WO1997031011A1 - Microsatellite markers for identifying canine genetic diseases or traits - Google Patents

Abstract

Microsatellite markers are provided which are useful in identifying linked markers for canine genetic diseases and traits. The microsatellite markers are derived from regions of genomic DNA which contain a repeat motif, flanked by unique sequences. The number of units contained within the repeat motif is variable, such that various different alleles are present in any given population. The microsatellite markers and their progeny are especially useful in detecting genetic diseases not phenotypically visible and identifying carriers of recessive diseases, as illustrated in the figure. In a preferred embodiment, microsatellite markers are provided which may be used to detect the canine copper toxicosis gene.

Description

MICROSATELLITE MARKERS FOR IDENTIFYING

CANINE GENETIC DISEASES OR TRAITS

FIELD OF THE INVENTION

This invention relates generally to genetic markers and methods of making and using such markers, and more particularly, to a microsatellite marker that may be used to detect copper toxicosis in canines.

BACKGROUND OF THE INVENTION

Due to inbreeding and the relatively shallow gene pool, a large number of genetic diseases are present in dogs (Clark, R. D. et al Medical and Genetic Aspects of Purebred Dogs (Forum Publications, Fairway, KS) (1994) and Robinson, R. , Canine Pract. 16: 29-34 (1991)) . Some of these genetic diseases such as copper toxicosis in the Bedlington terrier breed, are so prevalent in a particular breed that the mutant allele frequency may be higher than that of the normal allele (Herrtage, M . E. et al ., J. Small Anim. 28: 1141-1151 (1987); and Yuzbasiyan-Gurkan, V. et al. , Genomics 15: 86-90 (1993)). Other genetic diseases cross many breeds, as exemplified by progressive retinal atrophy causing blindness (Barnett, K. C. , Adv. Vet. Sci. Comp. Med. 20: 9-67 (1976)) and hip dysplasia resulting in painful and crippling arthritis (Corley, E . A. , Small Anim. Pract. 22: 570-593 (1992)).

Canine copper toxicosis (CT) is an autosomal recessive genetic disorder of copper accumulation which results in severe liver damage. Unless specific anti- copper treatment is instituted, affected dogs die by three to seven years of age. While reported in several breeds, it is best characterized in Bedlington terners with the frequency of the defective gene being estimated at 50%. The disease is also prevalent in the West Highland White Terrier and Keeshond.

Currently, the only method for diagnosing affected CT dogs is by a quantitative liver copper assay in a liver biopsy sample, after one year of age. Unfortunately, heterozygous and homozygous normal animals are indistinguishable from each other by this test. In order to determine if a dog is a heterozygous earner, test-breeding strategies must be employed which require that there be a dog of a known genotype to breed against the potential earner. This process is very costly and results in the birth of many affected individuals. It is therefore impractical for breeders to identify breeding stock free of the gene and currently earners of the gene are only identified after they are found to be the parents of an affected dog.

Because like CT, many of the canine genetic diseases are recessive, various methods have been investigated which would identify, on a molecular level, phenotypically normal carriers. One method that has been employed is the whole gene subtraction method. This approach requires the sorting out of differences between DNA from those with or without the disease or trait with molecular manipulation methods. Unfortunately, this technique is somewhat impractical and requires that all variability within individuals with the trait as well as the variability within those without the trait independent of the trait, be differentiable from the one or few that are dependent on the trait. Furthermore, this method has only been demonstrated on very simple organisms such as yeast, and while this technique appears theoretically possible for higher species, it rapidly becomes impractical, as it requires many breeding studies of large numbers of affected animals.

An alternative method, the use of restriction fragment Iength polymorphisms

(RFLP), is extremely labor intensive and expensive with respect to both characterization and analysis. Furthermore, this technique requires large quantities of DNA, generally is limited to only two alleles, and only a few loci have thus far been characterized for the canine genome. It appears that with this method, a separate genetic system must be generated for each breed of dog, and such a library may not be sufficiently variable in most situations of interest.

The randomly amplified DNA fragment Iength polymorphism (RAPD) approach uses random primers to amplify fragments of genomic DNA that vary from individual to individual within a species. While the primers are relatively easy to generate, the method is unreliable with minor experimental changes resulting in the resolution of different DNA band patterns. Furthermore, only a few such bands have been characterized for the canine genome.

