BACKGROUND
the horse  was one of the earliest domesticated species and has played numerous important roles in human societies: acting as a source of food, a means of transport, for draught and agricultural work, and for sport, hunting, and warfare  <cit> .
horse domestication is believed to have started in the western asian steppes approximately  <dig>  years ago, and quickly spread across the eurasian continent, with herds being augmented by the recruitment of local wild horses  <cit> .
domestication in the iberian peninsula might have represented an additional independent episode, involving horses that survived in a steppe refuge following the reforestation of central europe during the holocene  <cit> .
the horse reference genome has provided fundamental genomic information on the equine lineage and has been used for improving the health and performance of horses  <cit> .
horses exhibit  <dig> genetic traits and/or diseases that are similar to those of humans  <cit> .
to date, several horse whole genomes have been sequenced and analyzed  <cit> .
in  <dig>  the first whole genome re-sequencing analysis was conducted on the quarter horse breed to identify novel genetic variants  <cit> .
in  <dig>  divergence times among horse fossils, donkey, przewalski's horse, and several domestic horses were estimated, together with their demographic history  <cit> .
however, currently available whole genome sequences of modern horses only comprise western eurasian breeds.
over the centuries, more than  <dig> distinct horse breeds have been established by genetic selection for a wide number of desired phenotypic traits  <cit> .
the marwari  horse is a rare breed from the marwar region of india, and is one of six distinct horse breeds of india.
they are believed to be descended from native indian ponies, which were crossed with arabian horses beginning around the 12th century, possibly with some mongolian influence  <cit> .
the marwari horses were trained to perform complex prancing and leaping movements for ceremonial purposes  <cit> .
the marwari population in india deteriorated in the early 1900s due to improper management of the breeding stock, and only a few thousand purebred marwari horses remain  <cit> .
here, we report the first whole genome sequence of a male marwari horse as one of the asian breeds and characterize its genetic variations, including single nucleotide variations , small insertions/deletions , and copy number variations .
to investigate relationships among different horse breeds, we carried out a genome-wide comparative analysis using previously reported whole genome sequences of six western eurasian breeds  <cit> , and single nucleotide polymorphism  array data of  <dig> horses from  <dig> worldwide breeds  <cit> .
our results provide insights into its genetic background and origin, and identify genotypes associated with the marwari-specific phenotypes.
RESULTS
whole genome sequencing and variation detection
genomic dna was obtained from a blood sample of a male marwari horse  and was sequenced using an illumina hiseq <dig> sequencer.
a total of  <dig> gb of paired-end sequence data were produced with a read length of  <dig> bp and insert sizes of  <dig> and  <dig> bp from two genomic libraries .
a total of  <dig> , <dig>  reads remained after filtering, and  <dig> , <dig> reads were mapped to the horse reference genome  with a mapping rate of  <dig> %.
.
a total of  <dig> , <dig> reads were identified as duplicates and were removed from further analyses .
to enhance the mapping quality, we applied the indelrealigner algorithm to the de-duplicated reads.
a total of  <dig> , <dig>  reads were realigned, and the average mapping quality increased from  <dig>  to  <dig>  .
the whole genome sequences covered  <dig> % of the reference genome at  <dig> × or greater depth.
to identify novel genomic sequences, we performed a de novo assembly using the unmapped reads  to the horse reference genome.
a total of  <dig>  contigs  were assembled.
after mapping the contigs to the reference genome, we found that  <dig>  contigs  did not match the reference sequences; indicating that they may be novel regions specific to the marwari horse breed .
to identify the biological functions of these novel regions, the un-matched contigs were further analyzed by blast searches using the ncbi protein database.
however, none of the contigs significantly matched the known protein database .
comparing the marwari sequence to the reference genome, approximately  <dig>  million snvs and  <dig>  million indels were identified .
estimates of snp rate and heterozygosity of the marwari were similar to those of other horse breeds  .
we assessed the mutational frequency at the single nucleotide level in the marwari and compared it to estimates from other breeds .
interestingly, we found that the prevalent mutation types were not consistent among horse breeds.
the mutation spectrum of the marwari was dominated by c>t  transitions; a pattern which was also observed in the icelandic, norwegian fjord, and quarter horses.
conversely, the genomes of the arabian, standardbred, and thoroughbred horses were dominated by a>g  transitions.
a significant association between the mutation spectrum and horse breed  was found when we applied a chi-square test using spss  <cit>  to statistically compare the differences in the mutation spectrums among the breeds.
the marwari genome consisted of  <dig> , <dig>  homozygous and  <dig> , <dig>  heterozygous snvs .
