Exceptional conservation of horse–human order on X revealed by high-resolution radiation hybrid mapping

Terje Raudsepp*†, Eun-Joon Lee*†, Srinivas R. Kata‡, Candice Brinkmeyer*, James R. Mickelson§, Loren C. Skow*, James E. Womack‡, and Bhanu P. Chowdhary*¶ʈ

*Department of Veterinary Anatomy and Public Health, ‡Department of Veterinary Pathobiology, College of Veterinary Medicine, and ¶Department of Animal Science, College of Agriculture and Life Science, Texas A&M University, College Station, TX 77843; and §Department of Veterinary Pathobiology, University of Minnesota, 295f AS͞VM, St. Paul, MN 55108

Contributed by James E. Womack, December 30, 2003 Development of a dense map of the horse genome is key to efforts ciated with the traits, once they are mapped by genetic linkage aimed at identifying controlling health, reproduction, and analyses with highly polymorphic markers. performance. We herein report a high-resolution gene map of the The is the most conserved mammalian chro- horse (Equus caballus) X chromosome (ECAX) generated by devel- mosome (18, 19). Extensive comparisons of structure, organi- oping and typing 116 gene-specific and 12 short tandem repeat zation, and gene content of this chromosome in evolutionarily -markers on the 5,000-rad horse ؋ hamster whole-genome radia- diverse mammals have revealed a remarkable degree of conser tion hybrid panel and mapping 29 gene loci by fluorescence in situ vation (20–22). Until now, the chromosome has been best hybridization. The human X chromosome sequence was used as a studied in humans and mice, where the focus of research has template to select genes at 1-Mb intervals to develop equine been the intriguing patterns of X inactivation and the involve- orthologs. Coupled with our previous data, the new map comprises ment of various X specific genes in genetic diseases (23, 24), a total of 175 markers (139 genes and 36 short tandem repeats, of female and male fertility (25–27), and embryonic development (28). Relatively very little applied research has been conducted which 53 are fluorescence in situ hybridization mapped) distrib- ͞ uted on average at Ϸ880-kb intervals along the chromosome. This in livestock and companion pet species, which is primarily is the densest and most uniformly distributed chromosomal map attributable to the limited information available through the presently available in any mammalian species other than humans medium- to low-resolution X chromosome maps currently avail- able in pigs (29, 30), cattle (31, 32), cats (21, 33), dogs (34–38), and rodents. Comparison of the horse and human X chromosome ͞ ͞ maps shows remarkable conservation of gene order along the and sheep goats buffalo (31, 39, 40), which are insufficient to permit comprehensive studies aimed at dissecting traits of entire span of the , including the location of the interest. centromere. An overview of the status of the horse map in relation The current ECA X chromosome (ECAX) map is comparable to mouse, livestock, and companion animal species is also pro- to those of other livestock and companion species. Beginning vided. The map will be instrumental for analysis of X linked health with the physical assignment of three gene-specific loci, i.e., and fertility traits in horses by facilitating identification of targeted G6PD, HGPRT, and PGK, during the early 1970s (41), the chromosomal regions for isolation of polymorphic markers, build- ECAX map developed mainly through synteny (42, 43) and ing bacterial artificial chromosome contigs, or sequencing. genetic linkage mapping approaches (3). During recent years, the map has benefited considerably from the availability of the gene mapping ͉ comparative map ͉ mouse RH panel (44). The current ECAX map comprises 26 gene and 17 short tandem repeat loci (5, 22), offering a basic RH and quine genome analysis has proceeded at an unprecedented comparative map. However, the resolution of this map is inad- pace during recent years. From the initial horse [Equus equate to facilitate identification of economically important E Ϸ caballus (ECA)] gene map 6–7 years ago, organized interna- genes over the 153 Mbp of the chromosome (5). tional efforts have led to a 10-fold expansion in the map. The Building