Linkage map of seven polymorphic markers on rat Chromosome 18

A genetic linkage map of seven polymorphic markers was created with F₂ intercross progeny of F344/N and LEW/N rats and assigned to rat Chromosome (Chr) 18. Five of the markers described were defined by simple sequence length polymorphisms (SSLPs) associated with five genes: transthyretin (TTR), trypsin inhibitor-like protein (TILP), 2 adrenergic receptor (ADRB2), olfactory neuron-specific G protein (OLF), and gap junction protein (GJA1). One marker was defined by a restriction fragment length polymorphism (RFLP) detected with a probe for the human colony stimulating factor 1 receptor (CSF1R) gene. The D18N1R locus was defined by an anonymous DNA fragment amplified by the randomly amplified polymorphic DNA (RAPD) technique with a single short primer. These seven DNA loci formed a single genetic linkage group 30.4 cM in length with the following order: TTR-6.8 cM-D18N1R-9.1 cM-TILP-4.3 cM-CSF1R-0 cM-ADRB2-10.2 cM-OLF-0 cM-GJA1. The five SSLP markers were highly polymorphic. In a total of 13 inbred rat strains analyzed (F344/ N, LEW/N, LOU/MN, WBB1/N, WBB2/N, MR/N, MNR/N, ACI/N, SHR/N, WKY/N, BN/SsN, BUF/N, and LER/N), three to six alleles were detected for each marker. Remarkable linkage conservation was detected between the region of rat Chr 18 mapped and a region of mouse Chr 18. However, genes associated with these markers have been mapped to three different human chromosomes (Chrs 5, 6, and 18). The markers described here should be useful for genetic mapping studies and genetic monitoring of inbred rat strains.     

SstII polymorphic sites in the promoter region of the human cystatin C gene

Summary By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85 bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.     

Map of seven polymorphic markers on rat Chromosome 14: linkage conservation with human Chromosome 4

Seven polymorphic markers identified by polymerase chain reaction (PCR) amplification, including markers for six genes—DRD1L (dopamine receptor, D1-like-2), GLUKA (glucokinase), PF4 (platelet factor 4), ALB (albumin), AFP (-fetoprotein), and BSP (bone sialoprotein)—and one anonymous locus (D14N52), were mapped to a single 67-cM linkage group with F₂ intercross progeny of F344/N and LEW/N inbred rat strains. Two of these markers, ALB and AFP, have previously been assigned to rat Chromosome (Chr) 14, allowing assignment of this entire linkage group. Five of the markers—DRD1L, PF4, ALB, AFP, and PBSP—have been physically mapped to a large region of human Chr 4 encompassing the p arm and the q arm to band q28. Homologs of two of the markers, ALB and AFP, have been mapped to Chr 5 in the mouse. Comparison of human Chr 4 with the homologous regions on Chr 14 of the rat and Chr 5 of the mouse indicated that linkage conservation with human Chr 4 extends over a greater region in the rat than in the mouse. The markers described here were found to be highly polymorphic in twelve inbred strains (F344/N, LEW/N, ACI/N, BUF/N, BN/SsN, LOU/MN, MNR/N, MR/N, SHR/N, WBB1/N, WBB2/N, and WKY/N). These polymorphic markers should be useful in genetic linkage studies of important phenotypes in rats.     

The polymorphism of the plasma inter-α-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212

We investigated the ITI protein polymorphism in linkage analysis, usingDraI andSstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI^*1 and ITI^*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented byDraI 4.0 kb andDraI 2.4 + 1.6 kb, and bySstI 6.7 kb andSstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and byDraI 4.0/2.4 + 1.6 kb andSstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1–2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI^*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI^*1 and ITI^*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

Development of a deer mouse whole-genome radiation hybrid panel and comparative mapping of Mus chromosome 11 loci

A 5000-rad whole-genome radiation hybrid cell panel (BW5000) was developed for mapping the deer mouse (Peromyscus maniculatus bairdii) genome. The panel consists of 103 cell lines and has an estimated marker retention frequency of 63.9% (range, 28%–88%) based on PCR typing of 30 Type I (coding gene) and 25 Type II (microsatellite) markers. Using the composite Mus map, Type I markers were selected from six Mus chromosomes, 22 of which are on Mus Chr 11. Fifteen of the Mus Chr 11 markers were simultaneously mapped on an interspecific (P. maniculatus × P. polionotus) backcross panel to test the utility of the radiation hybrid panel, create a framework map, and help establish gene order. The radiation hybrids have effectively detected linkage in the deer mouse genome between markers as far apart as 6.7 cM and resolved markers that are, in the Mus genome, as close as 0.2 Mb. Combined results from both panels have indicated a high degree of gene order conservation of the telomeric 64 cM of Mus Chr 11 in the deer mouse genome. The remaining centromeric portion also shows gene order conservation with the deer mouse but as a separate linkage group. This indicates a translocation of that portion of Mus Chr 11 in P. maniculatus and is consistent with rearrangement breakpoints observed between Mus and other mammalian genomes, including rat and human. Furthermore, this separate linkage group is likely to reside in a chromosomal region of inversion polymorphism between P. maniculatus and P. polionotus.     

