The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:276-279
Brief Communication |
Microsatellite Polymorphism in Closely Related Dogs
From the Unitat de Genètica i Millora, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Edifici V, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Address correspondence to Laura Altet at the address above or e-mail: Laura.Altet{at}uab.es.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The effectiveness of microsatellites in parentage testing and individual identification has been proven in many species, including dogs. However, the use of these markers has not been extended to control for pedigrees in large populations of closely related animals. We have analyzed polymorphism in a set of 10 microsatellites over three generations of 360 pedigree rottweilers. Results were compared with two pure-bred populations of unrelated animals and with one population constituted by unrelated dogs of mixed breeds to measure polymorphism variation. We optimized this set of microsatellites to be analyzed by a semiautomated capillary electrophoresis method after amplification in two multiplex polymerase chain reactions (PCRs). The mean polymorphism information content (PIC) value in the rottweiler pedigree is 0.401 and the combined paternity exclusion probability (CPE) is 95.6%. These values are similar to those obtained in pure-bred populations of unrelated animals, and although polymorphism is reduced in relation to the pool population, we solved all paternity exclusions. In only a few cases did we have to use two additional microsatellites to solve individual identification of full-sib dogs.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Microsatellites have been proven as a useful tool in parentage testing and individual identification in many species, owing to their high levels of polymorphism (Hammond et al. 1994; Tautz 1989). However, these levels of polymorphism have an intrinsic limitation when analyzing dogs. Although there are more than 300 different breeds described all over the world, there is high genetic homogeneity within each breed because they have been inbred to select for characteristic traits. Therefore different lines of the same breed often share common ancestors and this results in decreased genetic variability within those breeds.
Large numbers of microsatellites have been described in dogs to date (Francisco et al. 1996; Holmes et al. 1993, 1995; Mellersh et al. 1994; Ostrander et al. 1993, 1995; Primmer and Mathews 1993; Thomas et al. 1997), however, genetic data has been pooled and analyzed mostly from mixed populations. In pure-bred populations, data about allele frequencies and polymorphism indexes are scarce (Fredholm and Wintero 1995; Koskinen and Bredbacka 1999; Sutton et al. 1998; Zajc et al. 1997; Zajc and Sampson 1999). In these pure breeds, where only unrelated animals have been analyzed, intrabreed variation is reduced in relation to pooled populations. Moreover, breeders who rely on dog parentage testing and individual identification commonly use a few selected animals to produce salable pups. This creates difficulties, since microsatellite polymorphism analysis has not been utilized to test for the paternity of closely related dogs. We have addressed this issue and present microsatellite polymorphism and allele frequencies in 360 pedigree rottweilers over the course of three generations. This population reached a maximum inbreeding coefficient of 16%. Our main purpose was to compare this microsatellite polymorphism with two different types of dog populations: one constituted of unrelated pure-bred animals with no common grandparents (golden retrievers and Labrador retrievers) and the other one constituted of 95 unrelated dogs of mixed breeds (pool population). In this way we are able to detect if microsatellite variability is substantially reduced in an inbred population where most of the animals share recent ancestors. These data are particularly relevant to dog breeders, which usually mate closely related individuals and consequently need to use accurate and sensitive parentage tests. Two multiplex PCR reactions were optimized for this set of microsatellites using a semiautomated fluorescent genotyping protocol.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Animal Material
The three generations of pedigree rottweilers originate from 47 crosses among 49 dogs used as breeding animals. Twenty-seven of these breeding animals share recent common ancestors (at least grandparents) and 18 of the 47 crosses are inbred. The whole pedigree belongs to a single breeder and we consider it as a related pure-bred population. The pure-bred populations of unrelated dogs are composed of animals that have been collected from different breeders and have no common grandparents: 33 golden retrievers and 23 Labrador retrievers. The pool of 95 unrelated dogs includes animals from 24 different breeds: Newfoundland, Spanish greyhound, Belgian tervueren, German shepherd, Belgian groenendael shepherd, dachshund, Siberian husky, poodle, Yorkshire terrier, giant schnauzer, West Highland white terrier, Spanish mastiff, Neapolitan mastiff, boxer, basset hound, English cocker spaniel, Dalmatian, fila brasileiro, bullmastiff, Lhasa apso, Irish wolfhound, beagle, rottweiler, and American Staffordshire terrier. Dog genomic DNA was isolated as described elsewhere (Francino et al. 1997).