among them,  <dig>  were found to be nonsynonymous snvs .
when the marwari variants were compared to those previously reported from the genomes of other breeds  <cit>  and the horse snp database from the broad institute,  <dig> , <dig> snvs and  <dig>  indels were novel variants.
of these,  <dig>  variants  represented amino acid changes which were found in  <dig>  genes .
to annotate the variants using well-known functional databases, human orthologs were retrieved from the ensembl biomart utility.
a total of  <dig>  of the  <dig>  genes had human orthologs, and  <dig>  genes were annotated using the david bioinformatics resource  <dig>   <cit> .
the genes with nssnvs and/or indels in coding regions were highly enriched in olfactory functions .
copy number variations  were identified using the r library "readdepth package"  <cit> .
a total of  <dig>  cnvs, including  <dig> gain and  <dig>  loss blocks, were identified in the marwari genome.
the sizes ranged from  <dig> kb to  <dig>  mb with an average length of  <dig> kb.
the cnv region  contained  <dig>  genes which were duplicated  or deleted  .
from the functional enrichment analysis, we found that the duplicated genes were enriched in olfactory function, whereas the deleted genes were enriched in immune regulation and metabolic processes .
relatedness to other horse breeds
we constructed a phylogenetic tree using snvs found in the whole genome data of the seven horse breeds   <cit> .
we identified  <dig> , <dig> nucleotide positions that were commonly found in the seven horse genomes.
a total of  <dig>  nucleotide positions were used for phylogenetic analysis after filtering for minor allele frequency , genotyping rate, and linkage disequilibrium .
we found that the marwari horse is most closely related to the arabian breed , while the icelandic horse and norwegian fjord were the most distinct from the other breeds, all of which are known to descend from arabian horses  <cit> .
to further explore the relationships among breeds, we compared the marwari horse genome data with snp array data from  <dig> individual horses belonging to  <dig> domestic breeds  <cit> .
a total of  <dig>  nucleotide positions were shared across all individuals including the marwari horse.
after pruning as described above,  <dig>  nucleotide positions were used for the comparative analyses.
we calculated pairwise genetic distances and conducted multidimensional scaling  to visualize the relationships among the horse breeds .
the marwari horse fell together with iberian-lineage breeds, such as the andalusian, mangalarga paulista, peruvian paso, and morgan horse breeds, all of which are known to have an arabian ancestry  <cit> .
additionally, we found that the marwari horse fell between arabian and mongolian horses, indicating their dual genetic influences on the marwari horse as previously suggested  <cit> .
we applied the structure program  <cit>  to estimate the genetic composition of the asian horse breeds including the marwari horse.
for k =  <dig> groups, the arabian horses were strongly separated from mongolian horses, and the genetic composition of the marwari horse was composed of alleles clustering with both the mongolian horse  and the arabian horse  .
other asian breeds  also showed genetic admixture between arabian and mongolian horses.
from k =  <dig> to k =  <dig>  the marwari had high genetic components of both arabian and mongolian horses, whereas akhal teke and caspian horses were mostly assigned to other clusters.
these results indicate that the marwari is genetically closely related to the arabian and mongolian horses.
it is unclear whether the latter relationship represents direct genetic input from mongolian horses or whether these horses are the closest population to the indian ponies from which the marwari is thought to have descended  <cit> .
further analysis including indian ponies and marwari horses will be required to distinguish the relative importance of these two scenarios, which are not mutually exclusive.
phenotype association of the identified variants
to provide insight into the unique marwari phenotypes, we investigated amino acid changes specific to this breed compared to those of other breeds .
a total of  <dig> amino acid changes in  <dig> genes were unique to the marwari horse.
among the  <dig> genes,  <dig> genes included one or more amino acid changes predicted by the polyphen <dig> program to alter protein function  <cit>  .
interestingly, the teashirt zinc finger family member  <dig>  gene had a homozygous p.ala344>val variant .
tshz <dig> is involved in transcriptional regulation of developmental processes and is associated with congenital aural atresia in humans, a malformation of the ear occurring in approximately  <dig> in  <dig>  births  <cit> .
additionally, tshz1-deficient mice show malformations in the middle ear components  <cit> .
therefore, the a334v amino acid change in tshz <dig> is a strong candidate as the genetic factor responsible for the inward-turning ear tips characteristic of the marwari breed.
a future genomic comparison with the kathiawari horse, which also has inward-turning ear tips, might support to this prediction.
after annotating the marwari variants for their known disease and trait information  <cit>  , we found that this breed has a homozygous variant for the g.27991841a>g mutation in the scl26a <dig> gene, which causes autosomal recessive chondrodysplasia in equine.
other variants were associated with racing endurance in thoroughbred horses , horse size , and pattern of locomotion .