Microsatellite markers from a microdissected swine chromosome 6 genomic library

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.     

Segregation of random amplified DNA markers in F1 progeny of conifers

Summary The recently developed approach to deriving genetic markers via amplification of random DNA segments with single primers of arbitrary nucleotide sequence was tested for its utility in genetic linkage mapping studies with conifers. Reaction conditions were optimized to reproducibly yield clean and specific amplification products. Template DNA from several genotypes of Douglas-fir (Pseudotsuga menziesii) and white spruce (Picea glauca) were tested against eight ten-base oligonucleotide primers. Most of the tested primer/parent tree combinations yielded polymorphic PCR products (RAPD markers). Selected primers were then used in PCR reactions with template DNA isolated from offspring in Douglas-fir and black spruce diallel crosses among the same parental lines. The diallel study confirmed the appropriate inheritance of RAPD markers in the F₁ generation. The value of these dominant RAPD markers for genetic linkage mapping in trees was established from both theoretical and applied perspectives.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Isolation of region-specific probes from pig Chromosome 6 by coincidence cloning

Coincidence cloning is a technique that permits the isolation of sequences common to two independent sources of complex DNA, and this method has been used to isolate a set of probes from a region of porcine Chromosome (Chr) 6 containing the loci for glucosephosphate isomerase (GPI) and the skeletal muscle calcium release channel (CRC). Porcine DNA was specifically PCR-amplified from a pigxhamster hybrid cell line containing the centromere region (p1.2–q1.2) of pig Chr 6 and other pig chromosome fragments by use of a porcine SINE specific primer with an EcoRI site in the 5-end. Flow-sorted Chr 6 preparations were amplified with the same SINE primer, but with a SalI site in the 5-end. The products were digested with EcoRI and SalI respectively, combined, denatured, and reannealed. The heteroduplex molecules, containing both an EcoRI and a SalI cohensive end, were selected by cloning in SalI/EcoRI-digested pUC13. Approximately 40% of the primary clones contained a single SalI/EcoRI-insert, indicating that they are coincidence clones. The average insert size was 1.4 kb. Fluorescence in situ hybridization of a pool of 34 coincidence clones to pig chromosomes showed a preferential labeling of the centromere region and of the q2.5–q2.7 region of pig Chr 6. Nineteen coincidence clones were hybridized to SINE-PCR products from flow-sorted pig Chr 6 and to pigxrodent hybrid cell lines. Eighteen clones gave positive signals correlated with the GPI/CRC content of the source DNAs.     

A single linkage group comprising 11 polymorphic DNA markers on rat Chromosome 3

Eleven polymorphic DNA markers were mapped to rat Chromosome (Chr) 3 by linkage analysis of F₂ progeny of F344/N and LEW/N rat strains. The markers, including seven genes and four anonymous loci, formed a single linkage group covering approximately 112 cM with the following order: Ptgs1 (prostaglandin G/H synthase I)-D3Arb178-Scn2a (sodium channel, type II, -polypeptide)-D3Arb1-Cat (catalase)-Bdnf (brain-derived neurotrophic factor)-D3Arb219-D3Arb2-Sus2 (seminal vesicle secretion II protein)-Sdc4 (ryudocan/syndecan4)-Stnl (statin-like protein). Eight of these markers were analyzed for polymorphisms in 14 additional inbred rat strains. Three to five alleles were detected for each marker, suggesting that they are highly polymorphic and useful for genetic mapping studies with inbred rat strains. Chromosomal syntenic conservation among rats, mice and humans is also discussed.     

The positions of 12 simple sequence repeat markers relative to reference loci on mouse Chromosome 16

The genetic map positions of 12 simple sequence repeat (SSR) markers spanning mouse Chromosome (Chr) 16 were determined relative to reference markers on that chromosome. Interval mapping data were obtained with a panel of DNAs from two intersubspecific backcrosses. All but one of the markers were typed by use of nonradioactive polymerase chain reaction (PCR) products analyzed on agarose gels. The marker order was determined to be Prm-1, D16Mit9, Igl-1, D16Mit29, D16Mit1/D16Mit2, Smst, D16Mit4, D16Mit11, Gap43, D16Mit14, D16Mit30, D16Mit5, Pit-1, D16Mit27, D16H21S16 (formerly D21S16h), D16Mit19, App, D16Mit7, Sod-1. Two of these markers mapped to the known human Chr 21 (HSA21)/Chr 16 conserved linkage group. Nine additional SSR markers could not be typed because they were not polymorophic (four markers), did not amplify MOLD/Rk DNA (three markers), or failed to give PCR products under a range of conditions (two markers). A subset of the most robust SSRs provide a useful marker set for the analysis of previously unmapped crosses.     

