Characterization of newly developed SSLP markers for the rat

We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F₂ progeny of (OLETFxBN)F₂ crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species. Received: 8 July 1999 / Accepted: 3 December 1999     

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain

Abstract Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.     

Comparative analysis and development of microsatellite markers on swine (Sus scrofa) chromosome 1qter

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

A radiation hybrid map of chicken chromosome 15

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.     

A high-resolution comparative RH map of the telomeric end of bovine chromosome 2 with human chromosomes 1 and 2

High density livestock to human comparative maps are necessary for the implementation of comparative positional candidate gene cloning. We have constructed a high-density comparative radiation hybrid (RH) map of the telomeric end of bovine chromosome 2 (BTA2) using a 12,000-rad whole genome cattle-hamster radiation hybrid (WGRH) panel. Eighteen bovine EST markers with orthologues on human chromosomes 1 and 2 (HSA1 and HSA2), together with nine microsatellite markers, were typed against the 180 cell lines of the WGRH panel. Twenty-one markers were included in the multi-point framework map at LOD =3.0. The comparative analysis reveals a new segment of highly conserved synteny between HSA2 and BTA2.     

A 1.5-Mb-resolution radiation hybrid map of the cat genome and comparative analysis with the canine and human genomes

We report the construction of a 1.5-Mb-resolution radiation hybrid map of the domestic cat genome. This new map includes novel microsatellite loci and markers derived from the 2X genome sequence that target previous gaps in the feline-human comparative map. Ninety-six percent of the 1793 cat markers we mapped have identifiable orthologues in the canine and human genome sequences. The updated autosomal and X-chromosome comparative maps identify 152 cat-human and 134 cat-dog homologous synteny blocks. Comparative analysis shows the marked change in chromosomal evolution in the canid lineage relative to the felid lineage since divergence from their carnivoran ancestor. The canid lineage has a 30-fold difference in the number of interchromosomal rearrangements relative to felids, while the felid lineage has primarily undergone intrachromosomal rearrangements. We have also refined the pseudoautosomal region and boundary in the cat and show that it is markedly longer than those of human or mouse. This improved RH comparative map provides a useful tool to facilitate positional cloning studies in the feline model.     

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.     

Mapping of EST- and STS-markers in the human genome using a panel fo radiation hybrids

Ten DNA markers were localized in the human genome by a screening procedure against the radiation hybrid somatic cell panel (GeneBridge 4 RH Panel) using polymerase chain reaction (RH mapping method). DNA markers were developed to nucleotide sequences adjacent to NotI sites of human chromosome 3 (NotI-STS markers) and also to nucleotide sequences of human cDNA (EST markers). Three EST markers mapped (B10164, S16R and 18F5R) were localized in the human genome for the first time. Marker B10164 was found to be homologous to the nucleotide sequence of the BASP1 gene coding a major receptor protein. Markers S16R and 18F5R presumably tagged new genes, because no homologies were revealed among the nucleotide sequences presented in the databases. For four NotI-STS, more precise localization on human chromosome 3 was determined. On the basis of the data obtained, the NotI map may be integrated with other types of physical maps of human chromosome 3. RH mapping with a standard commercial panel of radiation hybrid somatic cells provided a chance to integrate the data obtained into international databases and existing integrated human chromosomal maps.     

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA X.     

Assignment of non-informative turkey genetic markers through comparative approaches

Molecular markers such as microsatellites, provide genetic signposts for navigating genomes. In general, genetic markers that are monomorphic or non-informative in mapping populations typically remain unmapped and as such are less likely to be included in future studies. The use of hybrid cell panels and in silico mapping via whole genome sequences allow for positional mapping of non-segregating markers. This study utilizes the INRA ChickRH6 whole-genome radiation hybrid panel and chicken whole-genome shotgun sequence to map microsatellite markers from the turkey (Meleagris gallopavo). Thirty-three of the 41 markers typed on the RH panel had significant linkage to at least one other marker and 83 of 100 sequences returned significant BLAST similarities. Positioning of these markers provides additional sequence tagged sites in the turkey genome and increases the potential use of these markers for future genetic studies.     

A comparative map of interstitial bovine chromosome 5 with human chromosomes 12 and 22

We have constructed a high-density comparative radiation hybrid map of the interstitial region of bovine chromosome 5 (BTA5) using a recently constructed 12,000-rad, whole-genome, cattle-hamster radiation hybrid (WGRH) panel. Sixty-two bovine EST markers were selected which have orthologous sequences on human chromosomes 12 and 22 (HSA12 and HSA22). Sixty markers were included in the multi-point framework map at LOD 3.0. Our comprehensive RH map contains more than twice as many markers (88) than previous generation maps. Because of a higher marker density and increased resolution of the RH(12,000) panel, all markers were placed into a single linkage group based on two-point analysis at a LOD score 6.0. As a result, this new comparative map reveals new blocks of synteny and extensive gene order alterations between species. Breakpoints of synteny are located with high accuracy. Overall, this work reveals widespread chromosomal rearrangements between bovine, human and mouse genomes.     

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     

Isolation and high-resolution mapping of new DNA markers from the pericentromeric region of chromosome 10.

The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including D10Z1, D10S94, D10S97, and D10S102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small subchromosomal fragments that include both D10S94 and D10Z1. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a lambda library made with pp11A DNA. We have completed regional mapping of 22 of those clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between D10S34 and RBP3. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A.     

Assessment of AFLP marker behaviour in enriching STS radiation hybrid maps

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP^® technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.     

A new 4016-marker radiation hybrid map for porcine-human genome analysis

We constructed a 5000-rad comprehensive radiation hybrid (RH) map of the porcine (Sus scrofa) genome and compared the results with the human genome. Of 4475 typed markers, 4016 (89.7%) had LOD >5 compared with the markers used in our previous RH map by means of two-point analysis and were grouped onto the 19 porcine chromosomes (SSCs). All mapped markers had LOD >3 as determined by RHMAPPER analysis. The current map comprised 430 microsatellite (MS) framework markers, 914 other MS markers, and 2672 expressed sequence tags (ESTs). The whole-genome map was 8822.1 cR in length, giving an average marker density of 0.342 Mb/cR. The average retention frequency was 35.8%. Using BLAST searches of porcine ESTs against the RefSeq human nucleotide and amino acid sequences (release 22), we constructed high-resolution comparative maps of each SSC and each human chromosome (HSA). The average distance between ESTs in the human genome was 1.38 Mb. SSC contained 50 human chromosomal syntenic groups, and SSC11, SSC12, and SSC16 were only derived from the HSA13q, HSA17, and HSA5 regions, respectively. Among 38 porcine terminal regions, we found that at least 20 regions have been conserved between the porcine and human genomes; we also found four paralogous regions for the major histocompatibility complex (MHC) on SSC7, SSC2, SSC4, and SSC1. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A high-resolution radiation hybrid map of bovine chromosome 5q1.3-q2.5 compared with human chromosome 12q

In this study we present a comprehensive 3000-rad radiation hybrid map on bovine chromosome 5 (BTA5) of a region between 12.8 and 74.0 cM according to the linkage map, which contains a quantitative trait loci for ovulation rate. We mapped 28 gene-associated sequence tagged site markers derived from sequences of bovine BAC clones and 10 microsatellite markers to the BTA5 region. In comparison with HSA12q, four blocks of conserved synteny were apparent showing three chromosomal breakpoints and two inversions in this segment of BTA5. Therefore, we have improved breakpoint resolution in the human-bovine comparative map, which enhances the determination of candidate genes underlying traits of interest mapped to BTA5.