Polymorphisms revealed by PCR with single,short-sized,arbitrary primers are reliable markers for mouse and rat gene mapping

Ten single, arbitrarily designed oligodeoxynucleotide primers, with 50–70% (G+C) content, were used to amplify by polymerase chain reaction (PCR) sequences with DNA templates from several mouse species (Mus spretus, Mus musculus musculus, and Mus musculus domesticus), as well as DNA from the laboratory rat (Rattus norvegicus). Eight of these ten primers, used either individually or associated in pairs, generated a total of 13 polymorphic products which were used as genetic markers. All of these polymorphic sequences but one were mapped to a particular mouse chromosome, by use of DNA panels prepared either from interspecific backcross progeny of the type (C57BL/6 x Mus spretus)F₁ x C57BL/6 or DNA samples prepared from two sets of recombinant inbred (RI) strains (AKXL and BXD). Six rat-specific DNA segments were also assigned to a particular chromosome with DNA panels prepared from 18 rat/mouse somatic cell hybrids segregating rat chromosomes. From these experiments we conclude that, under precisely standardized PCR conditions, the DNA molecules amplified with these arbitrarily designed primers are useful and reliable markers for genetic mapping in both mouse and rat.     

Gene flow of unique sequences between Mus musculus domesticus and Mus spretus

Allelic diversity has been examined from a variety of Mus musculus subspecies and Mus spretus strains by sequencing at a 453-bp unique sequence locus. One M. m. domesticus classic inbred strain, C57BL/KsJ, contained a sequence identical to that in the M. spretus wild-derived inbred strain SEG, and other wild M. spretus isolates. Such a result should have been precluded by the expected divergence between the species unless there has been interspecies gene flow. Examination of C57BL/KsJ for M. spretus-specific repetitive sequences shows that it is neither a mis-identified spretus strain nor a domesticus/spretus hybrid. Thus, in addition to the previously reported presence of small amounts of Mus spretus-specific repetitive DNA in M. m. domesticus, there is a detectable flow of unique sequence between the two species. There was also ancestral polymorphism observed among the spretus alleles. The difficulty of distinguishing ancestral polymorphism from horizontal transfer is discussed. Received: 14 May 1999 / Accepted: 5 November 1999     

In situ analysis of centromeric satellite DNA segregating inMus species crosses

In situ hybridization of biotin-labeled mouse major satellite DNA clone pMR196 was applied toMus domesticus andMus spretus chromosomes (Chr). The same karyotypes were counterstained with distamycin A-DAPI to identify AT-rich heterochromatin. Chromosomes from the laboratory mouse, C57BL/6Ros (BL/6;M. domesticus) were uniformly labeled at the centromere except for the Y, while chromosomes from the divergentMus speciesM. spretus showed little or no hybridization. Differences betweenMus species in copy number of the major satellite DNA sequence were used to identify chromosomes ofM. domesticus andM. spretus in their F₁ hybrids and to discriminatedomesticus andspretus centromeres in backross progeny. The distribution pattern of heterochromatic regions demonstrated by distamycin A-DAPI counterstaining was comparable with that of in situ hybridization with pMR196, suggesting that A-T rich heterochromatin inM. domesticus is mainly constructed by the pMR196-related sequence. The in situ technique was used to examine segregation ofdomesticus centromeres in backcross progeny obtained by mating F₁ hybrid females withM. domesticus orM. spretus males. The segregation of centromeres did not deviate from the expected among the backcross progeny from C57BL/6Ros males, whereas chromosomes withM. domesticus centromeres were prone to appear in the progeny from backcross matings byM. spretus males.     