on our recent success in developing high-resolution progress is evident from the recently published meiotic (1–3), gene maps for ECA17 (45), ECA22 (A. L. Gustafson, T.R., M. cytogenetic (4), and radiation hybrid (RH) maps (5) that provide L. Wagner, J.R.M., L.C.S., and B.P.C., unpublished work), and an array of polymorphic and gene-specific markers distributed a targeted region of ECA26 (46), we undertook development of over all equine autosomes and the X chromosome. Comparative a high-resolution gene map of ECAX by stepwise selection of information available through these maps is proving critical for gene-specific markers from the human and mouse X chromo- accurate alignment of the horse genome with the sequenced some sequence templates. This effort has resulted in a dense and comprehensive map second only to the detailed maps in humans genomes of human and mouse (4, 5) and with the gene maps of and mice and will lay the foundation for identifying and ana- other livestock species. The maps, most of which are of low to lyzing X linked genes involved in equine reproduction and medium density, have served as a starting point to initiate genetic disorders. research aimed at identifying genes responsible for valuable traits associated with equine biology, health, and performance, Materials and Methods including genes responsible for base coat color, overo lethal Marker Selection and Primer Design. The sequence white, hyperkalemic periodic paralysis, and severe combined data available from National Center for Biotechnology Infor- immunodeficiency (6–9). Further, marker-based studies are in progress to dissect molecular causes of various coat colors [e.g., gray (10–12) and appaloosa (13, 14)], genetic diseases [e.g., Abbreviations: ECA, Equus caballus; ECAX, ECA X chromosome; RH, radiation hybrid; FISH, exertional rhabdomyolysis (15, 16); polysaccharide storage my- fluorescence in situ hybridization; BAC, bacterial artificial chromosome; cR, centiRay; PAR, opathy (17)], and other traits of interest. The major impediment pseudoautosomal region. associated with the latter group of studies is the lack of adequate †T.R. and E.-J.L. contributed equally to this work. resolution maps for individual equine chromosomes. Such maps ʈTo whom correspondence should be addressed. E-mail: [email protected]. could facilitate rapid targeted hunts for candidate genes asso- © 2004 by The National Academy of Sciences of the USA

2386–2391 ͉ PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308513100 Downloaded by guest on September 27, 2021 mation build 34 of the human reference sequence (http:͞͞ amplification of horse DNA, multiple PCR products, or ampli- genome.ucsc.edu͞cgi-bin͞hgGateway) and ENSEMBL l version fication products of equal sizes for horse and hamster. Of the (also from July 2003, www.ensembl.org͞Homo࿝sapiens) were remaining 127 primer pairs, 28 were identified from equine EST jointly interrogated for known genes from the human X chro- or gene sequences, and the remaining 99 originated from mosome. The genes were selected at Ϸ1-Mb intervals, beginning multiple alignments of mammalian sequences (Table 1). Se- at 0 Mbp (distal tip of the short arm) and ending at 153 Mbp quencing the PCR amplification products of individual primer (distal end of the long arm). Whenever possible, human exonic pairs to verify the identity of the markers resulted in 11 primer sequences from selected genes were compared with orthologous pairs being discarded, because the sequences did not correspond sequences from other mammals by using BLASTN, NR, and to the expected genes. This yielded 116 equine orthologs for EST࿝OTHERS search engines (www.ncbi.nlm.nih.gov͞BLAST). human X chromosome genes and represented a Ϸ70% success This was followed by multiple alignment of the sequences in rate in developing horse-specific markers for use on the 5,000- CLUSTALW (http:͞͞bioweb.pasteur.fr͞seqanal͞interfaces͞ rad RH panel. clustalw-simple.html). The alignments were used to design het- erologous primers for PCR amplification of horse DNA in a Generation of a Composite RH Map. A total of 128 new markers (116 hamster DNA background, as described (45, 47, 48). Briefly, all genes and 12 microsatellites) were typed on