The candidate gene method is another alternative wherein one or more candidate genes is identified based on what is known about the biochemical and clinical or other phenotypic attributes of the disease or trait and information about similar conditions in another species where a gene has been identified for a similar trait. This approach was taken in evaluating genes linked to the Wilson's disease gene in humans, a disease similar to CT. Unfortunately, the genes linked to the Wilson's disease in humans were not linked to CT in dog (Yusbasiyan-Gurkan, V . et al., Genomics 15:86-90 (1993)). Thus, even under the best-case scenario, the candidate gene method is merely a guess and the approach is of course, further limited by the availability of identified genes.

Because canine pedigrees for various genetic disease are abundant, with several generations and two or more affected members present in many cases these pedigrees lend themselves to linkage studies, provided polymorphic markers are available. Since most of the breeding is controlled, identification of linked markers would allow concerned breeders to greatly reduce the incidence of these diseases in future generations.

One type of marker that has been developed consists of simple sequence Iength polymorphisms (SSLPs). SSLPs arise from a varying number of repeats of a simple sequence, such as a dinucleotide repeat at a given locus, and have been reported to be frequent in most eukaryotic genomes (Tautz, D. et al., Nucleic Acids Res. 12:4127-4138 (1984)). Such loci, also referred to as microsatellites (Tautz, D., EXS: DNA Fingerprinting: State of the Science 1 :21-28 (1993)), are best exemplified by those containing the (CA)_n motif and are found to be highly polymorphic in many species and are being successfully used in the construction of genetic maps of the human (Weissenbach, J. et al., Nature 359:794-801 (1992)), mouse (Dietrich, W. et al., Genetics 131 :423-477 (1992)), rat (Serikawa, T. et al., Genetics 131 :701-721 (1992)) and bovine (Barendse, W. et al., Nat. Genet. 6:227-235 (1994)) genomes. High polymorphic information content and amenability to analysis by polymerase chain reaction (PCR) and thus to possible automation, make microsatellites excellent linkage and mapping tools.

CA microsatellites from the canine genome have been identified and their polymorphism evaluated on sets of unrelated dogs (Holmes, N.G. et al., Anim. Genet. 24:289-292 (1992)) or mixed bred dogs and beagles (Ostrander, E.A. et al., Genomics. 16:207-213 (1993)). Presently there are about 150 SSLP-type markers for the canine genome available. Unfortunately, these known markers lack the ability to detect a linked marker for any genetic trait, because of the low probability of finding a linked marker sufficiently close to a given genetic locus, to ensure detection. Many purebred dog populations have a relatively high level of inbreeding which makes it important that such markers be very polymorphic. Further, important genetic diseases occur across many dozens of breeds, requiring the markers be polymorphic in most, if not all, breeds with many different breeds having varying sets of genetic problems.

It would thus be desirable to provide a method for identifying genetic diseases and traits in canines. It would also be desirable to provide a method for identifying genetic diseases and traits in canines which has high variability and low breed specificity. It would further be desirable to provide a method which allows breeders to select and breed for certain favorable characteristics, or conversely, to avoid unfavorable diseases and traits. It would further be desirable to provide a method which allows the detection and screening of a recessive genetic disease such as copper toxicosis, which is phenotypically undetectable in heterozygote carriers. It would further be desirable to provide a method for identifying a carrier of a genetic disease or trait and affected individuals without undergoing test-breeding experiments. It would also be desirable to provide genetic markers for the canine genome. It would further be desirable to provide a marker for the CT gene in canines .

SUMMARY OF THE INVENTION

A set of microsatellite markers are provided which are useful in identifying linked markers for canine genetic diseases and traits. In particular, five hundred and nineteen microsatellite DNA markers are provided which are highly variable within and across many breeds of dogs. The microsatellite markers are derived from regions of genomic DNA which contain a repeated motif e. g. , (CA)_n, flanked by unique sequences. The number of units contained within the repeat motif is variable, such that various different alleles are present in any given population. The unique flanking sequences may be used as polymerase chain reaction (PCR) primers which allows for the rapid amplification and characterization of each locus from a small amount of DNA. Thus, each microsatellite marker has a unique set of primers. The microsatellite markers and their progeny are especially useful in detecting genetic diseases not phenotypically visible and identifying earners of recessive diseases. In a preferred embodiment, microsatellite markers are provided which may be used to detect the canine copper toxicosis gene.

