Generation of expressed sequence tags and sequence-tagged sites as physical landmarks in the mosquito, Aedes aegypti, genome

Twenty-three clones were developed as expressed sequence tags (ESTs) and sequence-tagged sites (STSs) that provide broad coverage of the mosquito, Aedes aegypti, genome at an average spacing of about 7.2 cM. Each of these clones had been mapped previously as a restriction fragment length polymorphism (RFLP) marker. Nineteen of these clones represent anonymous cDNAs and 4 clones represent known genes. Forty-seven percent of the anonymous cDNAs showed significant deduced amino acid sequence similarity to previously described sequences from a wide variety of organisms. STSs developed from RFLPs will provide effective tools for rapid screening of YAC and cosmid libraries, and will therefore, provide anchor points for contig construction for any region in the A. aegypti genome. Also, they facilitate the simultaneous integration of physical and genetic mapping data for A. aegypti.     

Seven genes from human chromosome 18 map to chromosome 24 in the bovine

Bovine sequence tagged sites (STSs) were developed for seven genes and used for synteny mapping with a hybrid bovine x rodent cell line panel. The genes were thymidylate synthase (TYMS), pituitary adenylate cyclase activating peptide (ADCYAP1), and melanocortin-2 receptor (MC2R) from the short arm of human chromosome (HSA) 18 and N-cadherin (CDH2), transthyretin (TTR), gastrin-releasing peptide (GRP), and plasminogen activator inhibitor 2 (PAI2) from the long arm of HSA 18. Primers for these genes were designed with human, ovine, or bovine sequences aligned with a sequence from a second species. The bovine PCR product was cloned, and the fragment was sequenced to verify that the homologous gene was indeed amplified. A second set of bovine-specific PCR primers were developed for each gene from these sequences. These STSs were used for synteny mapping, and all seven genes were syntenic with markers of bovine chromosome (BTA) 24. The concordance with BTA 24 was at least 96.5% for all genes.     

A microsatellite map of wheat

Hexaploid bread wheat (Triticum aestivum L. em. Thell) is one of the world's most important crop plants and displays a very low level of intraspecific polymorphism. We report the development of highly polymorphic microsatellite markers using procedures optimized for the large wheat genome. The isolation of microsatellite-containing clones from hypomethylated regions of the wheat genome increased the proportion of useful markers almost twofold. The majority (80%) of primer sets developed are genome-specific and detect only a single locus in one of the three genomes of bread wheat (A, B, or D). Only 20% of the markers detect more than one locus. A total of 279 loci amplified by 230 primer sets were placed onto a genetic framework map composed of RFLPs previously mapped in the reference population of the International Triticeae Mapping Initiative (ITMI) Opata 85 x W7984. Sixty-five microsatellites were mapped at a LOD >2.5, and 214 microsatellites were assigned to the most likely intervals. Ninety-three loci were mapped to the A genome, 115 to the B genome, and 71 to the D genome. The markers are randomly distributed along the linkage map, with clustering in several centromeric regions.     

Initial characterization of a protochordate histocompatibility locus

The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.     

Fatty acid profiles in subcutaneous and mesenteric adipose tissues from Zucker rats after energy restriction. Influence of dietary fat

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.     