A locus on the X Chromosome is linked to body length in mice

Linkage between body length (anus to nose (AN) length) and three markers on the mouse X Chromosome was found in an interspecific backcross ((C57BL/6J×Mus spretus) F1 ×C57BL/6J), designated BSB. A cross of 409 mice were scored for 148 genetic markers distributed on all chromosomes except the Y Chromosome. Statistical analysis revealed highly significant linkage (LOD score 5.5) between body length and a locus in the middle portion of the X Chromosome, the nearest markers being the microsatellite marker DXMit73 and a farnesyl pyrophosphate locus (Fpsl9) 3.1 cM proximal to DXMit73. The locus explains 10% of the variance in AN length and affects both males and females to about the same extent. Received: 17 April 1995 / Accepted: 13 October 1995     

A novel mouse Chromosome 2 congenic strain with obesity phenotypes

Linkage studies have identified many chromosomal regions containing obesity genes in mice. However, only a few of these quantitative trait loci (QTLs) have been used to guide the production of congenic mouse strains that retain obesity phenotypes. We seek to identify chromosomal regions containing obesity genes in the BSB model of spontaneous obesity because the BSB model is a multigenic obesity model. Previous studies identified QTLs on Chromosomes (Chrs) 2, 6, 7,12, and 15. BSB mice are made by backcross of lean C57BL/6J × Mus spretus. F₁s were backcrossed to C57BL/6J mice to produce BSB progeny. We have constructed a new BSB cross and produced congenic mice with obesity phenotypes by marker-directed selection called B6.S–D2Mit194–D2Mit311. We found a highly significant QTL for percentage body lipid on Chr 2 just proximal to the Agouti locus. Chr 2 congenics were constructed to determine whether the main effects would be detectable. We observed highly significant linkage of the Chr 2 congenic containing Agouti and containing markers distal to D2Mit311 and proximal to D2Mit194. Thus, this congenic contains approximately 14.6 cM or 30 Mb (about 1.1% of the spretus mouse genome) and several hundred genes. The obesity phenotype of the QTL is retained in the congenic. The congenic can now be used to model the genetic and physiological basis for a relatively simple, perhaps monogenic, obesity.     

A High-Resolution Map in the Chromosomal Region Surrounding theLpsLocus

TheLpslocus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutantLpsallele (Lps^d) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify theLpsgene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus× C57BL/6J)F1 × C57BL/6J and two novel panels of 597 (DBA/2J × C3H/HeJ)F1 × C3H/HeJ and 748 (C57BL/6J × C3H/HeJ)F1 × C3H/HeJ segregating atLps.A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping theLpslocus. This positions theLpslocus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three known genes (Cd30l, Hxb,andAmbp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of theLpslocus is several centimorgans proximal to that previously assigned.     

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3 end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.     

PCR-analyzed microsatellites: Data concerning laboratory and wild-derived mouse inbred strains

We have investigated 67 primers designed by Dr. J. Todd and co-workers to amplify microsatellites sequences in the mouse. We report on additional polymorphisms concerning seven laboratory inbred strains, complementary to those already published. We include the survey of three independently derived strains of Mus spretus: SPE/Pas, SEG/Pas and SPR/Smh. SPE/Pas and SEG/Pas are very close (3% polymorphism), whereas the third one, (SPR/Smh), is very different from the other two strains (33% polymorphism). Seventy-four to 84% of the microsatellites analyzed in this study are polymorphic between C57BL/6Pas and Mus spretus strains. By comparison, 36–46% are polymorphic between laboratory inbred strains involved in established sets of recombinant inbred strains. A strain derived from Mus musculus musculus (PWK/Pas) was found to be very different from both C57BL/6Pas (70% polymorphism) and SPE/Pas (82% polymorphism). These results emphasize the interest of using Mus musculus musculus inbred strains to establish interspecific crosses, particularly when considering their breeding performances.     

Genetic analysis of testis weight and fertility in an interspecies hybrid congenic strain for Chromosome X

A hybrid congenic strain, C57BL/6J.SPRET-Hprt ^a , carrying 17 map units of Chromosome (Chr) X from Mus spretus on a background of C57BL/6J, has the novel phenotype of low fertility associated with small testis weight. In histological cross-section, many of the tubules in the testes of these congenic mice are empty except for Sertoli cells, while the other tubules appear to be normal. The gene, interspecific hybrid testis weight 1 (Ihtw1) causing this phenotype, has been fine mapped by using the strategy of generating subcongenic strains from recombinants within the congenic region. Genetic and phenotypic analysis of the subcongenic strains has defined a critical region of 1.8 map units for Ihtw1. This region of the genetic map is orthologous to the region on human Chr X containing the gene for the Borjeson-Forssman-Lehman syndrome, an inherited disease in which males show microorchidism. Received: 12 June 2000 / Accepted: 8 September 2000     

Mapping of the Glycoprotein 330 (Gp330) Gene to Mouse Chromosome 2

Glycoprotein 330 (Gp330) is a member of the low-density lipoprotein receptor gene family that is expressed in the kidney. We have mapped the Gp330 gene to mouse chromosome 2, 4.5 cM proximal to Acra, in an interspecific backcross of (C57BL/6J × Mus spretus) F1 × C57BL/6J.     