In addition to identifying canine genetic diseases such as copper toxicosis, the microsatellite markers may also be used to create a genetic map of the canine genome, generate specific breed profiles, settle parentage disputes and identify dogs by DNA fingerprinting. Pedigrees of affected individuals, their siblings, parent and progeny can also be created Breeders and owners can thus choose breeding stock thereby reducing and possibly eliminating the incidence of specific genetic diseases.

Additional objects, advantages, and features of the present invention will become apparent form the following description and claims taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and by referencing the following drawings in which:

Figure 1A is a bar graph showing the average and standard deviation of heterozygosity percentages across loci within a breed;

Figure 1B is a bar graph showing the average and standard deviation of heterozygosity percentages across breeds within a locus;

Figures 2A-2D are photographs of gels showing marker locus D02011 in various breeds; and

Figure 3 is a photograph of a gel showing segregation of alleles at the C04107 locus in a Bedlington terrier pedigree .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Five hundred and nineteen microsatellite markers from specific gene loci are provided which are highly variable within and across many breeds of dogs. The microsatellite markers of the present invention comprise a repeat motif e. g. , (CA)_n, found in the canine genomic DNA, flanked by unique sequences . The unique sequences (also referred to herein as primer pairs) may be used as PCR primers, allowing the rapid amplification and thus detection of the sequence of interest in a small DNA sample. Table 2A sets forth the microsatellite markers of the present invention . The microsatellite markers and their progeny are especially useful in detecting genetic diseases not phenotypically visible and identifying carriers of recessive diseases.

In a preferred embodiment, microsatellite markers are provided which may be used to detect a carrier of the canine copper toxicosis gene. As further set forth in Specific Example II below, marker locus C04107 may be used to predict the inheritance of alleles at the copper toxicosis locus. C04107 has also been used to locate two other marker loci C04107B and C04107C, which either singly, or as a group, may also be used to detect the copper toxicosis gene .

The method of the present invention is useful for identifying disease free individuals (homozygous normal), carriers (heterozygous) and affected individuals (homozygous affected) at any stage of development. While a single marker may fail to provide the required information in any particular pedigree, a series of progeny markers will, and thus such a family of progeny markers derived from the linked markers set forth herein, are also included in the invention.

