1) was generated using a higher density SNP array (~170,000 SNPs). This related to Boxers collected from different geographical locations to set A. In set B, 27 ROHs were found, which spanned 40.8 Mb (~1.7%) of the dog genome. Three regions on CFA X (Figure 1b) were discarded as these were not present when only female samples were analyzed (data not shown). Five ROHs were shared in both sets (Table 2). In general, these were notably shorter and/or split into two separated shorter regions when a higher number of samples and SNPs were genotyped (Additional file 1: Figure S1a-d, f-i). Conversely, the first 8 Mb of CFA 26 showed almost total loss of heterozygosity (average observed marker heterozygosity < 0.02) in both sets (Additional file 1: Figure S1e, j). There was a single SNP (BICF2G630807104, CFA 26:4,222,068 bp) with MAF = 0.5 within the region of extended homozygosity on CFA 26 (Additional file 1: Figure S1j), closer examination of which showed that heterozygous genotypes had been called for all Boxer samples. Possible explanations might be wrong genotype call from intensity data or a structural variation affecting that single SNP. To avoid the concern about SNPs significantly deviating from Hardy-Weinberg Equilibrium (HWE) affecting the identification of ROHs the analysis was repeated in set B after the removal of SNPs with HWE test p-value < 0.005, which resulted in similar results (Additional file 2 and Additional file 3). For the subsequent analyses we focused on the ROHs on CFA 1:58,710,420-61,801,815 bp and CFA 26:3,008,71811,914,284 bp because these had markedly larger size and lower levels of variation than other regions common inbothsets(Figure1,Table2).Finally,wefounda region of increased heterozygosity on CFA 9:19,826,59021,137,140 bp (Figure 1b), closer examination of which revealed a region of approximately 1.5 Mb showing a pattern of alternate heterozygous and homozygous genotypes indicating a CNV (Figure 2). Presence in other breeds Since reduction of genetic polymorphism in a region can result from strong selection and brachycephaly is a breeddefining trait in the Boxer, we evaluated the presence of the ROHs on CFA 1 and 26 in non-brachycephalic and brachycephalic breeds. Brachycephaly is characterized by severe shortening of the muzzle, and therefore the underlying bones, and a more modest shortening and widening of the skull [9]. For both selective sweeps on CFAs 1 and 26, normal levels of heterozygosity were observed in nonbrachycephalic dog breeds and the Iberian wolf (Figure 3), based on a first dataset containing 118 samples from 6 different dog breeds and 7 Iberian wolf samples genotyped Table 1 Samples genotyped with call rate > 90% Group # Samples BeadChip Boxer (denoted as set A) 25 Illumina’s CanineSNP20 (~20,000 SNPs) Iberian wolf 7 Shar pei 37 Cirneco dell’Etna 12 Canarian warren hound 13 Ibizan hound 39 Pharaoh hound 5 Labrador retriever 1 12 Illumina’s CanineHD (~170,000 SNPs) Boxer (denoted as set B) 273 German shepherd dog (GSD) 43 English bulldog 4 French bulldog 6 Pug 10 1 a subset of ~17,000 SNPs shared with the Illumina’s CanineSNP20 was used for this breed. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 2 of 12 using the same panel of SNPs as in set A and on a second dataset containing 43 samples from German shepherd dog genotyped using the same panel of SNPs as in set B (Table 1). The selective sweep on CFA 1 was present and showed allelic match with the Boxer (data not shown) in other brachycephalic breeds such as English bulldog, Pug and French bulldog although in the latter the reduction in heterozygosity was not as extended as in the other two breeds and seemed to be located slightly upstream in the chromosome (Figure 3). The selective sweep on CFA 26 was detected with allele-matching in English bulldog (CFA 26:6,785,609-11,282,297 bp; only 3 out of 368 SNPs with non-zero MAF) and French bulldog (CFA 26:8,548,679-9,227,043; 48 SNPs involved with MAF equal to zero) (Figure 3, Additional file 4). Although the ROH was not apparent in the Pug in Figure 3, in the segment of the ROH shared between Table 2 Regions of homozygosity (ROHs) detected in sets A and B. Set A Region ID CFA BP1 BP2 KB NSNP KB/SNP Avg. Het Group SetA_01 1 57,121,838 67,829,167 10,707 84 127.5 0.02 *1 SetA_02 1 86,903,752 95,179,544 