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.     

High-resolution cytogenetic-based physical map of human chromosome 16

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.     

High-resolution radiation hybrid map of wheat chromosome 1D

Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from ~3,341 cR_35,000 for five major linkage groups to 11,773 cR_35,000 for a comprehensive map. The mapping resolution was estimated to be ~199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.     

A map of 22 loci on human chromosome 22.

We constructed a genetic linkage map of the entire long arm of human chromosome 22 with 30 polymorphic markers, defining 22 loci. The map consists of a continuous linkage group 110 cM long, when male and female recombination fractions are combined; average distance between the loci is 5.2 cM. All loci were placed on the map with high support against alternative orders (odds in excess of 1000:1). The order of loci presented in our map is in full agreement with that of the previous linkage maps of chromosome 22 and with the physical assignment of markers. Two markers included in this map, KI-831 (D22S212) and pEFZ31 (D22S32), allowed us to better define the region of the (11;22) translocation breakpoint specific for Ewing sarcoma. Ten additional polymorphic markers were placed on the 22-loci map with odds lower than 1000:1 against alternative locations. In total, we have introduced 29 new markers on the linkage map of chromosome 22.     

Phylogenomic analysis of vertebrate thrombospondins reveals fish-specific paralogues, ancestral gene relationships and a tetrapod innovation

Background  

Thrombospondins (TSPs) are evolutionarily-conserved, extracellular, calcium-binding glycoproteins with important roles in cell-extracellular matrix interactions, angiogenesis, synaptogenesis and connective tissue organisation. Five TSPs, designated TSP-1 through TSP-5, are encoded in the human genome. All but one have known roles in acquired or inherited human diseases. To further understand the roles of TSPs in human physiology and pathology, it would be advantageous to extend the repertoire of relevant vertebrate models. In general the zebrafish is proving an excellent model organism for vertebrate biology, therefore we set out to evaluate the status of TSPs in zebrafish and two species of pufferfish.     

Reconstruction of a 450-My-old ancestral vertebrate protokaryotype

From recent work the putative eutherian karyotype from 100 Mya has been derived. Here, we have applied a new in silico technique, electronic chromosome painting (E-painting), on a large data set of genes whose positions are known in human, chicken, zebrafish and pufferfish. E-painting identifies conserved syntenies in the data set, and it enables a stepwise reconstruction of the ancestral vertebrate protokaryotype comprising 11 protochromosomes. During karyotype evolution in land vertebrates interchromosomal rearrangements by translocation are relatively frequent, whereas the karyotypes of birds and fish are much more conserved. Although the human karyotype is one of the most conserved in eutherians, it can no longer be considered highly conserved from a vertebrate-wide perspective.     

High-resolution genetic map of bovine chromosome 29 through focused marker development

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.     

Systematic evaluation of map quality: human chromosome 22

Marker positions on nine genetic linkage, radiation hybrid, and integrated maps of human chromosome 22 were compared with their corresponding positions in the completed DNA sequence. The proportion of markers whose map position is <250 kb from their respective sequence positions ranges from 100% to 35%. Several discordant markers were identified, as well as four regions that show common inconsistencies across multiple maps. These shared discordant regions surround duplicated DNA segments and may indicate mapping or assembly errors due to sequence homology. Recombination-rate distributions along the chromosome were also evaluated, with male and female meioses showing significantly different patterns of recombination, including an 8-Mb male recombination desert. The distributions of radiation-induced chromosome breakage for the GB4 and the G3 radiation hybrid panels were also evaluated. Both panels show fluctuations in breakage intensity, with different regions of significantly elevated rates of breakage. These results provide support for the common assumption that radiation-induced breaks are generally randomly distributed. The present studies detail the limitations of these important map resources and should prove useful for clarifying potential problems in the human maps and sequence assemblies, as well as for mapping and sequencing projects in and across other species.     

A high-resolution comparative RH map of the proximal part of bovine chromosome 1

Current comparative maps between human chromosome 21 and the proximal part of cattle chromosome 1 are insufficient to define chromosomal rearrangements because of the low density of mapped genes in the bovine genome. The recently completed sequence of human chromosome 21 facilitates the detailed comparative analysis of corresponding segments on BTA1. In this study eight bovine bacterial artificial chromosome (BAC) clones containing bovine orthologues of human chromosome 21 genes, i.e. GRIK1, CLDN8, TIAM1, HUNK, SYNJ1, OLIG2, IL10RB, and KCNE2 were physically assigned by fluorescence in situ hybridization (FISH) to BTA1q12.1-q12.2. Sequence tagged site (STS) markers derived from these clones were mapped on the 3000 rad Roslin/Cambridge bovine radiation hybrid (RH) panel. In addition to these eight novel markers, 17 known markers from previously published BTA1 linkage or RH maps were also mapped on the Roslin/Cambridge bovine RH panel resulting in an integrated map with 25 markers of 355.4 cR(3000) length. The human-cattle genome comparison revealed the existence of three chromosomal breakpoints and two probable inversions in this region.     

Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species.

Chromosome homologies between the Japanese raccoon dog (Nectereutes procyonoides viverrinus, 2n = 39 + 2-4 B chromosomes) and domestic dog (Canis familiaris, 2n = 78) have been established by hybridizing a complete set of canine paint probes onto high-resolution G-banded chromosomes of the raccoon dog. Dog chromosomes 1, 13, and 19 each correspond to two raccoon dog chromosome segments, while the remaining 35 dog autosomes each correspond to a single segment. In total, 38 dog autosome paints revealed 41 conserved segments in the raccoon dog. The use of dog painting probes has enabled integration of the raccoon dog chromosomes into the previously established comparative map for the domestic dog, Arctic fox (Alopex lagopus), and red fox (Vulpes vulpes). Extensive chromosome arm homologies were found among chromosomes of the red fox, Arctic fox, and raccoon dog. Contradicting previous findings, our results show that the raccoon dog does not share a single biarmed autosome in common with the Arctic fox, red fox, or domestic cat. Comparative analysis of the distribution patterns of conserved chromosome segments revealed by dog paints in the genomes of the canids, cats, and human reveals 38 ancestral autosome segments. These segments could represent the ancestral chromosome arms in the karyotype of the most recent ancestor of the Canidae family, which we suggest could have had a low diploid number, based on comparisons with outgroup species.     

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.     

Search for putative suppressor genes in meningioma: significance of chromosome 22

Summary Cytogenetic and molecular studies of various solid tumors have indicated that a series of different chromosomal regions may be deleted in the tumor genome. Usually, losses of heterozygosity are observed and, from this finding, the presence of specific genes acting as tumor suppressors has been deduced. In particular tumors, however, only a single chromosome site appears to be affected. Therefore, we have carried out a study of human meningioma, investigating 7 such putative suppressor regions by applying twelve site-specific DNA markers. In 6 out of 19 tumors, we exclusively found loss of heterozygosity for markers of the long arm of chromosome 22; none of the tumors showed statistically significant additional allelic losses for the regions 1p, 3p, 5p, 5q, 11p, 13q, 17p. Our data support the long-standing observation that only losses of or within chromosome 22 are associated with the development of meningiomas. Other suppressor regions are apparently not involved.     

A genetic linkage map of the long arm of human chromosome 22

We have used a recombinant phage library enriched for chromosome 22 sequences to isolate and characterize eight anonymous DNA probes detecting restriction fragment length polymorphisms on this autosome. These were used in conjunction with eight previously reported loci, including the genes BCR, IGLV, and PDGFB, four anonymous DNA markers, and the P1 blood group antigen, to construct a linkage map for chromosome 22. The linkage group is surprisingly large, spanning 97 cM on the long arm of the chromosome. There are no large gaps in the map; the largest intermarker interval is 14 cM. Unlike several other chromosomes, little overall difference was observed for sex-specific recombination rates on chromosome 22. The availability of a genetic map will facilitate investigation of chromosome 22 rearrangements in such disorders as cat eye syndrome and DiGeorge syndrome, deletions in acoustic neuroma and meningioma, and translocations in Ewing sarcoma. This defined set of linked markers will also permit testing chromosome 22 for the presence of particular disease genes by family studies and should immediately support more precise mapping and identification of flanking markers for NF2, the defective gene causing bilateral acoustic neurofibromatosis.     

High-resolution physical and transcript map of the locus for venous malformations with glomus cells (VMGLOM) on chromosome 1p21-p22

Vascular anomalies are congenital lesions that usually occur sporadically, but can be inherited. Previously, we have described that venous malformations, localized bluish-purple skin lesions, are caused by an activating mutation in the TIE2/TEK receptor. Moreover, we mapped another locus to chromosome 1p21-p22, for venous malformations with glomus cells (VM-GLOM). Here we report a physical map, based on 18 overlapping YAC clones, spanning this 5-Mb VMGLOM locus, from marker GATA63C06 to D1S2664. In addition, we report a sequence-ready PAC map of 46 clones covering 1.48 Mb within the YAC contig, a region to which we have restricted VMGLOM. We describe 21 new STSs and nine novel CA repeats, seven of which are polymorphic. These data will enable positional cloning of genes for diseases mapped to this locus, including the VMGLOM gene, likely a currently unknown regulator of vasculogenesis and/or angiogenesis.     