SPECIFIC EXAMPLE I

Materials and Methods

Isolation and Characterization of Microsatellite Loci. Established protocols were used for the cloning and screening procedures as described (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual (2nd ed. Cold Springs Harbor. Cold Springs Harbor Laboratory Press) (1992)). Genomic DNA was isolated from a peripheral blood sample from a Labrador retriever and partially digested with Bam HI. Size selected fragments purified from agarose gels using QIAEX beads (Qiagen Corp., Chatsworth, CA) were cloned into the phagemid vector pBS (Stratagene, La Jolla, CA) to construct a library of average insert size of 600 bps and propagated in the host XL-1 blue. The library was plated at low density (about 500 colonies/plate) without amplification. Duplicate nitrocellulose colony lifts were prepared, denatured and hybridized with (CA)₁₆ oligomer, labeled with ³²P dCTP using terminal transferase. Positive colonies were picked with a sterile pipette tip and lysed in 50 μl of a solution consisting of 1% Triton X 100, 20 mM Tris and 2 mM EDTA. Using primers complementary to the T3 and T7 promoter sequences which flank the cloning site, the inserts were amplified from 1-2 μl of the colony lysate in polymerase chain reactions for 30 cycles of 94, 55 and 72°C at 1 , 2 and 3 min., respectively after an initial denaturation at 94°C for 4 min. The standard buffer, nucleotide and primer concentrations were 50 mM Tris-HCl (pH 8.3 at 25°C), 50 mM KCI, 1.5 mM MgCI₂, 200 μM dNTPs and 40 pmoles of each primer in 100 μl reactions. PCR reactions were carried out on either a Perkin-Elmer Cetus (Perkin Elmer, Corp. Norwalk, CT) or an MJR PTC-100 thermocycler (MJ Research, Watertown, MA). To carry out secondary screenings of the clones, aliquots of the amplification products were run on 1.5% agarose TBE gels (90 mM Tris, pH 8.3, 90 mM boric acid, 2 mM EDTA). Southern blot analysis was carried out on the gels after transfer to Gene-Screen Plus membranes (NEN, Boston, MA) using the alkaline transfer protocol. The membranes were probed with (CA)₁₆ oligomers, 3' end-labeled with digoxigenin-dUTP using terminal transferase. A chemiluminescence detection system based on Lumi-Phos 530 as a substrate was used to detect positive hybridization signals following the recommendations included in a commercial kit, Genius (Boehringer Mannheim Corp., Indianapolis, IN). The membranes were washed to a final stringency of 0.1 X SSC (1 X SSC = 15 mM sodium chloride, 1.5 mM sodium citrate) at 65°C. The blots were than processed for immunological detection as described by the manufacturer. Once a clone was confirmed to be positive, the corresponding amplification product was then purified using QIAEX beads (Qiagen Corp. , Chatsworth, CA) after electrophoresis on TAE gels (40 mM Tris acetate, pH 8. 3, 2 mM EDTA) and directly sequenced using cycle sequencing (Delta Taq 2 0 Cycle Sequencing Kit, United States Biochemical Corp ., Cleveland, OH). The sequencing reactions were carried out according to the manufacturer's instructions with the slight modification that T3 and T7 primers labeled at their 5' end with ³³P ATP (NEN, Boston, MA) using T4 polynucleotide kinase were used as sequencing primers Sequencing products were analyzed by electrophoresis on 6% polyacryiamide gels containing 8M urea. The gels were dried and exposed to X-OMAT X-ray film (Eastman Kodak, Rochester, NY) for 1-2 days and developed. Primers flanking the repeat motif in each insert were selected to minimize hetero- and homedimerization, occasionally, the computer program Oligo (National Biosciences, Plymouth, MN) was used to help in the primer selection. The primers were synthesized by the Michigan State University Macromolecular Structure Facility.

Dog DNA Panel To check the usefulness of microsatellite markers within and across different breeds of dogs, a dog DNA panel was established. The breeds to be included in the panel were chosen with consideration given to the diversity in origin and function of breeds that exist. Table I presents various characteristics of the breeds chosen for the dog panel (Alderton, D. , The Eyewitness Handbook of Dogs (New York Dorling Kindersley) (1993), American Kennel Club, The Complete Dog Book (17th ed New York Howell Book House) (1985); Clark, R . D. , Medical and Genetic Aspects of Purebred Dogs (Forum Publications, Fairway, KS (1994), Walkowitz, et al. , Successfuly Dog Breeding (2nd ed. , New York, Howel Book House) (1994), and Lee, M. P. , The Official Book of the Scottish Terrier (Neptune City, T.F. H. Publications p. 158) (1994)). Five to ten individual dogs from each breed were selected for inclusion in the panel. Pedigrees were investigated to ensure that only dogs that had no common ancestors through four generations were included for independent representation of alleles. Ten, apparently unrelated, mixed bred dogs were also sampled . DNA was isolated from peripheral blood as previously described (Sambrook, J et al. , Molecular Cloning. A Laboratory Manual (2nd ed., Cold Springs Harbor, Cold Springs Harbor Laboratory Press) (1989)).

