Development and applications of a molecular genetic linkage map of the mouse genome

Interspecific mouse backcrosses provide almost limitless genetic variation for gene mapping. We have used interspecific backcrosses to develop the first comprehensive molecular genetic linkage map of the mouse genome. More than 600 loci have been positioned on the map; the current average map resolution is less than 3 cM. Since all loci were mapped using a single backcross panel, gene order can be determined unambiguously. With this level of resolution, it is now possible to position any new locus on the linkage map with virtually 100% certainty. In this article, we review how interspecific linkage maps are constructed, the salient features of our linkage map, and some of the many applications of interspecific linkage maps, in general, for future research.     

A New Gene Mapping Resource: Interspecies Hybrids between Pere David''s Deer (Elaphurus Davidianus) and Red Deer (Cervus Elaphus)

Three male F(1) hybrids between Pere David's deer and red deer were mated to red deer to produce 143 backcross calves. The pedigrees are a rare example of a fertile between evolutionarily divergent species. We examined the use of these families for genetic mapping of evolutionarily conserved (Type I) loci by testing for genetic linkage between five species-specific protein variants and 12 conserved DNA probes. Two probes were homologous, and the remainder syntenic, to the protein coding loci in cattle or humans. Using six restriction enzymes, each DNA probe detected one or more restriction fragments specific to Pere David's deer. Linkage analyses among the species-specific variants placed the loci into four linkage groups within which linkage between adjacent loci and gene order was supported by a LOD > 3. The linkage groups were (HPX, HBB)-FSHB- ACP2, LDHA-CD5-IGF2, BMP3- (GC, ALB)-(KIT, PDGFRA) and LDLR-C3-FGF1. Southern and protein analysis of LDHA and ALB provided identical segregation data. These linkage groups were consistent with the cattle gene map and provide new information for comparing the gene maps of ruminants, humans and mice. The deer hybrids are an important new resource that can contribute to the comparative analysis of the mammalian genome.     

Mapping of body weight loci on mouse Chromosome X

Inheritance of overweight in humans appears to be under polygenic control. Study on the mouse model may help to determine candidate regions in human genome for the search of overweight genes. Inbred mouse strains showed wide variation in body weight and can provide an experimental model for the study of inheritance of overweight. By genetic linkage analysis, we report the mapping of two loci, named Bw1 and Bw2 (body weight 1 and 2), on Chromosome (Chr) X that strongly affect adult body weight in two interspecific testcross male populations (HSB and ASB) of mice. In addition, another locus, named Bw3, is also mapped on Chr X in ASB populations. These loci account for up to 24% of the phenotypic variation in both populations. Considering the conserved synteny between mouse and human Chr X, these results provide candidate regions on Chr X that can be tested for linkage with overweight in humans.     

Conserved vertebrate chromosome segments in the large salamander genome

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.     

A gene map of the rat derived from linkage analysis and related regions in the mouse and human genomes

We report the localization by linkage analysis in the rat genome of 148 new markers derived from 128 distinct known gene sequences, ESTs, and anonymous sequences selected in GenBank database on the basis of the presence of a repeated element. The composite linkage map of the rat contributed by our group integrates mapping information on a total of 370 different known genes, ESTs, and anonymous mouse or human sequences, and provides a valuable tool for comparative genome analysis. 206 and 254 homologous loci were identified in the mouse and human genomes respectively. Our linkage map, which combines both anonymous markers and gene markers, should facilitate the advancement of genetic studies for a wide variety of rat models characterized for complete phenotypes. The comparative genome mapping should define genetic regions in human likely to be homologous to susceptibility loci identified in rat and provide useful information for the identification of new potential candidates for genetic disorders. Received: 2 January 1999 / Accepted: 7 March 1999     

Genetic mapping through the use of synthetic tandem repeats in the mouse genome

Polymers of arbitrary oligonucleotides can be used to detect polymorphic loci in a wide range of vertebrate genomes. Using 60 such probes, we previously reported the selection of the most efficient STR probes for polymorphism detection in the set of genomes investigated. We now report the use of this selection for the mouse genome and its contribution to genetic mapping. Twenty-three synthetic tandem repeats (STRs) sequences were probed on a recombinant inbred panel C57B1/6 x DBA/2. The loci detected are distributed in 70 linkage groups; 42 of these groups, corresponding to about 100 different polymorphic loci, include reference markers. These linkage groups appear to be evenly distributed within all the 20 mouse chromosomes with apparently no bias of repartition towards telomeres or centromeres.     