the 5,000-rad primers were derived either from a single exon or from two horse ϫ hamster whole genome RH panel (44). When integrated adjacent exons, leaving an Ϸ500- to 700-bp intron between, and with 42 loci from the two previous RH maps (5, 22), the final map chosen for 100% sequence identity among human, cattle, pig, for the equine X chromosome comprised 169 markers (135 Type etc., orthologues but with one to three mismatches with the I and 34 Type II) uniformly distributed along the length of the rodent (mouse, rat) sequences. Primers were designed by using chromosome (Fig. 1). With the estimated size of ECAX at Ϸ153 PRIMER3 software (www-genome.wi.mit.edu͞cgi-bin͞primer͞ Mbp (5), the average marker density is 1 every 900 kbp, and the primer3࿝www.cgi). A total of 88 gene-specific markers were average gene marker density is 1 every Ϸ1 Mbp. The retention developed by using this approach. Additionally, species-specific frequency of the 169 markers in the RH panel ranged from 5.4% primers for all microsatellites (12) and some genes (28) were (BRIA3, BIRC4, and ODZ1) to 31.5% (Adlican), with an average designed from the available equine genomic or EST sequences. of Ϸ13% (Fig. 2, which is published as supporting information Detailed information on all new markers (128) is presented in on the PNAS web site). This retention frequency is satisfactory Table 1, which is published as supporting information on the considering the RH panel was made from a male horse. A PNAS web site. relatively high retention of markers was observed toward the proximal and distal end of the short arm, whereas retention was Primer Optimization and Sequencing. Horse and hamster genomic relatively lower than average in the distal part of the long arm DNAs were used to optimize the PCR conditions for individual (Fig. 2). primer pairs, such that only horse-specific DNA amplification At logarithm of odds score 7 (2PT-RHMAP), the 169 markers was obtained, and all equine PCR amplification products were clustered in six RH-linkage groups arranged tandemly from pter verified by sequencing. The identities of the sequences were to qter on the chromosome (LGI–LGVI; Fig. 3, which is pub- confirmed through BLAST (www.ncbi.nlm.nih.gov͞BLAST) and lished as supporting information on the PNAS web site), each BLAT (http:͞͞genome.ucsc.edu͞cgi-bin͞hgBlat?commandϭ comprising 48, 11, 15, 30, 31, and 34 markers, respectively (Fig. start) searches, as described (45), and all comparisons were 3). The proximal three linkage groups comprising 74 markers revalidated against the latest build of sequence data at University were located on the short arm, whereas the distal three linkage of California, Santa Cruz, and Ensembl. For RPS6KA3 (see groups comprising 95 markers were located on the long arm. The Table 1), new primers were designed by using sequence data order of markers within each RH linkage group was deduced by obtained from the first primer pair. using the equal retention probability and stepwise locus ordering models (RHMAXLIK). Twenty-nine loci served as frameworks RH Typing Analysis, Bacterial Artificial Chromosome (BAC) Library (odds of 1,000:1; shaded orange in Fig. 3). The comprehensive GENETICS Screening, and Fluorescence in Situ Hybridization (FISH) Mapping. map thus generated spans of 1,344 centiRay (cR)5000, uniformly PCR typing on the 5,000-rad horse ϫ hamster RH panel and data covering the entire length of ECAX (see Fig. 3 legend for details analysis was performed as described (5). The TAMU and on map construction). The only two human X (HSAX) regions CHORI-241 equine genomic BAC libraries were screened by (each spanning Ϸ5–6 Mbp) not represented on ECAX were the PCR to obtain clones specific for 29 ECAX genes. These clones centromeric region and the HSAXp21.1 region (spanning be- ensured physical anchors for the RH map at regular intervals tween 54–62 and 31–37 Mb positions, respectively, on the human along the entire length of the chromosome. Individual BAC sequence map). Otherwise, the representation͞alignment of clones were labeled with biotin and͞or digoxigenin by using HSAX on ECAX was quite uniform, averaging one gene-specific BIO- and DIG-Nick Translation Mixes (Roche Molecular Bio- marker every Ϸ1-Mbp