<dig>  <cit> ,
selection in the equid lineage
we assessed the signatures of selection in the equid lineage using the dn/ds  ratio  <cit> .
a consensus horse  sequence was constructed by integrating all of the available breed genomes  in an attempt to remove breed specificity and to include an asian breed component via the central asian heritage of the marwari .
a total of  <dig>  out of  <dig>  genes in the horse reference genome were substituted by the consensus sequences.
using the protein sequences of seven non-horse genomes ,  <dig>  orthologous gene families were constructed using orthomcl  <cit> .
using alignments of these gene families to estimate dn/ds, we identified  <dig> genes under selection in the horse genome .
the selected genes were particularly enriched in immune response  and possible motor ability  functions .
over evolutionary time, the horse has developed increased speed and its musculature has become specialized for efficient strides  <cit> .
it is therefore possible that the motor activity-associated genes we identified to be under positive selection have contributed to the muscular efficiency seen in modern horses.
CONCLUSIONS
our study provides the first whole genome sequences and analyses of the marwari, an asian horse breed.
comparing the marwari genome to the horse reference genome, approximately  <dig>  million snvs and  <dig>  million indels, including  <dig>  variants that cause amino acid changes, were identified.
we found a clear arabian and mongolian component in the marwari genome, although further work is needed to confirm whether modern marwari horses also descended from indian ponies.
we analyzed the marwari variants and found a candidate snv determining its characteristic inward-turning ear tips.
additionally, we investigated selection in the horse genome through comparisons with other mammalian genomes.
by creating a consensus sequence that included information on an asian breed, we found a number of genetic signatures of selection, providing insights into possible evolutionary and environmental adaptations in the equid lineage.
the whole genome sequencing data from the marwari horse provides a rich and diverse genomic resource that can be used to improve our understanding of animal domestication and will likely be useful in future studies of phenotypes and disease.

 the horse was one of the earliest domesticated species and has played numerous important roles in human societies: acting as a source of food, a means of transport, for draught and agricultural work, and for sport, hunting, and warfare. horse domestication is believed to have started in the western asian steppes approximately years ago, and quickly spread across the eurasian continent, with herds being augmented by the recruitment of local wild horses. domestication in the iberian peninsula might have represented an additional independent episode, involving horses that survived in a steppe refuge following the reforestation of central europe during the holocene. the horse reference genome has provided fundamental genomic information on the equine lineage and has been used for improving the health and performance of horses. horses exhibit genetic traits and/or diseases that are similar to those of humans. to date, several horse whole genomes have been sequenced and analyzed. in the first whole genome re-sequencing analysis was conducted on the quarter horse breed to identify novel genetic variants. in divergence times among horse fossils, donkey, przewalski's horse, and several domestic horses were estimated, together with their demographic history. however, currently available whole genome sequences of modern horses only comprise western eurasian breeds. over the centuries, more than distinct horse breeds have been established by genetic selection for a wide number of desired phenotypic traits. the marwari horse is a rare breed from the marwar region of india, and is one of six distinct horse breeds of india. they are believed to be descended from native indian ponies, which were crossed with arabian horses beginning around the 12th century, possibly with some mongolian influence. the marwari horses were trained to perform complex prancing and leaping movements for ceremonial purposes. the marwari population in india deteriorated in the early 1900s due to improper management of the breeding stock, and only a few thousand purebred marwari horses remain. here, we report the first whole genome sequence of a male marwari horse as one of the asian breeds and characterize its genetic variations, including single nucleotide variations, small insertions/deletions, and copy number variations. to investigate relationships among different horse breeds, we carried out a genome-wide comparative analysis using previously reported whole genome sequences of six western eurasian breeds, and single nucleotide polymorphism array data of horses from worldwide breeds. our results provide insights into its genetic background and origin, and identify genotypes associated with the marwari-specific phenotypes.  