Genetic mapping and QTL analysis of fiber-related traits in cotton (<Emphasis Type="Italic">Gossypium</Emphasis>)

Cotton, the leading natural fiber crop, is largely produced by two primary cultivated allotetraploid species known as Upland or American cotton (Gossypium hirsutum L.) and Pima or Egyptian cotton (G. barbadense L.). The allotetraploid species diverged from each other and from their diploid progenitors (A or D genome) through selection and domestication after polyploidization. To analyze cotton AD genomes and dissect agronomic traits, we have developed a genetic map in an F₂ population derived from interspecific hybrids between G. hirsutum L. cv. Acala-44 and G. barbadense L. cv. Pima S-7. A total of 392 genetic loci, including 333 amplified fragment length polymorphisms (AFLPs), 47 simple sequence repeats (SSRs), and 12 restriction fragment length polymorphisms (RFLPs), were mapped in 42 linkage groups, which span 3,287 cM and cover approximately 70% of the genome. Using chromosomal aneuploid interspecific hybrids and a set of 29 RFLP and SSR framework markers, we assigned 19 linkage groups involving 223 loci to 12 chromosomes. Comparing four pairs of homoeologous chromosomes, we found that with one exception linkage distances in the A-subgenome chromosomes were larger than those in their D-subgenome homoeologues, reflecting higher recombination frequencies and/or larger chromosomes in the A subgenome. Segregation distortion was observed in 30 out of 392 loci mapped in cotton. Moreover, approximately 29% of the RFLPs behaved as dominant loci, which may result from rapid genomic changes. The cotton genetic map was used for quantitative trait loci (QTL) analysis using composite interval mapping and permutation tests. We detected seven QTLs for six fiber-related traits; five of these were distributed among A-subgenome chromosomes, the genome donor of fiber traits. The detection of QTLs in both the A subgenome in this study and the D subgenome in a previous study suggests that fiber-related traits are controlled by the genes in homoeologous genomes, which are subjected to selection and domestication. Some chromosomes contain clusters of QTLs and presumably contribute to the large amount of phenotypic variation that is present for fiber-related traits.Communicated by J. Dvorak     

A molecular genetic linkage map of mouse chromosome 4 including the localization of several proto-oncogenes

We have constructed a 64-cM molecular genetic linkage map of mouse chromosome 4 using interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. Several proto-oncogenes and common sites of viral integration have been assigned regional locations on chromosome 4 including Mos, Lyn, Jun, Lmyc, Lck, Fgr, and Dsi-1. Additional loci mapped in this study to chromosome 4 were Tsha, Mup-1, Rrm2-ps1, Ifa, and Anf. A comparison of our mapping data with inbred strain mapping data did not show any evidence for inversions or deletions on chromosome 4. New regions of synteny were defined between mouse chromosome 4 and human chromosomes 1 and 8; a region of homology was found between mouse chromosome 4 and human chromosome 6. This linkage map will provide a framework for identifying homologous genes in mice and humans that may be involved in various disease processes.     

Detailed comparative gene map of rat chromosome 1 with mouse and human genomes and physical mapping of an evolutionary chromosomal breakpoint

We report the localization of 92 new gene-based markers assigned to rat chromosome 1 by linkage or radiation hybrid mapping. The markers were chosen to enrich gene mapping data in a region of the rat chromosome known to contain several of the principal quantitative trait loci in rodent models of human multifactorial disease. The composite map reported here provides map information on a total of 139 known genes, including 80 that have been localized in mouse and 109 that have been localized in human, and integrates the gene-based markers with anonymous microsatellites. The evolutionary breakpoints identifying 16 segments that are homologous regions in the human genome are defined. These data will facilitate genetic and comparative mapping studies and identification of novel candidate genes for the quantitative trait loci that have been localized to the region.     

A Genetic Linkage Map of the Mouse Using Restriction Landmark Genomic Scanning (Rlgs)

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyxed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it used end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping.     

Construction of a genetic linkage map in man using restriction fragment length polymorphisms.