interval. chemicals) and separately hybridized to horse metaphase chro- Seven genes from the upper terminal part of the RH group 1 mosomes to confirm probe origin and determine precise physical (a region corresponding to ECAXpter) produced PCR patterns locations. Additionally, nine closely positioned or overlapping characteristic to both the X and Y chromosomes. These genes markers in the RH map were cohybridized in differently labeled gave the same-size PCR amplification products in males and pairs or triplets on metaphase and interphase chromatin to refine females and also amplified in RH cell lines known to be Y their relative physical order. In situ hybridization, signal detec- specific (results not shown). Redesign of primers for most of tion, microscopy, and image analysis were carried out as de- these genes did not change the PCR typing pattern. However, the scribed (5). second set of primers generated by using sequence data from the first amplification products of STS produced an X specific Results pattern, as indicated with two adjacent map positions for this Generation of Gene-Specific Markers on ECAX. A total of 167 equine gene: STSX and STSXY (Fig. 3). gene-specific primer pairs were designed by using the human X chromosome sequence map (Ensembl, www.ensembl.org; FISH Map. Equine BAC clones containing 29 selected genes FISH Human Genome Browser, http:͞͞genome.ucsc.edu͞index. mapped to the expected chromosomal location (Table 1) based html?orgϭHuman&dbϭhg16&hgsidϭ27764832). After the first on the RH map and the previously FISH mapped loci (22). These round of optimization, 40 primer sets were excluded due to weak localizations bring the total number of cytogenetically mapped

Raudsepp et al. PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 ͉ 2387 Downloaded by guest on September 27, 2021 Fig. 1. High-resolution RH and comparative map of the long arm of horse X chromosome (ECAXq). A complete map and detailed figure legend are published as Fig. 3. From left to right are a schematic drawing of ECAXq, FISH localizations, RH map showing the distance and order of loci (ECAXqRH), and comparative location of orthologs in human (HSA) and mouse (MMU), followed by comparative map information on orthologs mapped in cattle (BTA), pig (SSC), dog (CFA), cat (FCA), goat (CHI), sheep (OAR), and river buffalo (BBU).

2388 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308513100 Raudsepp et al. Downloaded by guest on September 27, 2021 markers on ECAX to 53 (46 gene-specific and 7 short tandem Ϸ1-Mb intervals, this is the densest and most uniformly distrib- repeats; Fig. 3). The FISH markers are fairly uniformly distrib- uted map of the X chromosome presently available in any of the uted along the chromosome, from Xpter to Xqter (Fig. 3 and Fig. mammalian species other than humans and mice. 4, which is published as supporting information on the PNAS The currently available X chromosome maps of various live- web site), except in band Xp21. Two-color FISH on metaphase stock and pet͞companion animals show a range of 12 (sheep͞ chromosomes provided the following physical order for over- goat͞buffalo) to 92 (pig) genes mapped using different ap- lapping loci:. Adlican-TMSB4 (pterfcen; Fig. 4h), CHM- proaches [cattle, Ϸ37 genes (31, 32, 39, 51); pig, Ϸ92 genes (29, DIAPH2 (cenfqter; Fig. 4f) and FMR1-MTM1 (cenfqter; Fig. 30, 52–57); dog, 22 genes (34, 35, 37); cat, 30 genes (21, 33); 4h). Further, interphase FISH with combinations of differently sheep͞goat͞buffalo, 12 genes (31, 39, 40)]. In most of these labeled probes helped refine relative order of three loci (Adlican, species except cat (33) and dog (36), either the different maps are NLGN4, and TMSB4) located in the terminal region of the short not integrated and physically ordered into a single map or the arm (Xpter). The interphase-FISH order for the three loci was distribution of the loci is not uniform. In contrast, the 137 gene pterfAdlicanfNLGN4fTMSB4fcen (Fig. 4i). All FISH re- human–horse comparative map presented in this study is 2- to sults were in agreement with the physical order of loci deduced 4-fold more informative for gene content and is conspicuously by the RH map. However, there were minor exceptions: although integrated into a single physically ordered map. This is evident double-color FISH showed that NUDT11 is distal to MAGEH1 also from the comparative status of the X chromosome map for on ECAXp13 (Fig. 4e), RH analysis gave a reversed order. all ECAX genes hitherto mapped in different species (see Fig. 3, Reexamination of typing results of these and adjacent loci did right half of each sheet). Last, despite containing only Ϸ10% of not show any genotyping͞scoring errors. Last, our FISH assign- the expected X homologs from human (1,695 genes http:͞͞ ment of LAMP2 is in agreement with our RH map but does not genome.ucsc.edu͞cgi-bin͞hgGateway) or mouse (1,116 genes; concur with earlier localization (4). draft sequence, www.ensembl.org͞Mus࿝musculus), the map pro- vides excellent alignment of ECAX with the X chromosome map Comparative Mapping. The mapping of 137 equine orthologs of of the two species. HSAX genes demonstrated complete synteny conservation be- Despite a total of 169 markers, the ECAX RH map was tween the X chromosomes of the two species (Fig. 3). Of these, divided into six RH linkage groups, due to the threshold chosen 114 loci have also been mapped in mouse: 113 to MMUX and (logarithm of odds, 7 RHMAP2PT) to develop the linkage one (CLCN4) to MMU7 (49). The results reiterate conservation groups. Assuming the linkage groups to be separated by at least Ϸ of the X chromosome across three evolutionarily distantly 50 cR5000, the five gaps add 250 cR to the current map of 1,344 related mammals, with a remarkably higher degree of conser- cR, thus increasing the effective map size to Ϸ1,600 cR. This vation between horse and human than between horse and mouse implies that with the size of ECAX as 153 Mbp, the average span or human and mouse. As yet no mouse orthologs have been of the map for this chromosome in our 5,000-rad panel is Ϸ10 found for 23 of the horse͞human X specific genes. Of these, nine cR͞Mbp. Further, of the 153 Mbp, Ϸ2–3 Mbp spans the cen- genes are from the human pseudoautosomal regions (PAR), tromeric region, whereas 5–6 Mbp spans the intercalary hetero- PAR1 and -2 (see Fig. 3). chromatic region on the long arm of the chromosome (Xq21). It To obtain a refined comparative map among horse, human, is presumed that these regions are gene poor. These assumptions and mouse X chromosomes, we determined the precise sequence stem from the fact that gene density in the predominantly location of human and mouse orthologs for all 133 physically heterochromatic regions of HSA1, -9, and -16 is zero for ordered equine genes from the available genomic draft se- stretches spanning 20, 16, and 9 Mbp of heterochromatin, quences (see Materials and Methods). Four FISH-mapped genes respectively (http:͞͞bioinformatics.weizmann.ac.il͞cards). A (ANT3, MG61, PLP1, and F9) not mapped by RH analysis were similar condition was also seen for satellite-bearing chromosome also included in the comparison by placing them on the com- HSA22, where the proximal Ϸ14 Mbp are barren, and HSAY, parative map based on adjacent FISH markers, increasing the where the distal Ϸ20 Mbp do not contain any genes (except for number of comparative loci to 137. As described earlier (5), if three genes in the PAR2 region). GENETICS sequence locations of a group of human or mouse orthologs indicated conservation of gene order in relation to the derived XY Amplification and the Pseudoautosomal Region. Of significant order of equine genes, the data were clustered in boxes (see Fig. interest in the ECAX map is the distal͞terminal region of the 3). A total of seven clusters were observed in human and 13 in short arm. Seven markers from this region (APXL, DXYS155E, mouse. These clusters, referred to as conserved linkages [max- ASMT, GYG2, Adlican, NLGN4, and STS) demonstrate both X imally contiguous chromosomal region with identical gene con- and Y specific amplification with RH cell line DNA. In humans, tent and order (50)], showed groups of genes with similar all seven genes are known to have both X and Y homologs with physical order in horse–human and horse–mouse. The clustering sequence identities ranging from 60% to 96% [(58) GeneCards, also highlighted smaller evolutionarily conserved segments