High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.     

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

Construction of a medium-density horse gene map

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.     

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse × hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19—a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution ofancestral HSA19 segments in eight mammalian orders involving about 50 species.     

High-resolution RH map of horse chromosome 22 reveals a putative ancestral vertebrate chromosome

High-resolution gene maps of individual equine chromosomes are essential to identify genes governing traits of economic importance in the horse. In pursuit of this goal we herein report the generation of a dense map of horse chromosome 22 (ECA22) comprising 83 markers, of which 52 represent specific genes and 31 are microsatellites. The map spans 831 cR over an estimated 64 Mb of physical length of the chromosome, thus providing markers at approximately 770 kb or 10 cR intervals. Overall, the resolution of the map is to date the densest in the horse and is the highest for any of the domesticated animal species for which annotated sequence data are not yet available. Comparative analysis showed that ECA22 shares remarkable conservation of gene order along the entire length of dog chromosome 24, something not yet found for an autosome in evolutionarily diverged species. Comparison with human, mouse, and rat homologues shows that ECA22 can be traced as two conserved linkage blocks, each related to individual arms of the human homologue-HSA20. Extending the comparison to the chicken genome showed that one of the ECA22 blocks that corresponds to HSA20q shares synteny conservation with chicken chromosome 20, suggesting the segment to be ancestral in mammals and birds.     

High-resolution comparative physical mapping of mouse Chromosome 10 in the region of homology with human Chromosome 21

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10. It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10. The average marker density for this region is 1 marker/69 kb. Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences. The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers. YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig. Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21. Received: 22 July 1998 / Accepted: 13 November 1998     

Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.     

Towards a Drosophila genome map.

A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes. Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped. More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb). On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.     

Integrated physical,genetic and genome map of chickpea (Cicer arietinum L.)

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.     

Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation

An integrated large-insert clone map of the region Xq11-q12 is presented. A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb. The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers. Overall marker density across this region is approximately 1 STS/60 kb. Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2). Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints. In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation.     

Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers

A horse BAC library was constructed with about 40, 000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented—LG3, LG4, LG5, LG8, and LG12—leaving only three groups unassigned. This work showed how this library makes an integrated map a realistic objective for the near future and how it can make comparative mapping more efficient in a search for candidate genes of interest. Received: 9 January 1998 / Accepted: 13 April 1998     

A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues

Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse x hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR(5000). The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.     

Homology-driven assembly of a sequence-ready mouse BAC contig map spanning regions related to the 46-Mb gene-rich euchromatic segments of human chromosome 19

Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.     

A physical map of the polytenized region (101EF-102F) of chromosome 4 in Drosophila melanogaster

Chromosome 4, the smallest autosome ( approximately 5 Mb in length) in Drosophila melanogaster contains two major regions. The centromeric domain ( approximately 4 Mb) is heterochromatic and consists primarily of short, satellite repeats. The remaining approximately 1.2 Mb, which constitutes the banded region (101E-102F) on salivary gland polytene chromosomes and contains the identified genes, is the region mapped in this study. Chromosome walking was hindered by the abundance of moderately repeated sequences dispersed along the chromosome, so we used many entry points to recover overlapping cosmid and BAC clones. In situ hybridization of probes from the two ends of the map to polytene chromosomes confirmed that the cloned region had spanned the 101E-102F interval. Our BAC clones comprised three contigs; one gap was positioned distally in 102EF and the other was located proximally at 102B. Twenty-three genes, representing about half of our revised estimate of the total number of genes on chromosome 4, were positioned on the BAC contigs. A minimal tiling set of the clones we have mapped will facilitate both the assembly of the DNA sequence of the chromosome and a functional analysis of its genes.     

Equine synteny mapping of comparative anchor tagged sequences (CATS) from human Chromosome 5

Comparative anchor tagged sequences (CATS) from human Chromosome 5 (HSA5) were used as PCR primers to produce molecular markers for synteny mapping in the horse. Primer sets for 21 genes yielded eight horse-specific markers, which were mapped with the UC Davis horse–mouse somatic cell hybrid panel into two synteny groups: UCD14 and UCD21. These data, in conjunction with earlier human chromosome painting studies of the horse karyotype and synteny mapping of horse microsatellite markers physically mapped by FISH, confirm the assignment of UCD21 to ECA21 and suggest that UCD14 is located on ECA14. In addition, our results can be used to substantiate previously published data which indicate that ECA21 contains material orthologous to HSA5p and HSA5q, and to propose an approximate region for an evolutionary chromosomal rearrangement event. Received: 1 February 1999 / Accepted: 12 July 1999     

A high-resolution comparative radiation hybrid map of equine chromosome 4q12-q22

In this study, we present a comprehensive 5000-rad radiation hybrid map of a 40-cM region on equine chromosome 4 (ECA4) that contains quantitative trait loci for equine osteochondrosis. We mapped 29 gene-associated sequence tagged site markers using primers designed from equine expressed sequence tags or BAC clones in the ECA4q12-q22 region. Three blocks of conserved synteny, showing two chromosomal breakpoints, were identified in the segment of ECA4q12-q22. Markers from other segments of HSA7q mapped to ECA13p and ECA4p, and a region of HSA7p was homologous to ECA13p. Therefore, we have improved the resolution of the human-equine comparative map, which allows the identification of candidate genes underlying traits of interest.     

An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin

Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig.     

A rapid and accurate strategy for rice contig map construction by combination of fingerprinting and hybridization

A rapid and accurate strategy for rice contig map construction was described. Rice BAC library with average insert of 120 kb in length was used as building materials in contig mapping. The contigs of varied lengths ranging from 500 kb to several megabases with sufficient redundancy to ensure the accuracy of the joining between individual BACs were formed by fingerprinting. The contigs were then assigned to and ordered along the chromosomes by various molecular markers through their hybridization against the whole rice genomic library. The accuracy of clone overlaps in contig was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones. He contigs thus obtained covered nearly the rice genome.     

Assignment of 115 genes from HSA9 and HSA14 to SSC1q by RH mapping to generate a dense human-pig comparative map

A large number of significant QTL for economically important traits including average daily gain have been located on SSC1q, which, as shown by chromosome painting, corresponds to four human chromosomes (HSA9, 14, 15 and 18). To provide a comprehensive comparative map for efficient selection of candidate genes, 81 and 34 genes localized on HSA9 and HSA14 respectively were mapped to SSC1q using a porcine 7000-rad radiation hybrid panel (IMpRH). This study, together with the cytogenetic map (http://www2.toulouse.inra.fr/lgc/pig/cyto/genmar/htm/1GM.HTM), demonstrates that SSC1q2.1-q2.13 corresponds to the region ranging from 44.6 to 63.2 Mb on HSA14q21.1-q23.1, the region from 86.5 to 86.8 Mb on HSA15q24-q25, the region from 0.9 to 27.2 Mb on HSA9p24.3-p21, the region from 35.1 to 38.0 Mb on HSA9p13, the region from 70.3 to 79.3 Mb on HSA9q13-q21 and the region from 96.4 to 140.0 Mb on HSA9q22.3-q34. The conserved synteny between HSA9 and SSC1q is interrupted by at least six sites, and the synteny between HSA14 and SSC1q is interrupted by at least one site.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.