Microsatellite Markers
A total of 10 unlinked markers have been studied (Mellersh et al. 1997, 2000), seven dinucleotide markers—CPH5 and CPH9 (Fredholm and Wintero 1995) and CXX 366, CXX410, CXX442, CXX459, and CXX474 (Ostrander et al. 1995)—and three tetranucleotide markers—CXX2001, CXX2010, and CXX2054 (Francisco et al. 1996). We have used two more 4 bp microsatellites—CXX2130 and CXX2158 (Francisco et al. 1996)—to solve the genetic identity in some cases of full sibs.
These 10 microsatellites were amplified using two multiplex PCR reactions with five markers in each: multiplex-1 (CPH5, CXX366, CPH9, CXX474, and CXX459) and multiplex-2 (CXX2001, CXX2010, CXX2054, CXX410, and CXX442). The two multiplex reactions were carried out in 10 µl final reaction mixture containing PCR buffer (1x), 1.5 mM MgCl2, 0.2 mM of each dNTP (PE Biosystems), and 30–40 ng of dog genomic DNA. Primer concentration was optimized for each marker: 0.2 µM for 4 bp markers, 0.3 µM for CPH5, CPH9, and CXX366 markers, and 0.4 µM for the other 2 bp markers. One primer from each pair was fluorescently labeled with 6-FAM, TET, or HEX. Taq polymerase (Life Technologies Inc.) was used at a final concentration of 0.075 U/µl and 0.1 U/µl in multiplex-1 and multiplex-2, respectively. Thermocycling conditions were 3 min at 94°C followed by 25 cycles of 94°C (30 s), 58°C for multiplex-1 and 55°C for multiplex-2 (30 s) and 72°C (30 s), followed by a final extension of 15 min at 72°C in an MJ Research Hot-Bonnet. The two additional microsatellites were used either together or were added to multiplex-1, using 0.4 µM of each primer.
PCR reactions were analyzed by capillary electrophoresis in an ABI 310 Genetic Analyzer (Applied Biosystems, PE) and labeled PCR products were automatically sized relative to the internal standard (PRISM GENESCAN-350TM TAMRA) with the GeneScanTM Analysis 2.0 software.
Computation and Analysis
Allele frequencies and heterozygosity values were calculated with the Biosys-1 version 1.7 software package (Swofford and Selander 1989). The exclusion probability (PE) was calculated on the basis of the estimated allele frequencies (Jamieson 1994). Polymorphism information content (PIC) and PE values were calculated assuming that the genotypes of both parents were known (Botstein et al. 1980; Jamieson 1994). We compared the mean PIC values per population using the Student's t test with a significance level of 99.9% using the SAS package (SAS Institute 1995).
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Microsatellite allele frequencies and PIC values tend generally to be described in populations of unrelated animals. We were interested in measuring how these PIC and PE values are reduced when analyzing inbred populations. In such populations where most of the animals share recent ancestors, 10 microsatellites might be considered insufficient in order to distinguish closely related animals. Indeed, Sutton et al. (1998) found it necessary to use two typing systems (DNA fingerprinting and microsatellites, or DNA fingerprinting alone) in order to elucidate such problems. In this study we have analyzed 10 microsatellite polymorphism in 360 pedigree rottweilers over three generations (R). Results were compared with two pure-bred populations of unrelated animals [golden retriever (G) and Labrador retriever (L)] and also with a pool of 95 dogs from 24 different breeds (P). Allele frequencies for each microsatellite are shown in Table 1.
|
It is interesting to note that the mean PIC value obtained for these 10 microsatellites in R show no significant differences with the pure-bred populations of unrelated dogs we have analyzed (G and L) (shown in Table 2). These PIC values are similar to the ones previously described analyzing 19 microsatellites in three populations of unrelated pure-bred dogs (Zajc et al. 1997, 1999). The sustained polymorphism in R may be explained in two ways. First, the breeding animals possessed a relatively high level of heterozygosity. Second, despite an inbreeding coefficient of 16% in some crosses and a number of backcrosses among the R pedigree progeny, the breeder used a sufficient number of breeding animals to maintain variability.
