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

~'~EVIEWS

T h e construction of genetic linkage maps is a crucial first step in the structural and functional characterization of mammalian genomes. A recent composite mouse linkage map compiled by Davisson and Roderick 1 shows the chromosomal location of 965 loci and is the result of cumulative efforts of many investigators spanning more than 75 years. The 965 loci represent the locations of phenotypic and biochemical variants, cloned genes and anonymous DNA markers. Until recently, gene assignments in the mouse have tended to rely on meiotic mapping involving the use of two- and three-point; crosses between genetically nonidentical laboratory strains or recombinant inbred (RI) strains 2. Both of these mapping approaches are, however, limited by the difficulty encountered in identifying allelic differences among laboratory strains. This problem has recently been overcome by using interspecific crosses, which exploit the genetic diversity inherent among wild mouse species (reviewed in Ref. 3). Mus spretus represents one of the most distantly related Mus species that interbreeds with c o m m o n laboratory mice and produces fertile F 1 hybrid animals3. Because of this genetic divergence, backcrosses between M. spretus and c o m m o n laboratory mice are rapidly becoming the method of choice of mouse geneticists for generatirrg multilocus linkage maps of the mouse genome (see, for example, Refs 4-18). Almost any desired map resolution can nov," be obtained using interspecific mouse backcrosses. Since multiple loci can now be mapped in relation to each other in the same backcross panel, gene orders and map distances can easily be determined. This overcomes one of the major limitations of the intraspecific composite map where gene order and map distance can often only be inferred, resulting in many unavoidable inaccuracies in the map. One problem that has not yet been fully resolved is how to combine mapping data generated using different interspecific mapping panels. A partial solution to the problem is to include a c o m m o n set of anchor loci among the probes mapped on each panel. Mapping data can then be combined with respect to the anchor loci; however, once again the order of the genes falling between anchor loci can only be inferred w h e n combining data. Our aim here is not to try to combine interspecific mapping data being generated by various different laboratories into a single consensus map: at present, this is an almost impossible task and one that is better left to mouse chromosome committees that have recently been formed for this purpose. Rather, we review" the progress our laboratory has made in the past few years using the interspecific mapping approach to develop a comprehensive molecular genetic linkage map of the mouse genome. Finally, we review some of the many applications for interspecific linkage maps, in general, for future research.

Development of an interspecific linkage map of the mouse genome The interspecific backcross mapping panel we generated was produced from crosses of C57BL/6J and M spretus mice (Fig. 1) C57BL/6J was chosen as the

Development and applications of a molecular genetic linkage map of the mouse gen0me NEAL G. COPELANDAND NANCYA. JENKINS Interspecific mouse backcrosses provide almost limitless genetic variation for gene mapping. We have used interspeciflc backcrosses to develop the flrst 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 pane~ gene order can be determined unambiguously. With this level of resolatiog it is now possible to position any new locus on the linkage map with virtually 10096 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 interspeciflc linkage maps, in genera~ for future research. laboratory strain parent because it is one of the most genetically well-characterized inbred mouse strains. The M. spretus mice we used were nearly inbred, greatly simplifying the mapping of polymorphic markers in backcross (N z) mice. F z animals were produced from crosses of C57BL/6J females and M. spretl~s males, since tile reciprocal cross is less efficienl ;it pr(,ducing hybrid animals. The resulting F 1 males are sterile, but F 1 females are fertile and can bc used to establish the backcross generation. F~ hybrid fiqnales were backcrossed to C57BL/6J males to produce the backcross animals we used for gene mapping (Fig. 1 ). F 1 hybrid females can also be backcrossed to M. sprutus males, but once again this reciprocal cross was found to be less efficient at producing backcross mice. Any number of backcross animals can be produced l~t mapping. For our mapping panel, we produced 20~ backcross mice. Genomic DNA was then isolated tr(,m each mouse for mapping studies. Once backcross DNAs are obtained, how i~ a m<)l ecular genetic linkage map of the mouse gcnomc de,eloped? In our case, we simply obtained a collection (~I probes and sequentially followed their inheritance in backcross progeny by Southern blot hybridization and restriction fragment length polymorphisnl (RFI.P) analysis. Probes to known genes were mapped because they provide the maximum amount of biological information. as described below. Since all backcross animals carrx at least one autosomal C57BL/6J allele for each gene i Fig. 1), RFLPs specific to C57BL/6J can only be scored by hybridization intensity. For this reason, only RFLPs specific to M. spretus were followed in backcr(~s,, :mi reals. In practice, backcross animals were first typed l~r a series of loci that have already been accurately placed on the mouse linkage map by several independunt conventional crosses. These loci served as anchorb 1(,~ placing new genes on the evolving interspccific re:q,

