Journal of Heredity Advance Access originally published online on June 15, 2005
Journal of Heredity 2005 96(7):843-846; doi:10.1093/jhered/esi090
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Fifty-Four New Gene-Based Canine Microsatellite Markers
From the Department of Medicine, Division of Cardiology (Bestwick, Winther, and Jakobs) and Molecular and Medical Genetics (Litt) at Oregon Heath and Science University, L103A, 3181 SW Sam Jackson Park Road, Portland, OR 97239
Address correspondence to Petra Jakobs at the address above, or e-mail: jakobsp{at}ohsu.edu.
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Abstract |
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Fifty-four new markers were developed to fill in gaps in the current map of canine microsatellites and to complement existing markers that may not be sufficiently informative in highly inbred canine pedigrees. Canine genes contained on the radiation hybrid map were used to obtain the sequence of the human homolog. A BLAST search versus the canine whole genome shotgun (wgs) sequence resource was used to obtain the sequence of the canine genomic contigs containing the homolog of the corresponding human gene. Canine sequences that contained microsatellites and mapped back to the correct location in the human genome were used to design primers for amplification of the microsatellites from canine genomic DNA. Heterozygosities of the markers were tested by genotyping grandparental DNAs obtained from the Nestle Purina Reference family DNA distribution center plus DNAs from unrelated Bouviers and Irish wolfhounds. Canine map positions of markers on the July 2004 freeze of the canine genome assembly were determined by in silico PCR or BLAST.
The canine genome project has recently advanced to a stage where mapping genes that cause inherited diseases in purebred dogs is possible (Dukes-McEwan and Jackson 2002; Kirkness et al. 2003). Genome resources have already been used to map the canine genes for several inherited canine diseases (Sutter and Ostrander 2004). A recent study on a late-onset heart disease, dilated cardiomyopathy (DCM), that occurs naturally in Newfoundland dogs failed to detect linkage after a 200-marker genome-wide screen (Dukes-McEwan and Jackson 2002). The same inherited condition, DCM, occurs also naturally in pedigrees kept as pets and breeding stock in the Irish wolfhounds (IWs) that our group studies. Our working pedigree consists of 142 dogs, 43 of which are affected. An initial 240 markers with an average heterozygosity of 0.46 were genotyped by the Marshfield genotype service. Since then we increased the number of markers to > 400, including some of the 54 novel markers described here. Currently, we have completed two- and multipoint Lod scores for all chromosomes. Assuming a canine genome size of 2,596 Mb and a recombination rate of 1 cM/Mb, we have excluded 67% of the genome. A genome-wide linkage study in IW has yet to detect linkage. We believe that refinement of the canine genetic linkage map may be essential to successful completion of this project.
The current RH map with 3,200 markers provides a good estimate of the order and physical spacing (i.e., in base pairs) of markers along canine chromosomes (Guyon et al. 2003) and was recently complemented by the construction of a 4,249-marker integrated canine genome RH map that consists of 900 genes, 1,589 microsatellites, and 1,760 BAC end markers (Breen et al. 2004).
A limiting factor for canine linkage studies is the relatively small number of meiotically mapped canine markers compared to those available for humans or mice as well as the low informativeness of many markers in purebred dogs. This is expected to leave gaps in exclusion maps, which will need to be filled by the development of additional highly polymorphic markers. Originally, markers had to be developed via laboratory-based methods, using a resource such as the canine BAC library (Li et al. 1999). With the availability of the draft sequence of the dog (Kirkness et al. 2003), markers can be designed using bioinformatics approaches as shown in this study.
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Materials and Methods |
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DNA Isolation
DNA from Bouvier de Flanders and IWs was extracted from whole blood by a salting-out procedure (Miller et al. 1988). In addition, we obtained DNA from the canine reference families (Nestle-Purina distribution center) that consist of one large multigeneration pedigree (CF-1) composed of 16 sibships described previously (Mellersh and Ostrander 1997).