|
The mean PIC value for P shows significant differences in relation to the pure breeds (G, L, and R). This population contains a higher level of genetic heterogeneity since it comprises 24 different breeds with some characteristic alleles within each marker that result in a mean PIC value of 0.70. Otherwise this value does not guarantee sustained polymorphism levels when a certain pure breed is analyzed. For example, in our pure-bred populations (G, L and R), the PIC values range from 0.03 to 0.69, with only 40% of the PIC values being higher than 0.5 for the same microsatellites. Moreover, although there are some breed-specific alleles, the major differences between purebred populations are the relative allele frequencies at each individual loci, as has previously been reported (Fredholm and Wintero 1995; Zajc et al. 1997). CXX366 has the same number of alleles in G and in R, but shows an enormous difference in the PIC values (0.34 and 0.05, respectively) due to the different distribution of allele frequencies. Therefore it is important to describe allele frequencies of microsatellites in a certain pure breed of dogs in order to select a useful set of markers for parentage control in that particular breed.
Microsatellite sequence is also an important consideration when choosing markers. Although greater instability has been described for tetranucleotide motifs, making them more polymorphic than dinucleotide repeats, we have not found any correlation between the repeat motif and the polymorphism level of these microsatellites. The most informative markers for each breed were CPH5 (2 bp) in the R population, CXX2054 (4 bp) in the G and L populations, and CXX410 (2 bp) in the P population. It has also been reported that longer repeats can generate alternatively sized alleles more frequently than shorter ones (Francisco et al. 1996). However, the longest microsatellite marker we analyzed (CXX2010) was not the most polymorphic one in any of our breeds. Furthermore, our results suggest that polymorphism levels in dogs depend on the specific pure breed in which a microsatellite has been studied.
Markers CXX2001, CXX2054 and CXX410 do not always follow the above 2 bp or 4 bp repeat motifs, perhaps because of some variation contained within the repeat, as has been previously described for sequenced tetranucleotide markers (Francisco et al. 1996). Alternatively it may be due to variability in the flanking regions of the repeats (Grimaldi and Crouau-Roy 1997). Similar results have been reported by Sutton et al. (1998) for 4 bp repeat microsatellites, with alleles separated by 2 bp or less.
In conclusion, this work has addressed and substantiated the use of microsatellites for parentage testing and individual identification in a large population of closely related dogs. Although microsatellite polymorphism is reduced in the rottweiler pedigree if compared to the mixed-breed population, it is similar to the populations composed of unrelated pure-bred dogs. Therefore 10 microsatellites, multiplexed in two different PCR reactions, were enough to solve all paternity exclusions. Only a limited number of cases required the use of two additional microsatellites to allow individual identification of full-sib dogs. We also demonstrate the existence of allele-specific patterns in the G, L, and R breeds. Taken together, our results suggest that microsatellite polymorphism data obtained from heterogeneous populations cannot always be extrapolated to specific breeds. This fact must be taken into account when implementing microsatellite-based assay for parentage cases, especially in closely related dogs.
|
Acknowledgments |
|---|
We are thankful to the breeders Pere Pujals and Didier Schaer and to the Veterinary Clinic Hospital from the Universitat Autònoma de Barcelona for providing the samples used in this study. We are also thankful to Atilio Aranguren for SAS computation analysis and to Simon Boa and Marcel Amills for their critical reviews.