Characterization of microsatellites from flow-sorted porcine Chromosome 13

Porcine flow-sorted Chromosome (Chr) 13 was PCR amplified with primers based on porcine short interspersed element (SINE) sequences. The product was cloned, gridded in microtiter plates, and screened with a [GT]₁₀ oligonucleotide which gave 45 positive clones. Sequencing of these clones showed that 36 were unique, and 26 [GT]_n microsatellites were characterized. Six other simple repeat sequences, the majority of which were associated with the 3 end of the SINE sequence, were also detected. Twenty-one primers sets were selected, and 13 of these detected useful polymorphisms in the grandparents (n=26) of the European porcine mapping collaboration (PiGMaP) reference families. These 13 markers were mapped in the PiGMaP reference families, and a two-point linkage analysis was performed. The Lod scores indicated that three of the markers were not linked and the remaining 11 formed two linkage groups of two and nine markers respectively. The larger linkage group was also linked to the transferrin locus, permitting assignment of nine markers to porcine Chr 13.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers: X79915, 13N14F; X79916, 13N17R; X79917, 13N18R; X79918, 13N32R; X79919, 13R33R; X79920, 13R33U; X79921, 13R43U; X79922, 13R44R; X79923, 13N03R; X79924, 13N04R; X79925, 13N05R; X79926, 13N06R; X79927, 13N12U     

Mapping of rat Chromosome 2 markers generated from chromosome-sorted DNA

A quantitative trait locus (QTL) for blood pressure has recently been mapped to a region of roughly 30 cM on rat Chromosome (Chr) 2 by linkage and by the use of congenic strains. For further fine mapping of the QTL, however, closely linked chromosome markers residing in this 30-cM region are required. In the current work, 36 new markers were generated by screening rat Chr 2-sorted DNA libraries and subsequently mapped using five F₂ populations. Combining new and existing markers, the marker density for the 30-cM region approaches, on average, one marker per 1.1 cM. Received: 11 April 1997 / Accepted: 12 May 1997     

A genetic linkage map of papaya based on randomly amplified polymorphic DNA markers

A genetic linkage map of papaya (Carica papaya L.) was constructed using randomly amplified polymorphic DNA (RAPD) markers and a F₂ population derived from a University of Hawaii UH breeding line 356 x Sunrise cross. A total of 596 10-mer primers were screened, and 96 polymorphisms were detected. At LOD 4.0, 62 of these markers mapped to 11 linkage groups comprising 999.3 cM. About 80% of the markers conformed to expected Mendelian segregation ratios. We have mapped the locus that determines sex to a 14-cM region flanked by RAPD markers. The results demonstrate the usefulness of RAPD markers for developing a basic genetic linkage map in papaya.Journal series No. 4146 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Mouse genes encoding DNA topoisomerase I

We have initiated a genetic analysis of the physiologically important enzyme type I DNA topoisomerase in mouse. The exon-intron structures of the 5 part and the 3 part of the active gene, Top-1, were determined and shown to be quite similar to those of the previously determined human gene TOP1. The active mouse gene was mapped to the distal Chromosome (Chr) 2. In addition, the mouse genome contains one truncated processed topoisomerase-I-related pseudogene (retroposon), Top-1ps, on Chr 16. The Top-1ps locus, together with the immunoglobulin-lambda-light-chain locus, defines and additional conserved linkage group common to murine Chr 16 and human Chr 22, the site of the human pseudogene TOP1P2. The mapping data suggest that the pseudogene was established before mammalian radiation. Structural features, shared by the mouse and the human pseudogene, support this possibility.     

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations

Twenty-two highly variable SSR markers were developed in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] from five SSR-enriched genomic libraries. Fifteen PCR primer pairs amplified a single codominant locus, while seven primer pairs occasionally amplified two loci. The Mendelian inheritance of all 22 SSRs was confirmed via segregation analyses in several Douglas-fir families. The mean observed heterozygosity and the mean number of alleles per locus were 0.855 (SE=0.020) and 23 (SE=1.6), respectively. Twenty markers were used in genetic linkage analysis and mapped to ten known linkage groups. Because of their high polymorphism and unambiguous phenotypes, 15 single-locus markers were selected as the most suitable for DNA fingerprinting and parentage analysis. Only three SSRs were sufficient to achieve an average probability of exclusion from paternity of 0.998 in a Douglas-fir seed orchard block consisting of 59 parents.Communicated by O. Savolainen     

Deleted Chromosome 20 from a patient with Alagille syndrome isolated in a cell hybrid through leucine transport selection: study of three candidate genes

Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-l) deficient in-leucyl-tRNA synthetase, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, adenosine deaminase (ADA), and Prion protein (PRIP); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes.The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 (HNF3), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.     

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target—recognizing that we would not produce a single chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1:29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome (Chr) 1q12–q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43–q46, near the terminal end.