Analysis of Microsatellite Vanability Amplification of the correct target was verified by comparing the product obtained from genomic DNA to that obtained from the reference clone. The variability at each locus was tested by amplification of DNA from the dog panel. PCR conditions were 35 cycles of 94°C, optimal annealing temperature (50-60°C) and 72°C at 1 , 1 , and 2 min. , respectively after an initial denaturation at 94°C for 4 min. in the standard PCR buffer conditions described above 100 ng of genomic DNA was used as template in each reaction. 10 μl of the PCR products were analyzed by vertical electrophoresis using a modification of a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) protocol (Laemmli, U. K., Nature 227:680-685 (1970)) as described previously (Tas, S ., Anal. Biochem. 188: 33-37 (1992)). An HSI SE600 vertical slab gel electrophoresis system (Hoeffer Scientific Instruments, San Francisco, CA) connected to a cooling unit was used. The gels were poured between 16 x 16 cm plates using 1 mm spacers. 1 .5% acrylamide stacking gels of 2-3 cm were used on top of 12.5% acrylamide separating gels with 30 0 8 acrylamide to bis-acryiamide ratio . The gels were run at 40 mA through the stacking gel and than at 70 mA thorough the separating gel until the bromophenol blue dye reached the end of the plates, for approximately 4 hours. The amplification products were visualized after silver staining with the Silver Staining Kit (Bio-Rad Laboratories, Richmond, CA). This procedure resolved differences greater than or equal to 4 bps in the size of amplification products in the 75-250 bp range .

Results

Screening 110 plates resulted in the isolation of 1064 independent clones that were confirmed to be positive on secondary screening . Using 600 bps as the average insert size and 500 as the average colony number per plate, it was calculated that 1064 positives reflected an estimated incidence of one CA repeat clone every 31 kilobases in the dog genome.

The first 14 CA repeat loci for which primers were designed are presented in Table 2 together with the optimal annealing temperatures.

The complete set of microsatellite markers is set forth in Table 2A below. These markers were identified and the primers designed as described above.

Amplification reactions were carried out under standard PCR conditions described above using the annealing temperature indicated for each locus or a touchdown PCR protocol (Don, R.H. et al., Nucleic Acids Res. 19:4008 (1991)) was established. The variability of these loci were evaluated using the dog panel. For each locus, 5-10 dogs were studied in each breed. The number of alleles observed are presented in Tables 3A and 3B.

In general, all of the microsatellite loci tested displayed variability within and across breeds. While 9 cells out of 140 (6.4%) in Tables 3A and 3B were monomorphic, these were scattered though 6 different microsatellite loci, which were quite polymorphic in other breeds. The maximum number of alleles detectable by this analysis for a locus in a given breed was 8, in the case of locus D3908 in the Siberian Husky. The percent heterozygosity observed at each locus in each breed is presented in Tables 4A and 4B.

No heterozygotes were observed in only 21 out of 140 (15%) of the loci/breed combinations studied. At the same time, 30 out of 140 (21%) cells showed 100% heterozygosity. The mean and standard deviation of heterozygosity observed for each locus across different breeds, as well as the mean and standard deviation of heterozygosity observed within each breed across different loci are shown in Figures 1A and 1B, respectively. The breeds studied show a mean heterozygosity ranging from 36 to 60% across different microsatellite loci with considerable standard deviations. Among the loci studied D03908, D01902, D03709 and D00401 showed the highest mean heterozygosity across breeds of 87, 81 , 80 and 75%, respectively. The number of repeats in the reference clone in these loci were 16,18,12 and 22. The least informative loci across breeds were D02202 and D02012 at 5 and 19% mean heterozygosity, respectively. The number of repeats in the reference clone in these loci are 12 and 15, respectively. Correlation analysis did not reveal any significant linear relationship between the number of repeats at a locus and its overall observed heterozygosity (r=0.22).

Figures 2A-2D show the results from typical gels used to evaluate the alleles in gathering the data as described above. Amplification products of DNA from various different breeds at the locus D02011 are shown. Figures 2A-2D represent different gels, run under similar conditions. Note that the molecular weight marker identified in lanes marked M is the 246 bp band of the 123 bp ladder (Gibco-BRL, Gaithersburg, MD). The size of the amplification product in the reference clone was 238. The different alleles are easily identified, with PCR products separating in sharp and well resolved bands, near and below the 246 bp marker. Some non-specific amplification products can be observed, especially in cases with higher template DNA concentrations; however, these do not interfere with correct typing.

The results indicate that microsatellite loci containing CA repeats are abundant and highly polymorphic markers for the canine genome. These findings indicate that such markers hold great potential for use as linked markers for genetic defects in pure bred dogs.