Integrated mapping analysis of the Werner syndrome region of chromosome 8

The Werner syndrome locus (WRN) is located at 8p11-p12. To facilitate eventual cloning of the WRN gene, a 10,000-rad radiation-reduced hybrid (RH) cell panel was generated to map genetic markers, sequence-tagged sites (STSs), and genes in this region. A hamster cell line carrying an intact human chromosome 8 was fused with another hamster cell line. Two sets of hybrid cell panels from 2 separate fusions were generated; each panel consisted of 50 independent clones; 33 and 34 cell lines from the 2 fusions retained human chromsome material as determined by inter-Alu PCR. The combined panel was genotyped for 52 markers spanning the entire chromosome, including 10 genes, 29 anonymous polymorphic loci, and 13 STSs. Seventeen of these markers have not been previously described. Markers near the centromere were retained at a higher frequency than more distal markers. Fluorescence in situ hybridization was also used to localize and order a subset of the markers. A RH map of the WRN region was constructed using a maximum likelihood method, giving the following most likely order: D8S131-D8S339 (GSR)-D8S124-D8S278-D8S259-(D8S71)-D8S283- D8S87-D8S105-D8S135 (FGFR1)-D8S135PB-D8S255-ANK1. A genetic map of 15 short tandem repeat polymorphic loci in the WRN region was also constructed. The marker orders from the genetic and RH maps were consistent. In addition, an integrated map of 24 loci in the WRN region was generated using information from both genetic and RH mapping methods. A 1000:1 framework map for 6 loci (LPL-D8S136-D8S137-D8S87-FGFR1-ANK1) was determined by genetic mapping, and the resulting locus order was fixed during analysis of the RH genotype data. The resulting integrated map contained more markers than could confidently be ordered by either genetic or RH mapping alone.     

Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated high throughput rat genetic markers

Linkage mapping of quantitative trait loci requires analysis of a large number of animals. Although genetic markers isolated by representational difference analysis (RDA) and its modifications meet the needs, the number of these markers has been limited. In the present study, we established the arbitrarily primed (AP)-RDA method to isolate virtually an unlimited number of the high throughput genetic markers. A representation of the genome, an AP-amplicon, was prepared by AP-PCR with a single primer or with a combination of primers using genomic DNA of the ACI/N (ACI) or BUF/Nac (BUF) rat as a template. By subtracting the AP-amplicon of ACI from that of BUF, a total of 40 polymorphic and independent markers were isolated in seven series of AP-RDA using a single primer. Two series of AP-RDA with primer combination yielded seven additional independent markers. All of the markers gave clear positive/negative signals by hybridization of a filter where AP-amplicons from F2 rats of ACI and BUF were dot-blotted at a high density without any concentration or purification. All of the 47 independent markers were mapped to unique chromosomal positions by linkage analysis, even though some arbitrary primers had very similar sequences. The markers were also informative between other strains of rats. Simultaneous hybridization of multiple filters made it possible to genotype a large number of rats simultaneously for multiple genetic loci. The AP-RDA method promises isolation of a large number of high throughput genetic markers in any species and is expected to facilitate linkage mapping of subtle quantitative trait loci.     

Overlapping genomic sequences: a treasure trove of single-nucleotide polymorphisms

An efficient strategy to develop a dense set of single-nucleotide polymorphism (SNP) markers is to take advantage of the human genome sequencing effort currently under way. Our approach is based on the fact that bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) used in long-range sequencing projects come from diploid libraries. If the overlapping clones sequenced are from different lineages, one is comparing the sequences from 2 homologous chromosomes in the overlapping region. We have analyzed in detail every SNP identified while sequencing three sets of overlapping clones found on chromosome 5p15.2, 7q21-7q22, and 13q12-13q13. In the 200.6 kb of DNA sequence analyzed in these overlaps, 153 SNPs were identified. Computer analysis for repetitive elements and suitability for STS development yielded 44 STSs containing 68 SNPs for further study. All 68 SNPs were confirmed to be present in at least one of the three (Caucasian, African-American, Hispanic) populations studied. Furthermore, 42 of the SNPs tested (62%) were informative in at least one population, 32 (47%) were informative in two or more populations, and 23 (34%) were informative in all three populations. These results clearly indicate that developing SNP markers from overlapping genomic sequence is highly efficient and cost effective, requiring only the two simple steps of developing STSs around the known SNPs and characterizing them in the appropriate populations.     

Linkage mapping in Papio baboons: conservation of a syntenic group of six markers on human chromosome 1

We have established multipoint genetic linkage among six loci in baboons (Papio hamadryas). Published PCR primers designed to amplify five human microsatellite loci were used to amplify homologous loci in 229 pedigreed baboons. Southern blotting was used to type two RFLPs in a functional gene (anti-thrombin III) in a subset of those animals. All six loci are known to map to human chromosome 1q, a region of the genome predicted by karyotype studies to be conserved in baboons. Pairwise recombination frequencies and lod scores indicate that the six loci are also linked in baboons. Recombination distances among the loci are similar to those reported for humans. Like humans, the baboons exhibit higher rates of recombination in females than in males. This study demonstrates that (1) microsatellite loci first described and characterized in the human genome can be effectively used for genetic linkage mapping in non-human primates, (2) a group of genetic loci known to be linked on human chromosome 1q are also linked in the baboon genome, and (3) sex differences in recombination frequencies among loci on human chromosome 1q are also observed in the genome of this Old World monkey. This constitutes the first reported multipoint linkage map in any nonhuman primate.     