Primer Design
As a starting point, we used canine genes contained on the 3,200-marker map (Guyon et al. 2003). From GenBank, we obtained the sequences of the human homolog of such canine genes. We used BLAST versus the short insert canine wgs resource (Kirkness et al. 2003) to obtain the sequences of canine genomic contigs containing homologs of the corresponding human genes. These canine sequences were screened for 23 common microsatellite sequences using DNA Pattern Find (Stothard 2000) (http://bioinformatics.org/sms). Long tetranucleotide repeats, based on the GAAA or TAAA motif tend to occur frequently and to be highly polymorphic (heterozygosities > 0.7) in dogs and hence are the preferred targets (Francisco et al. 1996). Dinucleotide repeats with at least 20 (CA), (GA), or (AT) units are also likely to have high heterozygosities. Those that contained microsatellites were BLASTed versus the GenBank human nr database to see if they mapped back to the correct location in the human genome. Sequences that mapped appropriately were used to design primers for amplification of the microsatellites from canine genomic DNA. In some cases, searching the short insert canine wgs resource with a human gene homologous to a mapped canine gene failed to produce a useful microsatellite marker. Therefore, in such cases, we tested human genes located within 2 Mb of the initial human homolog.
Microsatellite genotyping was accomplished by using two unlabeled, locus-specific primers, consisting of the forward primer with a M13 tail (5'-CACGACGTTGTAAAACGAC-3') and the reverse primer. A fluorescent dye was incorporated into the PCR product by using both unlabeled, locus-specific primers, together with one 5 prime fluorescent-labeled M13 primer (FAM, VIC, HEX, NED, or PET) (Boutin-Ganache et al. 2001).
Several of the markers described here were developed using the draft canine genome sequence, which became available in July 2004. The draft sequence consists of contigs, usually at least 100 kb in length that were obtained via a whole-genome shotgun approach. Many of these contigs are "anchored" to the 1Mb radiation hybrid map (Guyon et al. 2003) because they contain sequenced tagged sites (STSs) corresponding to mapped markers. In such cases, human homologs of canine genes or canine ESTs or STS with identifying GenBank accession numbers were BLASTed against the large insert canine wgs resource. This usually identified a canine genomic contig at least 100 kb in length, which was then analyzed with DNA Pattern Find as described.
Genotyping
Briefly, genomic DNA samples from 23 unrelated dogs were used as templates for polymerase chain reaction (PCR) to amplify short tandem, di- or tetra-repeat markers. Capillary gel electrophoresis (Applied Biosystems 310/3100 Genetic Analyzer) was used for the high-resolution electrophoretic separation. Allele sizes and genotypes were determined by using GeneScan and Genotyper software (Applied Biosystems).
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Results |
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Development of New Gene-Based Microsatellite Markers
The characteristics of 54 novel microsatellites ascertained as described are listed in Table 1. Newly developed markers were tested to determine their heterozygosities (HETs) in unrelated purebred dogs. Individuals with two different alleles were heterozygous for that marker and the calculated heterozygosity in 23 unrelated dogs is plotted against the length of the longest uninterrupted repeat in Figure 1. These plots show a wide variation in the heterozygosities for markers of a given repeat length but suggest that tetranucleotide repeats with at least 12 repeats tend to be more polymorphic than those with fewer than 12. The calculated correlation coefficient for heterozygosity versus repeat length is 0.29 for dinucleotide repeats and 0.25 for tetranucleotide repeats.
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The locations of most of the markers on the draft genome sequence were consistent with their locations on the radiation hybrid map. However, three markers—CREM, EST9A5, and PIK3C2B—included in the radiation hybrid map were localized to very different regions of the same chromosomes on the draft sequence (italicized markers in Table 1). Also shown in italics are five markers (MLH1, MCC, RGGRP1, FLJ20511, and SRP68) that mapped to different chromosomes on the draft sequence than those predicted from their positions in the human genome, using the synteny relations from Table S11 in Kirkness et al. (Kirkness et al. 2003). These five markers do not appear on the radiation hybrid map but are derived from human genes located within 2 Mb of human homologs to canine genes that are on the radiation hybrid map. Locations of the eight italicized markers need to be checked by radiation hybrid or meiotic mapping before they are used in linkage studies of canine traits.
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Discussion |
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Using bioinformatic tools, we have developed 54 new gene based microsatellite markers. These markers were placed on the physical map by using a comparative genomic approach.
Genotypes from 17 grandparental DNAs from the canine reference families and 6 unrelated dogs from our gene mapping projects were used to determine the heterozygosities of the markers. The correlation between repeat length and heterozygosity is weak. This is in marked contrast to the situation for human CA repeat polymorphisms, for which that correlation is quite strong (Weber 1990).