A radiation hybrid map of chromosome 1D reveals synteny conservation at a wheat speciation locus

The species cytoplasm specific (scs) genes affect nuclear-cytoplasmic interactions in interspecific hybrids. A radiation hybrid (RH) mapping population of 188 individuals was employed to refine the location of the scs ^ae locus on Triticum aestivum chromosome 1D. “Wheat Zapper,” a comparative genomics tool, was used to predict synteny between wheat chromosome 1D, Oryza sativa, Brachypodium distachyon, and Sorghum bicolor. A total of 57 markers were developed based on synteny or literature and genotyped to produce a RH map spanning 205.2 cR. A test-cross methodology was devised for phenotyping of RH progenies, and through forward genetic, the scs ^ae locus was pinpointed to a 1.1 Mb-segment containing eight genes. Further, the high resolution provided by RH mapping, combined with chromosome-wise synteny analysis, located the ancestral point of fusion between the telomeric and centromeric repeats of two paleochromosomes that originated chromosome 1D. Also, it indicated that the centromere of this chromosome is likely the result of a neocentromerization event, rather than the conservation of an ancestral centromere as previously believed. Interestingly, location of scs locus in the vicinity of paleofusion is not associated with the expected disruption of synteny, but rather with a good degree of conservation across grass species. Indeed, these observations advocate the evolutionary importance of this locus as suggested by “Maan’s scs hypothesis.”     

A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1

Background

The generation of BAC/PAC contigs in targeted genome regions is a powerful method to establish high-resolution physical maps. In domestic animal species the generation of such contigs is typically initiated with the screening of libraries with probes derived from human genes that are expected to be located in the region of interest by comparative mapping. However, in many instances the available gene-derived probes are too far apart to allow the cloning of BAC/PAC contigs larger than a few hundred kb. High resolution physical mapping allows to estimate the sizes of gaps and to control the orientation of the individual sub-contigs, which helps to avoid errors during the assembly of smaller contigs into final Mb-sized contigs. The recently constructed porcine IMNpRH2 panel allowed us to use this approach for the construction of high-resolution physical maps of SSC 6q1.2.

Results

Two sequence-ready BAC/PAC contigs of the gene-rich region on porcine chromosome 6q1.2 (SSC 6q1.2) containing the RYRl gene were constructed. The two contigs spanned about 1.2 Mb and 2.0 Mb respectively. The construction of these contigs was monitored by the results provided by the mapping of 15 markers on the IMpRH_7000rad and 35 markers on the IMNpRH2_12000rad radiation hybrid panels. Analyses on the IMpRH panel allowed us to globally link and orientate preliminary smaller contigs, whereas analyses on the high resolution IMNpRH2 panel allowed us to finally identify the order of genes and markers.

Conclusions

A framework map of 523 cR₁₂₀₀₀ was established covering the whole studied region. The order of markers on the framework 1000:1 RH map was found totally consistent with the data deduced from the contig map. The kb/cR ratio was very constant in the whole region, with an average value of 6.6 kb/cR. We estimate that the size of the remaining gap between the two contigs is of about 300 kb. The integrated physical and RH map of the investigated region on SSC 6q1.2 was used for a comparative analysis with respect to the syntenic regions on HSA 19q13.1 and MMU 7 and revealed a perfectly conserved gene order across the entire studied interval.

     

A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21

A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the region and span an estimated length of approximately 3.3 Mb. The region presently contains one mapped marker per 22 kb on average, which represents a major improvement over the previous resolution of one marker per 380 kb obtained through the generation of a dense RH map for this segment. Dual color fluorescence in situ hybridization on metaphase and interphase chromosomes verified the relative order of some of the BACs and helped to orient them accurately in the contigs. Despite having similar gene order and content, the equine region covered by the contigs appears to be distinctly smaller than the corresponding region in human (3.3 Mb vs. 5.5-6 Mb) because the latter harbors a host of repetitive elements and gene families unique to humans/primates. Considering limited representation of the region in the latest version of the horse whole genome sequence EquCab2, the dense map developed in this study will prove useful for the assembly and annotation of the sequence data on ECA21 and will be instrumental in rapid search and isolation of candidate genes for traits mapped to this region.     

High-resolution physical map for chromosome 16q12.1-q13, the Blau syndrome locus

Background  

The Blau syndrome (MIM 186580), an autosomal dominant granulomatous disease, was previously mapped to chromosome 16p12-q21. However, inconsistent physical maps of the region and consequently an unknown order of microsatellite markers, hampered us from further refining the genetic locus for the Blau syndrome. To address this problem, we constructed our own high-resolution physical map for the Blau susceptibility region.     

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.