Archaeological and genetic evidence concerning the time and mode of wild horse (Equus ferus) domestication is still debated. High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however, tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data reveal 18 major haplogroups (A-R) with radiation times that are mostly confined to the Neolithic and later periods and place the root of the phylogeny corresponding to the Ancestral Mare Mitogenome at ~130-160 thousand years ago. All haplogroups were detected in modern horses from Asia, but F was only found in E. przewalskii--the only remaining wild horse. Therefore, a wide range of matrilineal lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify well-preserved ancient remains,(ii)(re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the possible role of mtDNA backgrounds in racehorse performance.

Analysis of multiple sequence alignments can generate important, testable hypotheses about the phylogenetic history and cellular function of genomic sequences. We describe the MultiPipMaker server, which aligns multiple, long genomic DNA sequences quickly and with good sensitivity (available at http://bio.cse.psu.edu/ since May 2001). Alignments are computed between a contiguous reference sequence and one or more secondary sequences, which can be finished or draft sequence. The outputs include a stacked set of percent identity plots, called a MultiPip, comparing the reference sequence with subsequent sequences, and a nucleotide-level multiple alignment. New tools are provided to search MultiPipMaker output for conserved matches to a user-specified pattern and for conserved matches to position weight matrices that describe transcription factor binding sites (singly and in clusters). We illustrate the use of MultiPipMaker to identify candidate regulatory regions in WNT2 and then demonstrate by transfection assays that they are functional. Analysis of the alignments also confirms the phylogenetic inference that horses are more closely related to cats than to cows.

The place and date of the domestication of the horse has long been a matter for debate among archaeologists. To determine whether horses were domesticated from one or several ancestral horse populations, we sequenced the mitochondrial D-loop for 318 horses from 25 oriental and European breeds, including American mustangs. Adding these sequences to previously published data, the total comes to 652, the largest currently available database. From these sequences, a phylogenetic network was constructed that showed that most of the 93 different mitochondrial (mt)DNA types grouped into 17 distinct phylogenetic clusters. Several of the clusters correspond to breeds and/or geographic areas, notably cluster A2, which is specific to Przewalski's horses, cluster C1, which is distinctive for northern European ponies, and cluster D1, which is well represented in Iberian and northwest African breeds. A consideration of the horse mtDNA mutation rate together with the archaeological timeframe for domestication requires at least 77 successfully breeding mares recruited from the wild. The extensive genetic diversity of these 77 ancestral mares leads us to conclude that several distinct horse populations were involved in the domestication of the horse.

Population demarcation of eight horse breeds was investigated using genotype information of 306 horses from 26 microsatellite loci. The breeds include the indigenous Norwegian breeds Fjord Horse, Nordland/Lyngen Horse, Døle Horse and Coldblooded Trotter together with Icelandic Horse, Shetland Pony, Standardbred and Thoroughbred. Both phylogenetic analysis and a maximum likelihood method were applied to examine the potential for breed allocation of individual animals. The phylogenetic analysis utilizing simple allele sharing statistics revealed clear demarcation among the breeds; 95% of the individuals clustered together with animals of the same breed in the phylogenetic tree. Even breeds with a short history of divergence like Døle Horse and Coldblooded Trotter formed distinct clusters. Implementing the maximum likelihood method allocated 96% of the individuals to their source population, applying an assignment stringency of a log of the odds ratio larger than 2. Lower allocation stringency assigned nearly all the horses. Only three individuals were wrongly allocated a breed by both methods. In conclusion, the study demonstrates clear distinction among horse breeds, and by combining the two assignment methods breed allocation could be determined for more than 99% of the individuals.

Assignment tests have been utilized to investigate population classification, measure genetic diversity and to solve forensic questions. Using microsatellite data from 26 loci genotyped in eight horse breeds we examined how population differentiation, number of scored loci, number of scored animals per breed and loci variability affected individual assignment precision applying log likelihood methods. We found that both genetic differentiation and number of scored loci were highly important for recognizing the breed of origin. When comparing two and two breeds, a proportion of 95% of the most differentiated breeds (0.200 < or = FST < or = 0.259) could be identified scoring only three loci, while the corresponding number was six for the least differentiated breeds (0.080 < or = FST < or = 0.139). An identical proportion of simulated breed crosses, differentiated from their parental breeds by FST estimates in the range 0.050-0.069, was identified when scoring 12 loci. This level of source identification was not obtained for the less differentiated breed crosses. The current data further suggested that population sample size and locus variability were not critical for the assignment precision as long as moderately large sample sizes (> or = 20 animals per population) and fairly variable loci were used.

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.

Suspensory ligaments (SL) from 32 Thoroughbreds and 32 Standardbreds were collected to evaluate the variation in muscle content with respect to age, breed, sex, limb, and use. Six transverse sections, each 3 to 5 mm thick, were obtained from each SL. Four sections were taken from the body of the SL and 1 from the midportion of each branch. Sections were stained with van Gieson picric acid-fuchsin solution, then photographed, and black-and-white slides were made from the processed negatives. The transverse-sectional area of the SL and the contained muscle were determined by use of a computer with a color monitor and a digitizing device with its associated software. The percentage of muscle was then calculated for each section, for the entire ligament, and for each horse. Results were analyzed by multiple-regression analysis and Duncan multiple-range test, using the General Linear Model of SAS. Standardbreds had 40% more muscle in their SL than did Thoroughbreds. There was no significant difference in the percentage of SL muscle among sex, age, use, individual limb, or forelimb vs hind limb. For Standardbred horses, females had significantly greater muscle area content than intact males. Also, hind limb muscle area content was significantly greater than forelimb muscle content. Thoroughbred horses between 2 and 10 years of age not in training had significantly more muscle content than horses of the same age not in training. The reasons for these differences remain unclear.

Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5' untranslated region of one horse gene. Germline sequences included the 5' region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intron. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

The phylogenetic relationship between Equus przewalskii and E. caballus is often a matter of debate. Although these taxa have different chromosome numbers, they do not form monophyletic clades in a phylogenetic tree based on mtDNA sequences. Here we report sequence variation from five newly identified Y chromosome regions of the horse. Two fixed nucleotide differences on the Y chromosome clearly display Przewalski's horse and domestic horse as sister taxa. At both positions the Przewalski's horse haplotype shows the ancestral state, in common with the members of the zebra/ass lineage. We discuss the factors that may have led to the differences in mtDNA and Y-chromosomal observations.

The present study intends to survey the genetic variability of an endangered semiferal Portuguese native pony breed, the Garrano. Thirteen microsatellite markers were examined in 277 animals born in 1998, belonging to eight subpopulations corresponding to eight northern Portuguese geographic regions. Mean heterozygosity (H(o)) in the Garrano breed was 0.732, ranging from 0.531 to 0.857 across subpopulations. Allelic frequencies and diversity differed significantly between regions, suggesting the existence of genetic differentiation within the breed confirmed by the population differentiation estimator F(ST). Allele sharing genetic distance (Dps) was used to determine the relationship between the analyzed subpopulations, some of which are diverging significantly from the others. Relationships among six Iberian horse breeds (including the Garrano) were assessed through the restricted maximum likelihood method, which clusters the Garrano with another Celtic pony, the Spanish Asturcon, traditionally bred in geographical continuity with the Garrano. Results reveal a statistically significant deficit of heterozygotes within the Garrano breed (F(IT)= 0.031, p < 0.05). Regular monitoring should therefore be implemented so that the effect of genetic drift within subpopulations, enhanced by inbreeding, may be successfully minimized.