8,276 90 92.0 0.03 *2 SetA_03 1 112,558,604 120,632,074 8,073 71 113.7 0.05 *3 SetA_04 3 62,679,327 67,735,724 5,056 53 95.4 0.05 SetA_05 10 3,081,933 12,082,601 9,001 68 132.4 0.04 *4 SetA_06 12 48,950,221 55,687,381 6,737 63 106.9 0.04 SetA_07 26 3,116,745 12,410,004 9,293 107 86.9 0.01 *5 SetA_08 29 11,263,518 18,670,911 7,407 69 107.4 0.05 Set B Region ID CFA BP1 BP2 KB NSNP KB/SNP Avg. Het Group SetB_01 1 26,672,978 27,730,188 1,057 78 13.6 0.03 SetB_02 1 45,218,029 46,286,798 1,069 66 16.2 0.04 SetB_03 1 58,732,954 61,801,815 3,069 221 13.9 0.03 *1 SetB_04 1 62,722,220 65,190,321 2,468 129 19.1 0.03 *1 SetB_05 1 89,187,131 90,230,941 1,044 86 12.1 0.04 *2 SetB_06 1 102,454,189 103,320,473 866 51 17.0 0.04 SetB_07 1 116,688,554 118,107,497 1,419 97 14.6 0.03 *3 SetB_08 1 117,979,514 118,963,939 984 51 19.3 0.04 *3 SetB_09 2 22,715,411 24,024,566 1,309 78 16.8 0.04 SetB_10 3 3,030,299 3,903,071 873 59 14.8 0.04 SetB_11 5 4,697,408 6,247,457 1,550 105 14.8 0.04 SetB_12 6 25,815,666 26,601,998 786 52 15.1 0.04 SetB_13 6 42,437,711 43,955,158 1,517 60 25.3 0.04 SetB_14 6 58,580,737 59,356,441 776 62 12.5 0.04 SetB_15 9 3,529,583 4,304,182 775 58 13.4 0.04 SetB_16 10 5,626,769 6,702,961 1,076 51 21.1 0.04 *4 SetB_17 10 59,180,359 60,456,532 1,276 90 14.2 0.04 SetB_18 10 65,209,901 66,606,742 1,397 86 16.2 0.04 SetB_19 10 68,323,860 69,026,432 703 61 11.5 0.04 SetB_20 13 39,705,171 40,797,838 1,093 71 15.4 0.04 SetB_21 14 19,806,989 20,470,904 664 52 12.8 0.04 SetB_22 18 6,868,787 7,908,159 1,039 54 19.2 0.04 SetB_23 20 7,816,139 8,635,017 819 58 14.1 0.03 SetB_24 24 24,444,170 27,387,620 2,943 217 13.6 0.03 SetB_25 24 28,845,341 29,848,829 1,003 93 10.8 0.03 SetB_26 26 3,008,718 11,914,284 8,906 707 12.6 0.01 *5 SetB_27 30 38,126,268 38,689,821 564 52 10.8 0.04 CFA, Canis familiaris chromosome; BP1 and BP2, base pair positions of the first and last SNP involved in the ROH, respectively; NSNP, number of SNPs involved in the ROH; KB/SNP, SNP density in the ROH; Avg. Het, average observed heterozygosity. ROHs present in both sets are grouped numerically. Note that the endof SetB_07 overlaps with start of SetB_08. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 3 of 12 Figure 1 Genome-wide plot of the averaged observed heterozygosity with the 50-SNP sliding window for set A (a) and set B (b). The x-axis corresponds to SNPs sort by chromosomes (differently coloured) and position and the y-axis represents the 10-logarithm of the averaged observed heterozygosity calculated with sliding windows of 50 SNPs, for which 0.1 and 1.0% quantiles of the empirical distribution are displayed. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 4 of 12 Boxer and English and French bulldogs a good number of SNPs in Pug samples showed the same genotypes as the other three brachycephalic breeds. In addition, the SNPs comprised between 8,565,784 and 8,620,015 bp (~55 Kb) were nearly fixed in the Pug (Additional file 4). In contrast to the Boxer, a distribution of genotypes in HWE was seen for BICF2G630807104 (CFA 26:4,222,068 bp) in the bulldog breeds studied whereas it was fixed in the Pug (data not shown). Within the SNPs making up the Illumina’sCanineSNP20 only two covered the CNV on CFA 9 (Additional file 5), both of them highly monomorphic with the exception of the SNP at position 20,274,406 bp for the Shar pei samples for which an excess of heterozygous genotypes (HWE test p-value < 0.001) were observed. A pattern of excessive heterozygous genotypes was observed for the region corresponding to the CNV on CFA 9 in the breeds genotyped with the Illumina’s CanineHD Beadchip (Additional file 6). Genetic content and functional annotation analysis The region of decreased heterozygosity which was observed on CFA 1 in our study overlapped with a region previously associated with canine brachycephaly [7]. This was detected using dogs from brachycephalic breeds and non-brachycephalic breeds to perform across-breed association and selection mapping. Both strategies identified a region on CFA 1 at 59 Mb. The decrease in the averaged observed heterozygosity of brachycephalic dogs relative to non-brachycephalic dogs is indicative of a selective sweep at this position. Genes which have been associated with brachycephaly on CFA 1 include: THBS2 [Ensembl:ENSCAFG00000000874], which is expressed in bone and cartilage during development and in the AB heterozygou s BB homozygous d i v id ua l s AA homozygous I n d 4 48,789 8 26,590 3 7,140 5 0,647 Position on CFA 9 ( bp ) 19, 4 19, 8 21,1 3 21,9 5 Figure 2 Pattern of alternate heterozygous/homozygous genotypes in a 1.5-Mb region corresponding to a described CNV on CFA 9.In the x-axis, the start and end positions of both the chromosome region shown and the CNV are indicated. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 5 of 12 adult skeleton [10], and SMOC2 [Ensembl:ENSCAFG 00000000868], similar in sequence to BM-40 [Ensembl: ENSCAFG00000017855] which is expressed primarily during embryogenesis and in adult bone tissue [11]. The ROH in Boxer on CFA 26:3,008,718-11,914,284 bp contained 135 annotated elements of which 95 (71.9%) were genes with an associated name; this level was similar to that observed in the whole dog genome (69.7%). The ROH from CFA 26 mapped to two adjacent syntenic regions on Homo sapiens chromosome (Hs) 12 (Figure 4a). Advantage was taken of the fact that the region in the human genome syntenic to the region of interest on CFA 26 was better annotated and could be used to perform functional annotation analysis through the use of Ingenuity Pathways Analysis software [12]. One hundred and six dog to human orthologs annotated elements were Figure 3 ROHs on CFA 1 and CFA 26 in non-brachycephalic and brachycephalic breeds other than Boxer. In the top panels, MAF values for the pool of non-brachycephalic breeds (grey squares) and German shepherd dog (grey crosses) are shown. For CFA 1, the region associated with brachycephaly by Bannasch et al. (CFA 1:59,536,208-59,832,965 bp) [7] is indicated (red lines). For CFA 26, the red lines show the extent of the ROH in Boxer for comparison with the other breeds. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 6 of 12 A S B B S 1S2 Boxer English Bulldo g French Bulldog ~700Kb Figure 4 Genetic content of the ROH on CFA 26.(a) Annotated elements in the dog genome (CanFam2.0) as well as regions of synteny with H. sapiens and M. musculus are shown. CFA 26:3,008,718-11,914,284 bp maps to two syntenic regions in H. sapiens: Hs 12:121,547,433-133,784,108 bp (S1) and Hs 12:110,448,771-121,498,418 (S2). The end position for S2 is defined relative to the end of the ROH in dog though indeed synteny extends longer. Note that the order of the syntenic regions relative to the dog sequence indicates rearrangements events in the species; also, the different orientation of the sequences, indicated with arrows preceding the chromosome number in the compared species, designates an inversion in the orientation between H. sapiens and C. familiaris for S1. Regions of extended homozygosity for Boxer, English bulldog and French bulldog and the region of ~700 Kb shared in all three are indicated. (b) Averaged observed heterozygosity along CFA 26 in Boxer. Minimum values are shown for three window sizes: 10and 50-SNPs, CFA 26:8.86 Mb (x); 20-SNPs, CFA 26:10.44 Mb (+). Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 7 of 12 used as input, resulting in a list of biological processes and disease categories that would be enriched given the genes in the region of interest. Amongst functional categories related to biological processes, skeletal and muscular system development and function as well as tissue morphology were the two most significantly associated categories (Additional file 7). Also, the selective sweep on CFA 26 contained genes that are linked to inherited diseases overrepresented in the Boxer [4] (Table 3). Lymphoblastic lymphoma is a type of non-Hodgkin lymphoma characterised by uncontrolled growth of either Tor B-cells and associated with POLE [Ensembl: ENSCAFG00000006215]. The frequency of T-cell lymphoma in the Boxer is higher than in other breeds [13-15]. Dilated cardiomyopathy (CMD) [OMIA: 327] is a disorder characterised by cardiac enlargement (especially of the left ventricle), poor myocardial contractility, and congestive heart failure. MYL2 [Ensembl:ENSCA FG00000008524] is involved in the development of the sarcomere and muscle contraction and has also been associated with cardiomyopathy of the heart ventricle. CFA 26:8,548,679-9,227,043 bp defined the region where extended homozygosity was common in Boxer, English