The estimate that there is one useful CA repeat every 31 kb in the canine genome is in good agreement with one every 42 kb estimated recently by others (Rothuzien, J. et al., Theor. App. Genet. 89:403-406 (1994)). In the above-described study, a secondary screening was carried out and only very strong hybridization signals were accepted as positive, which resulted in elimination of about 20% of the primary positives. It thus appears that the estimate of the minimal CA microsatellites frequency in the canine genome is accurate. These estimates have practical implications particularly, since most cosmids have insert sizes in the 30-40 kb range, the likelihood of finding a useful CA repeat in a cosimd clone harboring a gene of interest is high.

SPECIFIC EXAMPLE II

Materials and Methods

Patients and pedigrees. The patients and pedigrees used were primarily those used and described earlier (Yuzbasiyan-Gurkan, V. et al., Genomics 15:86-90

(1993)). Briefly, pedigrees of American Kennel Club registered Bedlington terriers were associated with the help of Bedlington terrier (BT) breeders. While all of the pedigrees have a family history of CT, not all had a symptomatic proband at the time of pedigree ascertainment. Diagnosis of dogs as to whether they were affected or unaffected with CT was made in all cases by quantitative copper assay from liver biopsies performed at 1 year of age or older by criteria earlier described. DNA was extracted from peripheral blood samples collected in acid-citrate-dextrose as anticoagulant as described (Yuzbasiyan-Gurkan, V. et al., Genomics 15:86-90

(1993)).

Microsatellite analysis. The microsatellite markers used in this study were developed as described in Specific Example I. Standard conditions used to amplify each marker locus in polymerase chain reactions (PCR) were as follows: 25-50 ng of genomic DNA as template in 25 μ\ of PCR buffer (50 mM Tris HCl, pH 8.3 @ 25°C, 50 mM KCI, 1.5 mM MgCI₂), 200 μM dNTPs, 200 pM with respect to each primer and 1.5 U of Taq DNA polymerase. A touchdown PCR protocol (Don, R.H. et al., Nucleic Acids Res. 19:4008 (1991)) was established to facilitate the robust amplification of most markers under the same conditions. PCR was carried out at 94°C for 45 sec, 52°C for 30 sec, and 72°C for 1 min.

The microsatellite markers were initially evaluated in ten sets of parents from the BT pedigrees. Those markers for which at least one parent was heterozygous were then evaluated in all the dogs in the pedigree. Seven to twelve microliters of product were run on a 5% to 7% Hydrolink D600 acrylamide horizontal gel according to the manufacturer's instructions with the following modification. During the overnight runs, a plexiglas gel carrier was placed on top of the gel to prevent the swelling and distortion that was otherwise observed. Initially, electrophoresis was carried out from 4 to 5 hr. at 50 V in 1 X TBE (90 mM Tris, pH 8.3, 90 mM boric acid, 2 mM EDTA) with ethidium bromide. A photograph was taken and the gel electrophoresis then continued overnight at 35-40 volts depending on the fragment size of the product. A second photograph was taken and the results visually evaluated. It was found that two photographs were helpful in comparing different dogs with similar patterns. The alleles were then tabulated and used in linkage analysis.

Linkage analysis. Two point LOD (logarithm of odds) scores between CT and all the markers tested were generated using the MLINK program of the LINKAGE package (v5.1) (Lathrop, G.M. et al., PNAS (USA) 81 :3443-3446 (1984)). A gene frequency of 0.5 was assumed for CT.

Results

Two hundred thirteen microsatellite markers were evaluated in the process of finding linkage. Of these 213 markers, 181 provided scorable products in BTs using the touchdown protocol described above. Of these, 114 were informative in the pedigrees and were further evaluated.

Of all the markers tested for linkage to CT, only one yielded a significant LOD score. As shown in Table 5 below, marker number C04107 was found to be linked to the CT locus at a LOD score of 5.96, at a recombination fraction of zero. No recombinants were detected. Since a LOD score of 5.96 indicates that the odds of observing this linkage by chance is about 1 in a million, and since, a LOD score of greater than 3 or an odds ratio of 1 in 1000 is considered proof of linkage, the findings imply that the CT locus is indeed very close to the C04107 locus and thus can be used to predict the inheritance of alleles at the CT locus. No recombinants were detected in this study and thus a value can not be put on the genetic distance between these loci, except to say that they are very close.