http:͞͞bioinformatics.weizmann.ac.il͞cards]. Presently 31 such among the three species that could be observed within the larger genes are located on HSAY. These genes, referred to as X conserved linkages (Fig. 3, cream-shaded regions over human͞ degenerates, are considered as surviving relics of the ancestral mouse orthologs). autosome from which X and Y chromosomes evolved (58). Four more X degenerate genes (TMSB4X, USP9X, KAL1, and SMCX; Discussion Fig. 3) were also mapped in this study, but primers for these We herein report a physical map of the horse X chromosome genes show only X specific amplification. We suggest that, comprising 175 markers (139 genes and 36 microsatellites). The because the latter four loci are further away from the XY 128 markers mapped in this study by RH analysis, 90% of which recombining region, sequences of these genes might have had are functional genes, expand the existing ECAX map (5, 22) by more evolutionary time for X-Y divergence (59); however, this Ͼ4-fold. Further, a total of 53 FISH localizations substantially needs further investigation. Nevertheless, the identification of improve the number of physical anchor points between the equine X-Y pattern genes in horse is an important starting point chromosome and the RH map and concurrently provide excel- to analyze shared sequences between equine X and Y chromo- lent corroboration to the RH data. The map is the first high- somes, thus shedding light on their evolution. resolution integrated gene map for a horse chromosome. With Synaptonemal complex analysis in horses shows that the markers distributed on average at 880-kb intervals along the ECAXpter region is pseudoautosomal [PAR (60, 61)]. The three chromosome and gene-specific loci dispersed on average at main characteristics of the PAR genes are that they (i) represent

Raudsepp et al. PNAS ͉ February 24, 2004 ͉ vol. 101 ͉ no. 8 ͉ 2389 Downloaded by guest on September 27, 2021 functional homologs of genes present on both X and Y chro- region. Based on studies focused on the evolution of mammalian mosomes; (ii) escape X inactivation; and (iii) recombine during X chromosome, it is proposed that the human X chromosome is male meiosis (62–65). The human PAR1 spans Ϸ2.6 Mb and composed of different evolutionary strata. These are roughly contains 13 genes (62, 64–66). Of these, two, DXYS155E and divided into the X conserved region XCR, corresponding to the ASMT, are mapped to ECAX in this study. The human PAR2, proximal one-quarter of short arm and complete long arm of located terminally on HSAXq, spans 320 kb and contains four HSAX, and the recently added region XRA, corresponding to genes [HSPRY3, SYBL1, IL9R, and CXYorf1 (65)]. Of these, two distal three-quarters of the short arm of HSAX (59, 68). have been mapped in this study to the terminal end of ECAXq Rearrangements observed by us in the horse, human, and mouse (Fig. 3). Analysis hitherto carried out in a range of mammalian genomes are incidentally located close to the suggested ancestral species show significant species-specific differences in the PAR fusion point of the conserved and the ancestral regions on genes. A gene that is pseudoautosomal in one species may not be HSAXp11.23 (68). A detailed study of this region aimed at pseudoautosomal in the other. Of the 13 human PAR genes, identifying evolutionary breakpoints may also lead to discovery ͞ eight do not have a known mouse homolog (64). Further, STS is of signatures of these rearrangements in horse mouse, which in a PAR gene in cattle (67), pig (54), dog, sheep, and mouse but turn will be useful for understanding the origin of gene order not in human and primate (63, 64); KAL1 is pseudoautosomal in differences in this region across the three species. pig (54) and cattle (67) but not in human and primate. Surpris- ingly, this locus is not even found in mouse (64). The horse PAR Future Uses. The mammalian X chromosome contains a dispro- has not yet been defined for gene content. The genes mapped in portionately high number of genes influencing development, female͞male fertility, reproduction, and disease [Online Men- this study provide a foundation to begin investigating the Xpter ͞ ͞ region in horse for identification of prospective