bulldog and French bulldog (Figure 4a), which comprised six genes significant in the functional annotation analysis (Table 3). Of these genes, ATP6V0A2 [Ensembl:ENSCAFG00000007234] and EIF2B1 [Ensembl:ENSCAFG00000007434] are of special interest because they are involved in genetic disorders in humans. Various loss-of-function mutations of ATP6V0A2 [Ensembl:ENSCAFG00000007234], which encodes the alpha-2 subunit of the V-type H+ ATPase, resulting in impaired glycosilation of proteins during synthesis cause autosomal recessive cutis laxa (ARCL) type II [OMIM:219200] and some cases of wrinkly skin syndrome [OMIM:278250] [16]. Mutations in each of the five subunits of the translation initiation factor eIF2B, including eIF2a encoded by EIF2B1 [Ensembl: ENSCAFG00000007434], can cause leukoencephalopathy with vanishing white matter (VWM, [OMIM: 603896]) [17]. VWM is a neurological disorder manifesting progressive cerebellar ataxia, spasticity, Table 3 Dog genes and human orthologs within the selective sweep on CFA 26 Boxer Human gene Dog gene Start (bp) End (bp) Ensembl Gene ID Annotation HIP1R HIP1R 9,641,249 9,654,192 ENSCAFG00000007764 Development of vertebral column P2RX4 Q64F94_CANFA 10,917,230 10,933,173 ENSCAFG00000008363 Contraction (and relaxation) of cardiac muscle P2RX7 B0FLR1_CANFA 10,959,759 11,002,514 ENSCAFG00000008401 Bone mineral density of skeleton Formation of perichondral bone Resorption of trabecular bone ATP2A2 AT2A2_CANFA 11,167,290 11,232,956 ENSCAFG00000008434 Contraction (and relaxation) of cardiac muscle Contraction and relaxation of papillary muscle Elongation of cardiomyocytes POLE POLE 3,436,073 3,481,729 ENSCAFG00000006215 Lymphoblastic lymphoma MYL2 NP_001003069.1 11,482,695 11,650,673 ENSCAFG00000008524 Contraction of cardiac muscle Development of sarcomere Cardiomyopathy of heart ventricle Boxer, English and French bulldogs Human gene Dog gene Start (bp) End (bp) Ensembl Gene ID Annotation CCDC92 CCDC92 8,759,998 8,790,267 ENSCAFG00000006996 Amino Acid Metabolism Protein Synthesis Small Molecule Biochemistry ATP6V0A2 ATP6V0A2 8,929,687 8,968,952 ENSCAFG00000007234 Cutis laxa Wrinkly skin syndrome EIF2B1 EIF2B1 9,029,462 9,038,275 ENSCAFG00000007434 Gene Expression Leukoencephalopathy with vanishing white matter Protein Synthesis DDX55 DDX55 9,040,108 9,053,306 ENSCAFG00000007452 Cancer Infection Mechanism (by HIV) TMED2 TMED2 9,056,220 9,064,265 ENSCAFG00000007468 Cancer Infection Mechanism (by HIV) SETD8 9,188,951 9,202,238 ENSCAFG00000007493 Tissue development and cell cycle The upper part of the table (Boxer) refers to the selective sweep present in the Boxer only (CFA 26:3,008,718-11,914,284 bp). The lower part of the table (Boxer, English and French bulldogs) refers to the part of the selective sweep shared in the three breeds CFA 26:8,548,679-9,227,043 bp. Genomic positions in the dog genome and their annotated involvement in biological process and diseases (italic bold) are listed. Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 8 of 12 inconstant optic atrophy and relatively preserved mental abilities. SETD8 [Ensembl:ENSG00000183955] encodes a lysine methyltransferase that regulates tumor suppressor p53 protein [18]. To note, the region of ~55 Kb of nearly complete homozygosity in the Pug is both upstream of these six genes and within the region of extended homozygosity shared amongst the three breeds. Interestingly, (i) the genes involved in skeletal and muscular system development and tissue morphology that were significant in the functional annotation analysis in the Boxer only (with the exception of POLE [Ensembl:ENSCAFG00000006215]) and (ii) the region shared in Boxer and English and French bulldogs, are both located within the region on CFA 26 that showed the greatest decay in the averaged observed heterozygosity (Figure 4b). The region of alternate heterozygous/homozygous genotypes patterns observed for CFA 9 in the Boxers overlapped perfectly to a CNV previously described as being polymorphic in a number of dog breeds [19,20]. This 1.5Mb CNV region contained three protein coding genes, one of which had two reported transcript variants, and four non coding RNA genes (Additional file 8). Analysis of Gene Ontology Biological Process (GO BP) terms revealed that the two transcript variants of the protein coding