This study provides an example of the utility of the 1.5x canine genome draft sequence for identifying novel microsatellite markers for genetic linkage studies.
With the availability of the current (July 2004 freeze) dog genome sequence, the use of the UCSC Web site (Kent et al. 2002; genome.ucsc.edu) further simplifies the process of identifying new markers. The location of microsatellites within sequence contigs is directly indicated using the tandem repeat finder (TRF) (Benson 1999). Clicking on an individual repeat brings up a screen showing the length of the repeat block, the extent of mismatching and the percentage of insertions or deletions. Long tetranucleotide microsatellites, such as those based on the repeat motif GAAA, are often highly polymorphic (Francisco et al. 1996) and can be conveniently selected for further examination and primer design. The map locations of markers derived in this way must be regarded as tentative because the draft sequence contains gaps and probably also contains some misalignments. Marker selection from contigs that are anchored to the 2003 radiation hybrid map (Guyon et al. 2003) can mitigate this problem.
Many of the loci described here were chosen for their genomic location to facilitate the exclusion mapping for our IW linkage project. Some markers were also chosen for their telomeric locations so they could be used to test if recombination near telomeres is enhanced in canines as it is in humans (Kong et al. 2002).
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Acknowledgments |
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This work was supported by the National Institute of Health (NIH RO1-HL71096). The authors thank Ewen Kirkness for suggesting the methodology for finding microsatellite markers using the Celera/TIGR whole genome shotgun sequence resource and Steve Hannah from Nestle Purina Inc. for providing genomic DNA from the Canine Reference Families. This paper was delivered at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: Bernard van Oost
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References |
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Benson G, 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27:573–580.
Boutin-Ganache I, Raposo M, Raymond M, and Deschepper CF, 2001. M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. Biotechniques 31:24–26 28.[Web of Science][Medline]
Breen M, Hitte C, Lorentzen TD, Thomas R, Cadieu E, Sabacan L, Scott A, Evanno G, Parker HG, Kirkness EF, and others 2004. An integrated 4249 marker FISH/RH map of the canine genome. BMC Genomics 5:6.[CrossRef][Medline]
Dukes-McEwan J, and Jackson IJ, 2002. The promises and problems of linkage analysis by using the current canine genome map. Mamm Genome 13:667–672.[CrossRef][Web of Science][Medline]
Francisco LV, Langston AA, Mellersh CS, Neal CL, and Ostrander EA, 1996. A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mamm Genome 7:359–362.[CrossRef][Web of Science][Medline]
Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK, Renier C, Gelfenbeyn B, and others 2003. A 1-Mb resolution radiation hybrid map of the canine genome. Proc Natl Acad Sci USA 100:5296–5301.
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, and Haussler D, 2002. The human genome browser at UCSC. Genome Res 12:996–1006.
Kirkness EF, Bafna V, Halpern AL, Levy S, Remington K, Rusch DB, Delcher AL, Pop M, Wang W, Fraser CM, and Venter JC, 2003. The dog genome: survey sequencing and comparative analysis. Science 301:1898–1903.
Kong A, Gudbjartsson DF, Sainz J, Jonsdottir GM, Gudjonsson SA, Richardsson B, Sigurdardottir S, Barnard J, Hallbeck B, Masson G, and others 2002. A high-resolution recombination map of the human genome. Nat Genet 31:241–247.[CrossRef][Web of Science][Medline]
Li R, Mignot E, Faraco J, Kadotani H, Cantanese J, Zhao B, Lin X, Hinton L, Ostrander EA, Patterson DF, and de Jong PJ, 1999. Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library. Genomics 58:9–17.[CrossRef][Web of Science][Medline]
Mellersh CS, and Ostrander EA, 1997. The canine genome. Adv Vet Med 40:191–216.[Web of Science][Medline]
Miller SA, Dykes DD, and Polesky HF, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215.
Sutter NB, and Ostrander EA, 2004. Dog star rising: the canine genetic system. Nat Rev Genet 5:900–910.[CrossRef][Web of Science][Medline]
Stothard P, 2000. The sequence manipulation suite: JavaScriptprograms for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102–1104.[Web of Science][Medline]
Weber JL, 1990. Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms. Genomics 7:524–530.[CrossRef][Web of Science][Medline]
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