TIC, APRIL1991 VOL 7 .~O. 4

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[~EVIEWS As more and more probes are typed, a picture of the recombination events that occurred along each chrom o s o m e inherited from the (C57BL/6J x M. spretus) F 1 parent is gradually formed. As additional probes are mapped, gene order is easily determined by comparing new segregation patterns with known segregation patterns and minimizing the number of double or multiple crossovers required to explain the new probe distribution. This has been referred to as 'pedigree analysis'3.

Now that more than 600 loci have been placed on the linkage map, several conclusions can be drawn about the usefulness and validity of this approach for gene mapping. The DNA sequence divergence between the genomes of C57BL/6J and M. spretus is very high, so it is necessary to screen only a limited number of restriction endonucleases to detect RFLPs. Interspecific mapping is also fast and efficient. N~'lon filters of DNAs of the backcross progeny cut with each of the enzymes used to detect RFLPs are produced; Salient features of interspecific each filter can be reused at least 20 times. If a probe Over 600 loci have n o w been placed on our interhybridizes well to mouse DNA, it is possible to m a p a specific linkage map (see, for example, Refs 9-11, new probe in as little as five days. Many probes can 14-16, 18, 19). Approximately 45% of these loci have be m a p p e d simultaneously using different sets of been published; the rest should be published in the backcross filters. near future. The locations of these loci on the 19 autoIn general, gene orders and map distances observed somes and X chromosome that comprise the mouse in interspecific and intraspecific crosses do not differ, genetic linkage m a p are shown in Fig. 2. Each hatch suggesting that the genome organization of these two mark indicates the location of at least one locus. In species is very similar. The only documented structural some cases, two or more loci that failed to recombine difference 20 is a small inversion in the proximal region in the backcross panel are indicated by a single hatch of M. spretus chromosome 17 that is absent in laboramark. tory strain chromosome 17. While it is likely that other The total estimated haploid genetic length of the structural differences will be identified, the overall qualmouse genome is 1600 cM1; thus, the current average ity of the interspecific maps generated thus far suggests map resolution is less than 3 cM. The loci span more that these differences will not represent a serious limitathan 83% of the k n o w n genetic map and are fairly well tion to interspecific mapping. Genetic maps for regions distributed over all of the autosomes and the X chromoshowing structural differences can be generated from some (Fig. 2). This marker distribution provides essenintraspecific crosses or other interspecific crosses. tially 100% probability for mapping any new gene. Where measured, more single recombination events and fewer double and multiple recombination events than expected were detected. Recombination interC57BL/6J ~' X Mus spretus ~ ference has also been notedpreviously by others engaged in interspecific and intraspecific mapping 21-z3. The factors responsible for O0 mum recombination interference remain to nn be elucidated. Whatever the cause, mmmm 111 such interference simplifies even further the ordering of genes on the mouse linkage map, since it reduces F1 © C57BL/6J (~ X the probability of multiple crossovers. Finally, transmission ratio distortion was observed for loci on chromosomes 2, 4 and 10 (Refs 10, 16, O0 18). Transmission ratio distortion repIImn resents the nonmendelian segreganmnm ill tion of genes in backcross animals. It mmmm has also been noted previously by others engaged in interspecific backcross mapping13 (.J-L. Gu~net, pets. N2 N2 N2 N2 commun.; D.A. Stephenson and V.M. Chapman, pers. conmaun.), but the particular chromosomes showing C'O O0 • • 5 • transmission ratio distortion seem to ~m mm no differ among the various mapping mm mm panels employed. The factors renn ~m am sponsible for transmission ratio distortion are also not understood but bTGH may result from differential embryo Breeding scheme used to generate the interspecific mouse backcross mapping survival resulting from the action panel. For comparison, only one pair of chromosomes is shown for each parent. of different combinations of M. apretus alleles are indicated by write boxes; C57BL/6J alleles are indicated by C57BL/6J and M. spretus alleles dark boxes. Note that only recombination events occurring in the (C57BL/6J × M. spretus) F1 parent can be scored in N2 backcross mice. during embryonic development.