whole genome sequencing and variation detection genomic dna was obtained from a blood sample of a male marwari horse and was sequenced using an illumina hiseq sequencer. a total of gb of paired-end sequence data were produced with a read length of bp and insert sizes of and bp from two genomic libraries. a total of, reads remained after filtering, and, reads were mapped to the horse reference genome with a mapping rate of %.. a total of, reads were identified as duplicates and were removed from further analyses. to enhance the mapping quality, we applied the indelrealigner algorithm to the de-duplicated reads. a total of, reads were realigned, and the average mapping quality increased from to. the whole genome sequences covered % of the reference genome at × or greater depth. to identify novel genomic sequences, we performed a de novo assembly using the unmapped reads to the horse reference genome. a total of contigs were assembled. after mapping the contigs to the reference genome, we found that contigs did not match the reference sequences; indicating that they may be novel regions specific to the marwari horse breed. to identify the biological functions of these novel regions, the un-matched contigs were further analyzed by blast searches using the ncbi protein database. however, none of the contigs significantly matched the known protein database. comparing the marwari sequence to the reference genome, approximately million snvs and million indels were identified. estimates of snp rate and heterozygosity of the marwari were similar to those of other horse breeds. we assessed the mutational frequency at the single nucleotide level in the marwari and compared it to estimates from other breeds. interestingly, we found that the prevalent mutation types were not consistent among horse breeds. the mutation spectrum of the marwari was dominated by c>t transitions; a pattern which was also observed in the icelandic, norwegian fjord, and quarter horses. conversely, the genomes of the arabian, standardbred, and thoroughbred horses were dominated by a>g transitions. a significant association between the mutation spectrum and horse breed was found when we applied a chi-square test using spss to statistically compare the differences in the mutation spectrums among the breeds. the marwari genome consisted of, homozygous and, heterozygous snvs. among them, were found to be nonsynonymous snvs. when the marwari variants were compared to those previously reported from the genomes of other breeds and the horse snp database from the broad institute,, snvs and indels were novel variants. of these, variants represented amino acid changes which were found in genes. to annotate the variants using well-known functional databases, human orthologs were retrieved from the ensembl biomart utility. a total of of the genes had human orthologs, and genes were annotated using the david bioinformatics resource. the genes with nssnvs and/or indels in coding regions were highly enriched in olfactory functions. copy number variations were identified using the r library "readdepth package". a total of cnvs, including gain and loss blocks, were identified in the marwari genome. the sizes ranged from kb to mb with an average length of kb. the cnv region contained genes which were duplicated or deleted. from the functional enrichment analysis, we found that the duplicated genes were enriched in olfactory function, whereas the deleted genes were enriched in immune regulation and metabolic processes. relatedness to other horse breeds we constructed a phylogenetic tree using snvs found in the whole genome data of the seven horse breeds. we identified, nucleotide positions that were commonly found in the seven horse genomes. a total of nucleotide positions were used for phylogenetic analysis after filtering for minor allele frequency, genotyping rate, and linkage disequilibrium. we found that the marwari horse is most closely related to the arabian breed, while the icelandic horse and norwegian fjord were the most distinct from the other breeds, all of which are known to descend from arabian horses. to further explore the relationships among breeds, we compared the marwari horse genome data with snp array data from individual horses belonging to domestic breeds. a total of nucleotide positions were shared across all individuals including the marwari horse. after pruning as described above, nucleotide positions were used for the comparative analyses. we calculated pairwise genetic distances and conducted multidimensional scaling to visualize the relationships among the horse breeds. the marwari horse fell together with iberian-lineage breeds, such as the andalusian, mangalarga paulista, peruvian paso, and morgan horse breeds, all of which are known to have an arabian ancestry. additionally, we found that the marwari horse fell between arabian and mongolian horses, indicating their dual genetic influences on the marwari horse as previously suggested. we applied the structure program to estimate the genetic composition of the asian horse breeds including the marwari horse. for k = groups, the arabian horses were strongly separated from mongolian horses, and the genetic composition of the marwari horse was composed of alleles clustering with both the mongolian horse and the arabian horse. other asian breeds also showed genetic admixture between arabian and mongolian horses. from k = to k = the marwari had high genetic components of both arabian and mongolian horses, whereas akhal teke and caspian horses were mostly assigned to other clusters. these results indicate that the marwari is genetically closely related to the arabian and mongolian horses. it is unclear whether the latter relationship represents direct genetic input from mongolian horses or whether these horses are the closest population to the indian ponies from which the marwari is thought to have descended. further analysis including indian ponies and marwari horses will be required to distinguish the relative importance of these two scenarios, which are not mutually exclusive. phenotype association of the identified variants to provide insight into the unique marwari phenotypes, we investigated amino acid changes specific to this breed compared to those of other breeds. a total of amino acid changes in genes were unique to the marwari horse. among the genes, genes included one or more amino acid changes predicted by the polyphen program to alter protein function. interestingly, the teashirt zinc finger family member gene had a homozygous p.ala344>val variant. tshz is involved in transcriptional regulation of developmental processes and is associated with congenital aural atresia in humans, a malformation of the ear occurring in approximately