gene VPS13D [Ensembl:ENSCAFG00000016397] (CFA 9:21,079,541-21,164,823 bp) were associated with processes of protein localization and viral envelope fusion with host membrane (GO:0008104 and GO:0019064, respectively). This gene was also mapped to the Hs 1:12,290,124-12,572,099 bp but only the protein localization term was associated (GO:0008104). The remaining annotated elements had neither GO BP terms associated nor homology in H. sapiens. Discussion The substitution of a strongly selected mutation produces a selective sweep on the frequency of neutral alleles at linked loci characterised by a reduction of the local genetic variation [21-23]. Two selective sweeps detected on CFAs 1 and 26 in the Boxer genome were replicated in a larger sample size of the same breed obtained from a different geographical location and genotyped for a panel of SNPs of higher density. Assessing both the presence of these regions in other breeds and their genetic content can provide information on how they affect the phenotype and relate to the ancestral origin of breeds. In our study, the selective sweep previously associated with brachycephaly on CFA 1 [7] was replicated in a larger sample of Boxers and in samples from other brachycephalicbreeds.Moreover,samplesinthisstudywerefroma different geographic area compared to the previous work [7] (Europe and US, respectively), suggesting the selective sweep is shared in the two populations within each breed. The selective sweep on CFA 26 indicates strong artificial selection of a trait of interest in the Boxer, although the phenotypic trait resulting from this particular selective sweep is unknown. The sweep was not present in the Iberian wolf, one ancient breed (Shar pei), Labrador retrievers, German shepherd dogs or four hound breeds. On the other hand, it was present, although in shorter length, in English and French bulldogs, breeds that share with the Boxer the brachycephalic trait and a related breed creation process. Altogether, these results suggest that the selection of the sweep predated the formation of Boxer and both bulldog breeds. It is known that the English bulldog contributed to the breed creation of both Boxer and French bulldog breeds [24]. The Boxer is believed to have originated from a long-existing and now extinct German breed, the Bullenbeisser, which was crossed with a small number of English bulldog exemplars exported from the UK. Likewise, the French bulldog originated from toy varieties of English bulldog that were more popular in France. Moreover, it is interesting that the region of the selective sweep common in the three breeds coincides with the lowest reduction in the heterozygosity along the sequence (Figure 4). In selective sweeps, the reduction of genetic variation is lowest at the site of directional selection and not as great at distant sites due to recombination, although asymmetry in the valleys of reduced heterozygosity may provide imprecise information about the location of the sweep [25]. Based on our data, it is hard to assess whether the selective sweep on CFA 26 shared in Boxer and English and French bulldogs was also present in the Pug, also a brachycephalic breed, because only a short segment of reduced polymorphism within the sweep was observed in this breed (~55 Kb). Nonetheless, the history of the Pug differs from that of these three breeds mentioned before. The Pug dates to the ancient China and it is suggested that interbreeding with Pekingese, Japanese chin and possibly Shih tzu contributed to the breed creation process. Pugs were imported to Europe through Holland around 1,600s [24]. A possible scenario is that the standing neutral variation on CFA 26 present in the original English bulldog was passed to both Boxer and French bulldog during the breed creation process. Some variants would have been beneficial thereafter when selection of brachycephaly started, which is reasonable to think that happened during the breeds creation process since brachycephaly is a breed standard in these three types of dogs. Thus, strong selection of variants close to the position 8-10 Mb on CFA 26 contributing to brachycephaly might have swept nearby genetic variation. Variable selective sweep length Quilez et al.BMC Genomics 2011, 12:339 http://www.biomedcentral.com/1471-2164/12/339 Page 9 of 12