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Limitations o f interspecific backcross m a p p i n g While interspecific backcrosses involving M. spretus offer one of the most powerful genetic systems yet devised for mapping genes in the mouse, they nonetheless have a few limitations. First, laboratory strain x M. spretus F1 hybrid males are sterile. Consequently, recombination data from only female F 1 mice are obtained, and recombination in the X-Y pseudoautosomal pairing region cannot be measured. This limitation can be overcome if necessary by using other wild mouse species that are more closely related to laboratory strains and produce fertile F l hybrids of both sexes. Second, the amount of DNA that can be obtained from each backcross animal will ultimately limit the number of probes that can be mapped in a single panel. One solution is to derive immortal cell lines from backcross mice to be used as a renewable resource for future mapping studies. A similar approach is being used successfully for human gene mapping. However, except for occasional reports 2., immortal mouse cell lines that are karyotypically stable remain elusive. A second solution is to use one of the polymerase chain reaction (PCR)-based approaches for gene mapping that have recently been described25-E Any of these PCR-based approaches should make it possible to map virtually an unlimited number of loci in any single panel before existing I)NA stocks are depleted.

Applications for interspecific linkage maps Comparative mapping

One of the most important applications of motisc interspecific linkage maps is for comparative m a p p i n g In general, closely linked genes tend to remain linked during ew)lution. Once detailed linkage maps arc obtained for any two species, it is possible to obtain a clear picture of the linkages lhal were ('()nsclwed between the two species during evolution. One cxample of the type of mouse-human comparative maps we have generated cising ()tit panel is sh()~ n in Fig..4 for mouse chrom()somc 2. In till, 59 loci have bcen placed on chromosome 2; 29 loci have been mapped in humans. Tile 29 loci map t(~ six different htlmarl chlomosomcs. As expected, loci that :Ire closely linked in mouse tend to be linked in hulnans. The length ()[ the conserved linkage segments vaD': for chronlosomc 2 they range from 5.5 cM for the segment mapping to human chromosome 15, to 32.5 cM for the s0gment mapping to human chromosome 20 (Fig. 3). Nadeau and Reiner> have estimated that the mean length of all conserved autosomal segments in ~l~> mouse genome is 10.4 + 2.4 cM. and that 13q + 3q disruptions have occurred since the ctivergencc ~f lineages leading to mice and humans. To datc, approximately 90 consetxc.d segments have been identified3(, (N.G. Copeland et al.. unpublished) \X'hen all m o u s e - h u m a n conserved segments arc idcnliticd and characterized, it will be possible t(~ p r e d i c t ir~