PAR genes. delian Inheritance in Man, www.ncbi.nlm.nih.gov Sitemap ͞ index.html#OMIM; Online Mendelian Inheritance in Animals, Subsequent expression methylation studies of these genes can ͞͞ ͞ ͞ ͞ help validate their pseudoautosomal status and contribute to pre- http: morgan.angis.su.oz.au Databases BIRX omia (25, 26, cise characterization and physical demarcation the equine PAR. 69, 70)] that are also of significance in horses. The human and pig X chromosomes carry an unexpectedly high number of genes Comparative Map. The physically ordered ECAX map, comprising specifically expressed in the skeletal muscle (55, 71, 72). Analysis 139 equine genes, is in close agreement with our previous maps of these genes could have implications for the performance of horses. Structural and numerical aberrations of the X chromo- (5, 22) and provides a robust comparative map in relation to the some are the most common documented chromosome abnor- observed order of these genes in humans and mice. The most malities in the horse that invariably lead to reproductive failures, striking feature of the horse–human comparison is that, barring intersexuality, hermaphroditism, or sex reversal (73, 74). Addi- minor exceptions involving four to five interruptions on the short tionally, a number of X linked conditions͞diseases have been arm, the relative order of loci in the two species is exceptionally described in the horse, e.g., G6PD deficiency (75), X linked ter ter conserved from Xp to Xq . This degree of gene order severe immunodeficiency (76), fragile X (77), agammaglobu- conservation has not yet been observed for any other chromo- linemia (78), and hydrocephalus (79). Presently very little infor- some between humans and other nonprimate mammals. The mation is available concerning the underlying molecular causes horse–mouse comparison, however, shows noticeably less con- of these conditions. The high-resolution RH and comparative servation in gene order. The rearrangements are evident from map of ECAX presented in this study, in conjunction with the 13 conserved linkage blocks (boxes containing ordered loci) detailed map͞sequence information on human and mouse X originating from different regions of MMUX, some with re- chromosomes (23, 26, 27, 70), will serve as a basis to rapidly versed centromere–telomere orientation in relation to horse and converge on X specific genes significant in the horse. Efforts to human (Fig. 3). isolate BAC clones for all mapped markers on this chromosome The comparative map of the equine X chromosome presented will considerably facilitate localized isolation of polymorphic ͞ here helps to identify 13 blocks clusters of loci demonstrating markers, building BAC contigs in areas of interest and sequenc- conserved gene order across horse, human, and mouse (Fig. 3, ing targeted chromosomal regions. yellow shaded regions). These clusters potentially represent the most conserved X chromosome regions of the ancestor common We thank Dee Honeycutt for excellent management of the horse RH to horse, human, and mouse. Additionally, the blocks provide a panel and Glenda Goh and Kristi Hope for technical assistance. This quick comparative overview of smaller conserved linkages and project was funded by grants from the Texas Higher Education Board their relative organization in the three species. Although an (ARP 010366-0162-2001; ATP 000517-0306-2003 BPC, to J.E.W.), Na- ͞ exceptional degree of gene order conservation is observed tional Research Initiative Competitive Grants Program (NRICGP) U.S. between ECAX and HSAX, there are also minor rearrange- Department of Agriculture (USDA) Grant 2003-03687 (to B.P.C.), NRICGP͞USDA Grant 2000-03510 (to L.C.S.), Texas Equine Research ments. The most noticeable of these is a small segment corre- Foundation (to B.P.C. and L.C.S.), Link Endowment (to B.P.C. and sponding to ECAXp12–13 and the human sequence positions L.C.S.), American Quarter Horse Association, USDA–National 46–54 Mb (Fig. 3, red bracket). Interestingly, the horse and Research Support Projects-8 Coordinators’ Fund, and the Dorothy mouse genomes are also relatively more rearranged in the same Russell Havemeyer Foundation.

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