in births. additionally, tshz1-deficient mice show malformations in the middle ear components. therefore, the a334v amino acid change in tshz is a strong candidate as the genetic factor responsible for the inward-turning ear tips characteristic of the marwari breed. a future genomic comparison with the kathiawari horse, which also has inward-turning ear tips, might support to this prediction. after annotating the marwari variants for their known disease and trait information, we found that this breed has a homozygous variant for the g.27991841a>g mutation in the scl26a gene, which causes autosomal recessive chondrodysplasia in equine. other variants were associated with racing endurance in thoroughbred horses, horse size, and pattern of locomotion., selection in the equid lineage we assessed the signatures of selection in the equid lineage using the dn/ds ratio. a consensus horse sequence was constructed by integrating all of the available breed genomes in an attempt to remove breed specificity and to include an asian breed component via the central asian heritage of the marwari. a total of out of genes in the horse reference genome were substituted by the consensus sequences. using the protein sequences of seven non-horse genomes, orthologous gene families were constructed using orthomcl. using alignments of these gene families to estimate dn/ds, we identified genes under selection in the horse genome. the selected genes were particularly enriched in immune response and possible motor ability functions. over evolutionary time, the horse has developed increased speed and its musculature has become specialized for efficient strides. it is therefore possible that the motor activity-associated genes we identified to be under positive selection have contributed to the muscular efficiency seen in modern horses.  our study provides the first whole genome sequences and analyses of the marwari, an asian horse breed. comparing the marwari genome to the horse reference genome, approximately million snvs and million indels, including variants that cause amino acid changes, were identified. we found a clear arabian and mongolian component in the marwari genome, although further work is needed to confirm whether modern marwari horses also descended from indian ponies. we analyzed the marwari variants and found a candidate snv determining its characteristic inward-turning ear tips. additionally, we investigated selection in the horse genome through comparisons with other mammalian genomes. by creating a consensus sequence that included information on an asian breed, we found a number of genetic signatures of selection, providing insights into possible evolutionary and environmental adaptations in the equid lineage. the whole genome sequencing data from the marwari horse provides a rich and diverse genomic resource that can be used to improve our understanding of animal domestication and will likely be useful in future studies of phenotypes and disease. 
novel variants from the marwari sequences were annotated, and were found to be enriched in olfactory functions. the sequences were mapped to the horse reference genome at a mapping rate of ~98% and with ~95% of the genome having at least × coverage. in this study, we characterized the genome of the marwari horse, a rare breed with unique phenotypic characteristics, including inwardly turned ear tips. it is thought to have originated from the crossbreeding of local indian ponies with arabian horses beginning in the 12th century. a total of million single nucleotide variations, million small insertions or deletions, and copy number variation blocks were identified. the horse is one of the earliest domesticated species and has played an important role in the development of human societies over the past years. we generated gb of whole genome sequences from a marwari horse using the illumina hiseq sequencer. additionally, we suggest a potential functional genetic variant in the tshz gene associated with the inward-turning ear tip shape of the marwari horses. here, we present an analysis of the marwari horse genome. we confirmed a strong arabian and mongolian component in the marwari genome. this is the first genomic data for an asian breed, and is an invaluable resource for future studies of genetic variation associated with phenotypes and diseases in horses.
 the horse is one of the earliest domesticated species and has played an important role in the development of human societies over the past years. in this study, we characterized the genome of the marwari horse, a rare breed with unique phenotypic characteristics, including inwardly turned ear tips. it is thought to have originated from the crossbreeding of local indian ponies with arabian horses beginning in the 12th century.  we generated gb of whole genome sequences from a marwari horse using the illumina hiseq sequencer. the sequences were mapped to the horse reference genome at a mapping rate of ~98% and with ~95% of the genome having at least × coverage. a total of million single nucleotide variations, million small insertions or deletions, and copy number variation blocks were identified. we confirmed a strong arabian and mongolian component in the marwari genome. novel variants from the marwari sequences were annotated, and were found to be enriched in olfactory functions. additionally, we suggest a potential functional genetic variant in the tshz gene associated with the inward-turning ear tip shape of the marwari horses.  here, we present an analysis of the marwari horse genome. this is the first genomic data for an asian breed, and is an invaluable resource for future studies of genetic variation associated with phenotypes and diseases in horses. marwarihorseequus ferus caballuswhole-genome sequencinggenome july- august asia pacific bioinformatics network thirteenth international conference on bioinformatics sydney, australia 