The primer sequence and allele information about this marker are shown in Table 6. The allele frequencies were determined from alleles observed in apparently unrelated dogs.

Marker C04107 was used to locate markers C04107B and C04107C shown in Table 2A, which are close to C04107 and also contain repeats. This "family" of markers may be used to detect CT.

A typical pedigree illustrating linkage to C04107 is shown in Figure 3. In Figure 3, circles and squares depict females and males, respectively, and individuals affected with CT are indicated by the filled symbols. The asterisk in the figure indicates an individual not available for analysis. The bands are the negative image of amplification products obtained from the dogs indicated in the pedigree and analyzed individuals share the 2,2 genotype at this locus. In this pedigree, all dogs with the 1,1 genotype are predicted to be homozygous normal while those with the 1 ,2 genotype are predicted to be heterozygous, and thus carriers of the CT gene.

Given the finding of linkage and allowing for a small error for recombination, it is predicted that all the offspring with the 1, 1 genotype are clear of the CT gene i.e., homozygous normal, and that all 1 , 2 offspring are carriers in this pedigree.

Since data on the ESD and RB1 loci were available for most of the dogs from a previous study (Yuzbasiyan-Gurkan, V. et al., Genomics 15:86-90 (1993)), the linkage relationships of these loci with C04107 were was also evaluated. Neither ESD or RB1 were found to closely linked to C04107 (see Table 5).

As demonstrated by the pedigree illustrated in Figure 3, given an informative mating, it is now possible to identify all the genotypes in the offspring, distinguishing between the homozygous normal, homozygous affected and heterozygous dogs provided the genotype of one affected dog is available. However, C04107 is not extremely polymorphic in the BT population, showing only two alleles and a calculated heterozygosity of 0.43. Therefore, typing at the C04107 will not always yield information about the CT status of the offspring. Thus far, all affected dogs have been of the 2,2 genotype and the 2 allele is more common than the 1 allele (see Table 6). The matings which produce affected dogs will be found to be either between parents who are both 2,2 both 1 ,2 or one 1,2 and the other 2,2. In such cases, typing at the C04107 locus will only be useful in the second and third mating types. In the latter mating pairs, predictive information would only be available as to which dogs are affected. In order to make most pedigrees in the breed informative, additional polymorphic markers closely linked to C04107 are developed. It is predicted that a battery of three to five highly polymorphic markers will make almost every pedigree informative.

If strong linkage disequilibrium occurs at C04107 or nearby loci, the predictive power will be substantially improved. However, further studies of allele distributions in the BT population are needed to evaluate linkage disequilibrium, in any case, it should be possible to dramatically reduce the frequency of this serious disease within a very few generations.

As discussed above, canine copper toxicosis is present in the West Highland White Terrier and perhaps in several other breeds. (Thornburg, L.P. et al., Vet. Pathol. 27:81-88 (1990)). In the West Highland Terrier, it is clear that the phenotype is more complex, in that there is a spectrum of liver copper levels. This marker is evaluated in the West Highland White Terrier breed and it is determined whether there is segregation of high liver copper values with C04107.

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion and from the accompanying claims and drawings, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention.

All publications referred to herein are expressly incorporated by reference.

Claims

WE CLAIM:

1. A primer comprising a polynucleotide, wherein the polynucleotide has a sequence selected from the group consisting of the sequences of Table 2A.

2. The primer of Claim 1 , wherein the sequence is the Sns sequence of marker locus C04107 of Table 2A.

3. The primer of Claim 1 , wherein the sequence is the Asn sequence of marker locus C04107 of Table 2A.

4. The primer of Claim 1 , wherein the sequence is the Sns sequence of the marker locus C04107B of Table 2A.

5. The primer of Claim 1 , wherein the sequence is the Asn sequence of the marker locus C04107B of Table 2A.

6. A method for amplifying DNA, comprising the step of performing PCR with the DNA and a primer set selected from the group consisting of the primer sets of Table 2A.

7. The method of Claim 6, wherein the primer set is that shown as the

Sns sequence and Asn sequence of the marker locus C04107 of Table 2A.

8. The method of Claim 6, wherein the primer set is that shown as the Sns sequence and Asn sequence of the marker locus C04107B of Table 2A.