The development of sequence-tagged sites for human chromosome 4

As part of our efforts to construct a high-resolution physical map of human chromosome 4, we developed a systematic approach for efficiently generating large numbers of chromosome-specific sequence-tagged sites (STSs). In this paper, we describe how rate-limiting steps in our STS development were identified and overcome, and detail our current development strategy. We present information for 822 new human chromosome 4-specific STSs, including PCR amplification conditions and subchromosomal localization data, obtained by analysis of the STS with somatic cell hybrids containing different portions of human chromosome 4. Although most STSs presented here were developed from anonymous clones whose sequences were determined in this laboratory, several STSs were developed for genes and other DNA sequences that were previously mapped to chromosome 4. Our data indicate that the availability of DNA sequence for an STS locus, in addition to the sequences of the two PCR oligonucleotides, significantly increases the transfer of that STS by allowing investigators to select new oligonucleotides best suited to the standard conditions used in their laboratories.     

Detection of Methylobacterium species by 16S rRNA gene-targeted PCR

We designed PCR primers for specific amplification of the 16S rRNA genes of seven species of the genus Methylobacterium. All of the pairwise species tested were successfully differentiated by PCR detection with a combination of five primer sets, with the exception of M. extorquens and M. rhodesianum. These primers did not cross-react with closely related bacteria in the alpha subclass tested.     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.     

New microsatellite markers in chicken optimized for automated fluorescent genotyping

We have isolated and developed 180 new polymorphic chicken microsatellite markers. In addition, primers have been developed for 91 microsatellites derived from the GenBank sequence database (isolated by the laboratory of Terry Burke, Leicester University), of which 89 were polymorphic, and six existing polymorphic markers (HUJ) have been modified. The primer sequences were designed to allow optimal performance of the markers, in sets containing multiple microsatellites, on ABI sequencers. The average number of alleles for the 275 polymorphic markers described was 4.0. Of these markers, 93% were polymorphic in the Wageningen resource population whereas 57% of the markers were polymorphic in the East Lansing reference population and only 44% could be mapped in the Compton reference population. The microsatellite markers described in this paper, in combination with the microsatellite markers published previously, are particularly well suited for performing a total genome scan for the detection of quantitative trait loci (QTL).     

Studies on the eel sperm flagellum. 2. The kinematics of normal motility

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.     

Landmarks in the development of coronary artery bypass surgery

Genetic linkage maps have been produced for a wide range of organisms during the last decade, thanks to the increasing availability of molecular markers. The use of microsatellites (or Simple Sequence Repeats, SSRs) as genetic markers has led to the construction of "second-generation" genetic maps for humans, mouse and other organisms of major importance. We constructed a second-generation single-tree genetic linkage map of Norway spruce (Picea abies K.) using a panel of 72 haploid megagametophytes with a total of 447 segregating bands [366 Amplified Fragment Length Polymorphisms (AFLPs), 20 Selective Amplification of Microsatellite Polymorphic Loci (SAMPLs) and 61 SSRs, each single band being treated initially as a dominant marker]. Four hundred and thirteen markers were mapped in 29 linkage groups (including triplets and doublets) covering a genetic length of 2198.3 cM, which represents 77.4% of the estimated genome length of Picea abies (approximately 2839 cM). The map is still far from coalescing into the expected 12 chromosomal linkage groups of Norway spruce (2n = 2x = 24). A possible explanation for this comes from the observed non-random distribution of markers in the framework map. Thirty-eight SSR marker loci could be mapped onto 19 linkage groups. This set of highly informative Sequence Tagged Sites (STSs) can be used in many aspects of genetic analysis of forest trees, such as marker-assisted selection, QTL mapping, positional cloning, gene flow analysis, mating system analysis and genetic diversity studies.     

Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping

To facilitate large-scale genetic mapping of the human genome, we have developed chromosome-specific sets of microsatellite marker loci suitable for use with a fluorescence-based automated DNA fragment analyser. We present 254 dinucleotide repeat marker loci (80% from the Généthon genetic linkage map) arranged into 39 sets, covering all 22 autosomes and the X chromosome. The average distance between adjacent markers is 13 centiMorgans, and less than 4% of the genome lies more than 20 cM from the nearest marker. Each set of microsatellites consists of up to nine marker loci, with allele size ranges that do not overlap. We selected marker loci on the basis of their reliability in the polymerase chain reaction, polymorphism content, map position and the accuracy with which alleles can be scored automatically by the Genotyper program.     

Comparative mapping of 9 human chromosome 22q loci in the laboratory mouse

We present a comparative map of genes on human chromosome 22q and homologous loci in the mouse genome. Gene order in humans was established using a panel of somatic cell hybrids. Genetic maps spanning homologous segments on three mouse chromosomes were generated using an interspecific backcross. The conserved linkage between human chromosome 22 and mouse chromosome 16 includes two closely linked loci, Comt and IgI-1. The second conserved linkage involves human chromosome 22 and mouse chromosome 11 and contains two genetically and physically linked loci, Lif and Nfh. Finally, conserved synteny involving mouse chromosome 15 and human chromosome 22 spans 30 cM and contains five loci (Acr, Bzrp, Dia-1, Il2rb and Pdgfb). Loci within this conserved synteny have been sublocalized to different portions of human chromosome 22. The order of genes on mouse chromosome 15 and human chromosome 22 provides further evidence for chromosomal rearrangements within the conserved synteny that have occurred since the divergence of lineages leading to mice and humans.     

Sequence-tagged-site (STS) markers of arbitrary genes: development, characterization and analysis of linkage in black spruce

Sequence-tagged-site (STS) markers of arbitrary genes were investigated in black spruce [Picea mariana (Mill.) B.S.P.]. Thirty-nine pairs of PCR primers were used to screen diverse panels of haploid and diploid DNAs for variation that could be detected by standard agarose gel electrophoresis without further manipulation of amplification products. Codominant length polymorphisms were revealed at 15 loci. Three of these loci also had null amplification alleles as did 3 other loci that had no apparent product-length variation. Dominant length polymorphisms were observed at 2 other loci. Alleles of codominant markers differed in size by as little as 1 bp to as much as an estimated 175 bp with nearly all insertions/deletions found in noncoding regions. Polymorphisms at 3 loci involved large (33 bp to at least 114 bp) direct repeats and similar repeats were found in 7 of 51 cDNAs sequenced. Allelic segregation was in accordance with Mendelian inheritance and linkage was detected for 5 of 63 pairwise combinations of loci tested. Codominant STS markers of 12 loci revealed an average heterozygosity of 0.26 and an average of 2.8 alleles in a range-wide sample of 22 trees.     

Paired STSs amplified from radiation hybrids, and from associated YACs, identify highly polymorphic loci flanking the ataxia telangiectasia locus on chromosome 11q22-23

The high resolution mapping of the ataxia telangiectasia (A-T) locus on chromosome 11q22-23 requires the generation of new polymorphic markers specifically within the segment of 11q22-23 to which the locus has been assigned. We have made use of a library of Alu-PCR clones, amplified from a radiation reduced somatic cell hybrid containing the relevant chromosome 11 segment, to generate sequence tagged sites (STS) within the 11q22-23 region and have used YAC clones to extend the loci identified by these STSs. The identification of paired polymorphisms (from Alu-PCR and the associated YAC derived clone), which are physically linked, but which show minimal linkage disequilibrium, provides a highly informative haplotype for use in genetic linkage analysis in A-T families. We describe the characterisation of 2 such polymorphic loci, D11S535 and D11S611, which map between existing flanking markers, and which provide additional information on the location of the major A-T locus.