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III!EVIEWS almost every case the location of a gene in one species, solely from its location in the other species. One important application of comparative maps lies in their ability to predict the order of closely linked human genes. In humans, it is often difficult to order closely linked genes since each gene is polymorphic in only a subset of individuals. However, in the mouse it is easy to order closely linked genes since every interspecific backcross animal is polymorphic for every probe tested. Mapping information gained in the mouse can therefore be used to assign a tentative gene order in humans. O Physical mapping methods or other methods can then be used to confirm these predictions. 10p13 Comparative maps can also be used to identify new mouse models of human disease. Probes that are closely linked to known human genetic diseases can be 9q33-q34 - mapped in mouse to determine if 9q34, 9q34.1 they map in a conserved segment that contains a mouse 9q32-q34 mutation with phenotype similar to that produced by the human genetic disease. A number of 2qal-q32 mouse models of human disease have already been identified by this comparative mapping 2q36-q37 approach37,38.

is to map their chromosomal location to determine if they are allelic with any previously identified mouse mutation. Several applications of this approach have already been published39--44. A large number of interesting mutations have been identified and cloned in other model organisms. Many of the genes encoding these mutations are evolutionarily well conserved and can be used to identify .and clone related genes in the mouse. By mapping their chromosomal location in mouse, it should be possible to identify mouse mutations that are encoded by some of these genes. The usefulness of this approach was recently demonstrated when Vim it was shown that the mouse undulated mutation, which maps to mouse chromosome 2, is encoded by the mouse homologue of the Drosophila paired box gene Pax-1 (Ref. 45). Spna-2

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Mapping and cloning genes corresponding to known mutations Many hundreds of biologically interesting mutations have been identified in mouse, yet relatively few of these mutations have been cloned and studied at the molecular level. The development of high resolution mouse molecular genetic linkage maps will greatly increase the speed at which these mutations are cloned, either by identifying candidate genes for mouse mutations through cosegregation and allelism studies, or by providing closely linked molecular markers for chromosome walking or jumping experiments. Within the past few years, a number of laboratories have begun to use retroviral insertional mutagenesis or DNA microinjection to induce new developmental mutations in the mouse germ line. An important first step in characterizing these mutations

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Mapping new uncloned mouse mutations The chromosomal location of uncloned mouse mutations can be determined by crossing mutant laboratory strains with M. spretus and typing the backcross progeny with an optimally designed set of probes chosen from the many loci already typed in interspecific hybrids. The strain carrying the mutation does not have to be typed for polymorphic loci, since one follows only the segregation of known RFLPs specific to M. spretus and the mutant phenotype in interspecific backcross mice. Once the chromosomal location of the mutant gene is assigned, additional probes in the region can be used to refine further the position of the mutant gene, using the same set of interspecific DNAs. The molecular probes used to refine the map location may turn out to be .allelic with the new mutation or may map near enough to be used to initiate chromosome walks or jumps designed to clone the mutation of interest. This mapping approach can be augmented by the use of repetitive probes. Only a few repetitive probes are theoretically needed to detect linkage across

HGID lnterspecific backcross linkage map of mouse chromosome 2. Only loci that have been mapped in both human and mouse chromosomes are included. The map po~iitionof each locus in human chromosomes is shown on the left of the chromosome. Solid bars represent portions of chromosome 2 showing synteny with human chromosomes. Loci have been centered on the chromosome; no correlation be~een the centromere and most proximal marker or the telomere and most distal marker is implied. TIG APRIL1991 VOL. 7 ,XO. 4