Genetic mapping of 40 cDNA clones on the mouse genome by PCR

We recently proposed a new PCR-based genetic marker assay for the mouse genome that exploits sequence differences in the 3-untranslated region (UTR) of cDNAs between different mouse strains, called biallelic polymorphic expressed sequence tags (bESTs). The specific use of 3-UTR has several advantages: (1) frequent sequence polymorphism between different mouse strains, (2) most commonly uninterrupted by introns, (3) usually unique sequence even among closely related gene family members. In this paper, we identify additional genetic loci defined by bEST and determine their location on the mouse genetic map by using interspecific backross mapping panels between C57BL/6J and Mus spretus. Of 136 markers tested, 86 produced unique PCR products from C57BL/6J and M. spretus genomic DNAs. We then sequenced these 86 PCR products from C57BL/6J and M. spretus and found that 59 markers have sequence polymorphisms. Of these, we mapped 36 by restriction fragment length polymorphism (RFLP) of the PCR products and 4 by length polymorphism (LP) of the PCR products. We discuss the possibility of a large-scale application of this method for cDNA mapping.     

Fine mapping of Ath6, a quantitative trait locus for atherosclerosis in mice

Ath6 is a novel quantitative trait locus associated with differences in susceptibility to atherosclerosis between C57BL/6J (B6) and C57BLKS/J (BKS) inbred mouse strains. Combining data from an intercross and a backcross (1593 meioses) between mice from B6 and BKS strains and from The Jackson Laboratory interspecific backcross panels, (C57BL/6J ×Mus spretus) F₁× C57BL/6J and (C57BL/6J × SPRET/Ei) F₁× SPRET/Ei, we constructed a consensus genetic map and narrowed Ath6 to a 1.07 ± 0.26 cM interval between the anonymous DNA marker D12Pgn4 and the gene Nmyc1. This region is near the proximal end of murine Chromosome (Chr) 12, which is homologous to the human chromosomal region 2p24-p25. Marker order in the Ath6 region was concordant among the two crosses and The Jackson Laboratory interspecific backcross panels. This high resolution map rules out candidate genes encoding apolipoprotein B, syndecan 1, and Adam17. The two Ath6 crosses have a combined potential resolution of 0.06 cM. Received: 12 September 2000 / Accepted: 22 February 2001     

Mapping of mouse intracisternal A-particle proviral markers in an interspecific backcross

We present a linkage map of intracisternal A-particle (IAP) proviral loci. The IAP family consists of 2000 endogenous proviral elements that are widely dispersed in the mouse genome. The map was constructed by using an interspecific backcross and markers defined by oligonucleotide probes specific for subclasses of expressed IAP elements. In genomic DNA from C57BL/6J mouse, these probes each detected from 12 to 44 HindIII restriction fragments that represent junctions between proviral and 5-flanking DNA. The fragments have characteristic strain distribution patterns (SDPs) that are particularly polymorphic in the DNAs of C57BL/6J and Mus spretus mice used for the backcross. IAP loci were placed on the map by comparison of their distribution patterns with those of known genetic markers in the backcross. The map includes 51 IAP loci that have not been previously mapped and 23 IAP proviruses that had been previously mapped in recombinant inbred (RI) strains. Comparable map positions were obtained with the IAP markers in the interspecific backcross and the RI strains. The mapped IAP loci were widely dispersed on the X Chromosome (Chr) and all of the autosomes except Chrs 9 and 19, providing useful genetic markers for linkage studies.     