We describe a new basis for the construction of a genetic linkage map of the human genome. The basic principle of the mapping scheme is to develop, by recombinant DNA techniques, random single-copy DNA probes capable of detecting DNA sequence polymorphisms, when hybridized to restriction digests of an individual's DNA. Each of these probes will define a locus. Loci can be expanded or contracted to include more or less polymorphism by further application of recombinant DNA technology. Suitably polymorphic loci can be tested for linkage relationships in human pedigrees by established methods; and loci can be arranged into linkage groups to form a true genetic map of "DNA marker loci." Pedigrees in which inherited traits are known to be segregating can then be analyzed, making possible the mapping of the gene(s) responsible for the trait with respect to the DNA marker loci, without requiring direct access to a specified gene's DNA. For inherited diseases mapped in this way, linked DNA marker loci can be used predictively for genetic counseling.     

Isolation, chromosomal localization, and nucleotide sequence of the human HOX 1.4 homeobox

We have isolated a 14-kb DNA sequence containing a single homeobox from a low-stringency screen of a human genomic phage library by using heterologous homeobox sequences as probes. Chromosomal mapping of this clone using in situ hybridization to metaphase chromosomes and a panel of mouse x human somatic cell hybrids localized it to human chromosome 7p13-p15 in the region of the HOX 1 locus. We have sequenced the homeobox and show it has 100% identity to the deduced amino acid sequence of the mouse Hox-1.4 homeobox. We detect no restriction fragment length polymorphisms with the 14-kb clone, which is devoid of any moderately repetitive DNA sequences. This implies an inability of this region to tolerate change in sequence, consistent with a function highly conserved throughout evolution. The regions in the human genome where homeobox-containing loci reside share patterns of organization and sequence and have other gene loci in common, implying evolutionary constraints over these regions and providing clues on how they may have evolved.     

Loci affecting long-term hybrid survivorship in Louisiana irises: implications for reproductive isolation and introgression

Iris fulva and I. brevicaulis are long-lived plant species known to hybridize where they coexist in nature. Year-to-year survival contributes significantly to overall fitness for both species and their hybrid derivatives, and differences in hybrid survivability may have important consequences to interspecific gene flow in nature. We examined the genetic architecture of long-term survivorship of reciprocal backcross I. fulva x I. brevicaulis hybrids in a common-garden, greenhouse environment. Differences in mortality were found between the two backcross (BC1) hybrid classes, with hybrids crossed toward I. fulva (BCIF) revealing twice the mortality of those hybrids backcrossed toward I. brevicaulis (BCIB). Using genomic scans on two separate genetic linkage maps derived from the reciprocal hybrid populations, we found that hybrid survivorship is influenced by several genetic regions. Multiple interval mapping (MIM) revealed four quantitative trait loci (QTLs) in BCIF hybrids that were significantly associated with survivorship. Introgressed I. brevicaulis DNA increased survivorship at three of the four QTLs. For the fourth QTL, introgressed I. brevicaulis DNA was associated with decreased survivorship. No QTLs were detected in BCIB hybrids; however, single-marker analysis revealed five unlinked loci that were significantly associated with survivorship. At all five markers, survivorship was positively associated with introgressed I. fulva DNA. The present findings have important implications for the evolutionary dynamics of naturally occurring hybrid zones. Regions of the genome that increase survivorship when in a heterozygous (i.e., hybrid) state should have an increased likelihood of passing across species boundaries, whereas those that decrease survivorship will be less likely to introgress.     

Construction of a detailed molecular map of the mouse X chromosome by microcloning and interspecific crosses.

A large number of microclones obtained by microdissection of the mouse X chromosome have been mapped using an interspecific Mus domesticus/Mus spretus cross. Clones displaying close linkage to a number of loci of known phenotype but unknown gene product, such as mdx (X-linked muscular dystrophy), have been obtained. Over a central 30 cM span of the mouse X chromosome, 17 clones have been mapped and ordered at a sufficient density to contemplate the complete physical mapping of this region that will aid in the isolation of a number of unidentified genes. Some of the mapped microclones detect moderately repetitive sequences that were clustered in several discrete regions of the mouse X chromosome.     