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the entire mouse genome. A 9 10 1 2 3 4 5 6 7 8 number of such repetitive ql probes have already been )-Odc-I : T(Mtv'33)t Tpi-3 reported 4~>4~. The mapping -Tpi-4 panel we have established Tpi-5 t Mtv-3( provides a powerful tool for Tpi-e Tpi-Z identifying and mapping new Odc-4 repetitive probes, since all Tpi-2 rMtv-34I Odc-7 polymorphic loci identified with each new repetitive probe =oac-b I can be mapped using this panel. As an example, we have I Ode-2 recently used our mapping panel to identify arid chhracterize three repetitive probes that collectively identify 28 distinct loci. These 28 loci can 11 12 13 14 15 16 17 18 19 X detect linkage over 70% of the mouse genome in as few as Tpi-9 Mtv.36l -Odc 100 backcross micem (Fig. 4). These probes include a mouse Odc'l=~ l Tp*-8 mammary tumor vires (MMTV) envelope (env) probe, an .0dc-13 ornithine decarboxylase (Odc) Mtv-37 .Mtv-38 probe, and a triose-phosphate isomerase (Tp~) probe. One limitation o f . most repetitive probes is their limited strain distribution. For 10 cM example, the repetitive probes we have identified are only useful in the context of interLinkage map showing the approximate locations of Mtv, Ode and Tpi loci on mouse specific crosses involving M. chromosomes. The positions o f these loci on mouse chromosomes w a s determined as spretus. Likewise, proviral and described 19. Chromosomal regions ( _+40cM) swept by scoring 100 backcross animals for pseudogene probes are preslinkage ;ire indicated by thickened lines. The loci in parentheses were not fixed in the 11 ent in some strains and spretus parents at the time the interspecific backcross was performed t.llh,-29, .lltv-31 and species but not in others. MIv-33). (Reproduced, with permission, from Ref. 19.) Interspecific crosses will also facilitate the mapping and ckming of loci affecting polygenic traits. The only hemizygosity of polymorphic alleles in tumors criterion is that the inbred strain and wild mouse (reviewed in Ref. 51). In mice, this approach has been species differ in the polygenic trait to be analysed. The problematic, since most murine tumors studied to date power of this approach was recently demonstrated by have occurred on relatively uniform host genetic' back Paterson et al. t9 who used it to identify loci affecting grounds. This problem can be alleviated by taking the concentration of soluble solids and fruit pH in advantage of the genetic diversity inherent in inter interspecific crosses of tomato. specific crosses. By screening tumors that arise in interspecific F l hybrid mice for reduction of polymorphk Searching fi)r new proto-oncogenes in murine tumors alleles to hom
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~'~EVIEWS yeast artificial chromosome (YAC) contigs greater than 1.5 Mb 54, this resolution should provide sufficient marker density for initiating construction of an overlapping set of YAC contigs for the mouse genome. At the 95% confidence interval, 1 cM resolution requires that 300 backcross animals be typed for approximately 4800 loci, assuming that recombination breakpoints/loci are randomly distributed throughout the mouse genome. At the 99% confidence interval, 500 backcross animals need to be typed for 8000 loci. Either level of resolution is easily obtainable by interspecific mapping. The creation of a 1 cM mouse map is thus a readily attainable goal in the near furore. The development of a 1 cM linkage map will facilitate physical mapping of the mouse genome and the cloning of virtually any mutation in the mouse. By cloning genes encoding mouse mutations homologous to known human diseases, a plethora of new models for human disease will be developed. The isolation and ordering of a set of YAC contigs that span the genomc will facilitate both studies employing homologous recombination and embryonic stem cells to create new mutations in the mouse germ line, and the eventual sequencing of the mouse genome, All in all, the future for mouse genetics seems bright and we look forward to the many interesting biological discoveries that are likely to come from this research in the years to come.

Acknowledgements We thank M. Bedell, A. Buchberg, M. Justice, D. Kingsley, J. Mercer, L. Siracusa and S. Spence for their comments on the manuscript, D. Gilbert, B. Cho, D. Angle, D. Swing, J. Dietz and B. Eagleson for excellent technical assistance, and L. Bmbaker for typing the manuscript. We especially thank the hundreds of investigators who have generously shared their probes, which made this mapping project possible. This research was supported by the National Cancer Institute, DHHS, under Contract NO1-CO-74101 with ABL.

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15 Justice, M.J. et al. (1990) Genomics 6, 341-351 1 6 Justice, M.J. et al. (1990) Genetics 125, 855-866

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"rig APRIL1991 VOL. 7 NO. 4