Maps from two interspecific backcross DNA panels available as a community genetic mapping resource

We established two mouse interspecific backcross DNA panels, one containing 94 N2 animals from the cross (C57BL/6J × Mus spretus)F₁ × C57BL/6J, and another from 94 N2 animals from the reciprocal backcross (C57BL/6J × SPRET/Ei)F₁ × SPRET/Ei. We prepared large quantities of DNA from most tissues of each animal to create a community resource of interspecific backcross DNA for use by laboratories interested in mapping loci in the mouse. Initial characterization of the genetic maps of both panels has been completed. We used MIT SSLP markers, proviral loci, and several other sequence-defined genes to anchor our maps to other published maps. The BSB panel map (from the backcross to C57BL/6J) contains 215 loci and is anchored by 45 SSLP and 32 gene sequence loci. The BSS panel map (from the backcross to SPRET/Ei) contains 451 loci and is anchored by 49 SSLP loci, 43 proviral loci, and 60 gene sequence loci. To obtain a high density of markers, we used motif-primed PCR to fingerprint the panel DNAs. We constructed two maps, each representing one of the two panels. All new loci can be located with a high degree of certainty on the maps at current marker density. Segregation patterns in these data reveal several examples of transmission ratio distortion and permit analysis of the distribution of crossovers on individual chromosomes.     

Cardiac and skeletal muscle troponin I isoforms are encoded by a dispersed gene family on mouse Chromosomes 1 and 7

We mapped the locations of the genes encoding the slow skeletal muscle, fast skeletal muscle, and cardiac isoforms of troponin I (Tnni) in the mouse genome by interspecific hybrid backcross analysis of species-specific (C57BL/6 vs Mus spretus) restriction fragment length polymorphisms (RFLPs). The slow skeletal muscle troponin I locus (Tnni1) mapped to Chromosome (Chr) 1. The fast skeletal muscle troponin I locus (Tnni2), mapped to Chr 7, approximately 70 cM from the centromere. The cardiac troponin I locus (Tnni3) also mapped to Chr 7, approximately 5–10 cM from the centromere and unlinked to the fast skeletal muscle troponin I locus. Thus, the troponin I gene family is dispersed in the mouse genome. Received: 10 May 1995 / Accepted: 1 September 1995     

High-Resolution Linkage Map of Mouse Chromosome 13 in the Vicinity of the Host Resistance LocusLgn1

Natural resistance of inbred mouse strains to infection withLegionella pneumophilais controlled by the expression of a single dominant gene on chromosome 13, designatedLgn1.The genetic difference atLgn1is phenotypically expressed as the presence or absence of intracellular replication ofL. pneumophilain host macrophages. In our effort to identify theLgn1gene by positional cloning, we have generated a high-resolution linkage map of theLgn1chromosomal region. For this, we have carried out extensive segregation analysis in a total of 1270 (A/J × C57BL/6J) × A/J informative backcross mice segregating the resistance allele of C57BL/6J and the susceptibility allele of A/J. Additional segregation analyses were carried out in three preexisting panels of C57BL/6J ×Mus spretusinterspecific backcross mice. A total of 39 DNA markers were mapped within an interval of approximately 30 cM overlapping theLgn1region. Combined pedigree analyses for the 5.4-cM segment overlappingLgn1indicated the locus order and the interlocus distances (in cM):D13Mit128–(1.4)–D13Mit194–(0.1)–D13Mit147–(0.9)–D13Mit36–(0.9)–D13Mit146–(0.2)–Lgn1/D13Mit37–(1.0)–D13Mit70.Additional genetic linkage studies of markers not informative in the A/J × C57BL/6J cross positionedD13Mit30, -72, -195,and-203, D13Gor4, D13Hun35,andMtap5in the immediate vicinity of theLgn1locus. The marker density and resolution of this genetic linkage map should allow the construction of a physical map of the region and the isolation of YAC clones overlapping the gene.     

Additional microsatellite markers for mouse genome mapping

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.     