Mapping genetic factors controlling pollen viability in an interspecific cross in Helianthus sect. Helianthus

Segregation of 48 genetic markers, including one CMS restorer gene, one morphological character gene, six isozymes and 40 RAPD loci, was scored in a backcross progeny of an interspecific hybrid H. argophyllusxH. annuus cv RHA274. A linkage map was generated taking into account segregation distortions for 11 of the 48 loci in the frame of two different models considering locus-pair segregation in the context of either independent selection pressures or non-equilibrated parental classes. The map consists of nine linkage groups and nine isolated markers covering 390 cM. Approximately half of the plants of the BC1 were male fertile as expected for the segregation of one dominant male-fertility restorer gene; however, these displayed a large range of variation for pollen viability. About 80% of this variation was explained by three genomic regions located on linkage groups 1, 2 and 3. The observation of meiotic chromosomes revealed a significant rate of mispairing (rod bivalents and tetravalents) in tight correlation with pollen viability, indicating that chromosome rearrangements (translocations) are the preponderant factors reducing pollen viability in this progeny. Cytogenetic and mapping data suggest that the three genomic regions involved in pollen-viability variation are located close to translocation points which differentiate the parental-species karyotypes. Segregation distortion was observed for loci correlated with pollen-viability variation. These were most likely the result of two possible suggested mechanisms.     

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.     

Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus x M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11-15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.     

Genetic mapping in the region of the mouse X-inactivation center

The mouse X-inactivation center lies just distal to the T16H breakpoint. Utilizing pedigree analysis of backcross progeny from a Mus domesticus/Mus spretus interspecific cross, we have mapped a number of genetic loci, gene probes, microclones, and EagI linking clones distal to the T16H breakpoint. The genetic analysis provides a detailed genetic map in the vicinity of the mouse X-inactivation center. Comparative mapping data from the human X chromosome indicate that the most probable location of the mouse X-inactivation center is distal to Ccg-1 and in the region of the Pgk-1 locus. We report the assignment of two new loci, EM13 and DXSmh44, to the Ccg-1/Pgk-1 interval.     

Chromosome structural changes in diploid and tetraploid A genomes of Gossypium.

The genus Gossypium, which comprises a divergent group of diploid species and several recently formed allotetraploids, offers an excellent opportunity to study polyploid genome evolution. In this study, chromosome structural variation among the A, At, and D genomes of Gossypium was evaluated by comparative genetic linkage mapping. We constructed a fully resolved RFLP linkage map for the diploid A genome consisting of 275 loci using an F2 interspecific Gossypium arboreum x Gossypium herbaceum family. The 13 chromosomes of the A genome are represented by 12 large linkage groups in our map, reflecting an expected interchromosomal translocation between G. arboreum and G. herbaceum. The A-genome chromosomes are largely collinear with the D genomes, save for a few small inversions. Although the 2 diploid mapping parents represent the closest living relatives of the allotetraploid At-genome progenitor, 2 translocations and 7 inversions were observed between the A and At genomes. The recombination rates are similar between the 2 diploid genomes; however, the At genome shows a 93% increase in recombination relative to its diploid progenitors. Elevated recombination in the Dt genome was reported previously. These data on the At genome thus indicate that elevated recombination was a general property of allotetraploidy in cotton.     

Beyond pathways: genetic dissection of tocopherol content in maize kernels by combining linkage and association analyses

Although tocopherols play an important role in plants and animals, the genetic architecture of tocopherol content in maize kernels has remained largely unknown. In this study, linkage and association analyses were conducted to examine the genetic architecture of tocopherol content in maize kernels. Forty‐one unique quantitative trait loci (QTLs) were identified by linkage mapping in six populations of recombinant inbred lines (RILs). In addition, 32 significant loci were detected via genome‐wide association study (GWAS), 18 of which colocalized with the QTLs identified by linkage mapping. Fine mapping of a major QTL validated the accuracy of GWAS and QTL mapping results and suggested a role for nontocopherol pathway genes in the modulation of natural tocopherol variation. We provided genome‐wide evidence that genes involved in fatty acid metabolism, chlorophyll metabolism and chloroplast function may affect natural variation in tocopherols. These findings were confirmed through mutant analysis of a particular gene from the fatty acid pathway. In addition, the favourable alleles for many of the significant SNPs/QTLs represented rare alleles in natural populations. Together, our results revealed many novel genes that are potentially involved in the variation of tocopherol content in maize kernels. Pyramiding of the favourable alleles of the newly elucidated genes and the well‐known tocopherol pathway genes would greatly improve tocopherol content in maize.