Cytological and molecular characterization of centromeres in Mus domesticus and Mus spretus

We have applied EM in situ hybridization (EMISH) and pulsed field gel electrophoresis (PFGE) to samples from diploid primary cell cultures and an established cell line to examine in detail the relative organization of the major and minor satellite DNAs and telomere sequences in the genomes of Mus domesticus and Mus spretus. EMISH localizes the Mus domesticus minor satellite to a single site at the centromere-proximal end of each chromosome. Double label hybridizations with both minor satellite and telomere probes show that they are in close proximity and possibly are linked. In fact, PFGE of M. domesticus DNA digested with Sal I and Sfi I reveals the presence of fragments which hybridize to both probes and is consistent with the physical linkage of these two sequences. The M. domesticus minor satellite is the more abundant satellite in Mus spretus. Its distribution in M. spretus is characterized by diffuse labeling with no obvious concentration near chromosome ends. In addition to this repeat the M. spretus genome contains a small amount of DNA that hybridizes to a M. domesticus major satellite probe. Unlike the M. domesticus minor satellite, it is not telomere proximal but is confined to a domain at the border of the centromere and the long arm. Thus, although both species possess all three sequences, except for the telomeres, their distribution relative to one another is not conserved. Based on the results presented, we propose preliminary molecular maps of the centromere regions of Mus domesticus and Mus spretus.     

A growth QTL (<Emphasis Type="Italic">Pbwg1</Emphasis>) region of mouse chromosome 2 contains closely linked loci affecting growth and body composition

Previous QTL studies have identified 24 QTLs for body weight and growth from 3 to 10 weeks after birth in an intersubspecific backcross mouse population between C57BL/6J and wild Mus musculus castaneus that has 60% of the body size of C57BL/6J. The castaneus allele at the most potent QTL (Pbwg1) on proximal chromosome 2 retards growth. In this study we have developed a congenic strain with a 44.1-Mb interval containing the castaneus allele at Pbwg1 by recurrent backcrossing to C57BL/6J. The congenic mouse developed was characterized by significantly higher body weight gain between 1 and 3 weeks of age and lower weight of white fat pads at 10 weeks of age than C57BL/6J. However, no clear difference in body weight at 1–10 weeks of age was observed between congenic and C57BL/6J strains. QTL analysis with 269 F₂ mice between the two strains did not identify any QTLs for body weight at 1, 3, 6, and 10 weeks of age, but it discovered eight closely linked QTLs affecting body weight gain from 1 to 3 weeks of age, lean body weight, weight of white fat pads, and body length within the Pbwg1 region. The castaneus alleles at all fat pad QTLs reduced the phenotypes, whereas at the remaining growth and body composition QTLs, they increased the trait values. These results illustrate that Pbwg1, which initially appeared to be a single locus, was resolved into several loci with opposite effects on the composition traits of overall body weight. This gives a reason for the loss of the Pbwg1 effect found in the original backcross population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene-environment interaction: a significant diet-dependent obesity locus demonstrated in a congenic segment on mouse Chromosome 7

We have previously reported suggestive evidence for a locus on Chromosome (Chr) 7 that affects adiposity in F₂ mice from a CAST/Ei × C57BL/6J intercross fed a high-fat diet. Here we characterize the effect of a high-fat (32.6 Kcal% fat) diet on male and female congenic mice with a C57BL/6J background and a CAST/Ei-derived segment on Chr 7. Adiposity index (AI) and weights of certain fat pads were approximately 50% lower in both male and female congenic mice than in control C57BL/6J mice, and carcass fat content was significantly reduced. The reduction of fat depot weights was not seen, however, in congenic animals fed a low-fat chow diet (12 Kcal% fat). The congenic segment is approximately 25 cM in length, extending from D7Mit213 to D7Mit41, and includes the tub, Ucp2, and Ucp3, genes, all of which are candidate genes for this effect. Some polymorphisms have been found on comparing c-DNA sequences of the Ucp2 gene from C57BL/6J and CAST/Ei mice. These results suggest that one or more genes present in the congenic segment modulate the susceptibility to fat deposition on feeding a high-fat diet. We were unable to show any significant difference between the energy intakes of the congenic and the control C57BL/6J mice on the high-fat diet. Also, measurements of energy expenditure in male mice at 6 weeks of age, during the first 2 weeks of exposure to the high-fat diet, failed to show any differences between control and congenic animals. Received: 30 September 1998 / Accepted: 22 December 1998