|
Footnotes |
|---|
Corresponding Editor: Stephen J. O'Brien
Received March 27, 2000
Accepted October 31, 2000
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
-
Botstein D, White RL, Skolnick M, and Davis RW, 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331.[ISI][Medline]
Francino O, Amills M, and Sánchez A, 1997. Canine Mhc DRB1 phenotyping by PCR-RFLP analysis. Anim Genet 28:41–45.[ISI][Medline]
Francisco LV, Langston AA, Mellersh CS, Neal CL, and Ostrander EA, 1996. A class of highly polymorphic tetra-nucleotide repeats for the canine genetic mapping. Mammal Genome 7:359–362.[ISI][Medline]
Fredholm M and Wintero AK, 1995. Variation of short tandem repeats within and between species belonging to the Canidae family. Mammal Genome 6:11–18.[ISI][Medline]
Grimaldi M-C and Crouau-Roy B, 1997. Microsatellite allelic homoplasy due to variable flanking sequences. J Mol Evol 44:336–340.[ISI][Medline]
Hammond H, Jin L, Zhong Y, Caskey CT, and Chakraborty R, 1994. Evaluation of 13 short tandem repeat loci use in personal identification applications. Am J Hum Genet 55:175–189.[ISI][Medline]
Holmes NG, Dickens HF, Parker HL, Binns MM, Mellersh CS, and Sampson J, 1995. Eighteen canine microsatellites. Anim Genet 26:132–133.[ISI][Medline]
Holmes NG, Mellersh CS, Humphreys SJ, Binns MM, Holliman A, Curtis R, and Sampson J, 1993. Isolation and characterization of microsatellites from the canine genome. Anim Genet 24:289–292.[ISI][Medline]
Jamieson A, 1994. The effectiveness of using co-dominant polymorphic allelic series for (1) checking pedigrees and (2) distinguishing full-sib pair members. Anim Genet 25(suppl 1):37–44.
Koskinen MT and Bredbacka P, 1999. A convenient and efficient microsatellite-based assay for resolving parentages in dogs. Anim Genet 30:148–149.[ISI][Medline]
Mellersh C, Holmes N, Binns M, and Sampson J, 1994. Dinucleotide repeat polymorphisms at four canine loci (LEI 003, LEI 007, LEI 008 and LEI 015). Anim Genet 25:125.[ISI][Medline]
Mellersh CS, Hitte C, Richman M, Vignaux F, Priat C, Jouquand S, Werner P, Andr;aae C, DeRose S, Patterson DF, Ostrander EA and Galibert F, 2000. An integrated linkage-radiation hybrid map of the canine genome. Mammal Genome 11:120–130.[ISI][Medline]
Mellersh CS, Langston AA, Acland GM, Fleming MA, Ray K, Wiegand NA, Francisco LV, Gibbs M, Aguirre G, and Ostrander EA, 1997. A linkage map of the canine genome. Genomics 46:326–336.[ISI][Medline]
Ostrander EA, Sprague GF Jr, and Rine J, 1993. Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog. Genomics 16:207–213.[ISI][Medline]
Ostrander EA, Mapa FA, Yee M, and Rine J, 1995 One hundred and one simple sequence repeat-based markers for the canine genome. Mammal Genome 6:192–195.[ISI][Medline]
Primmer CR and Mathews ME, 1993. Canine tetra-nucleotide repeat polymorphism at VIAS-D10 locus. Anim Genet 24:332.[ISI][Medline]
SAS Institute, 1995. SAS user's guide: statistics, version 6.12. Cary, NC: SAS Institute.
Sutton MD, Holmes NG, Brennan FB, Binns MM, Kelly EP, and Duke EJ, 1998. A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites. Anim Genet 29:168–172.[ISI][Medline]
Swofford D and Selander RB, 1989. BIOSYS-1: a computer program for the analysis of allele variation in population genetics and biochemical systematics, release 1.7. Urbana: Illinois Natural History Survey.
Tautz D, 1989. Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 17:6463–6471.
Thomas R, Holmes NG, Fischer PE, Dickens HF, Breen M, Sampson J, and Binns MM, 1997. Eight canine microsatellites. Anim Genet 28:153–154.[ISI][Medline]
Zajc I, Mellersh CS, and Sampson J, 1997. Variability of canine microsatellites within and between different dog breeds. Mammal Genome 8:182–185.[ISI][Medline]
Zajc I and Sampson J, 1999. Utility of canine microsatellites in revealing the relationships of pure bred dogs. J Hered 90:104–107.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. N. Irion, A. L. Schaffer, T. R. Famula, M. L. Eggleston, S. S. Hughes, and N. C. Pedersen Analysis of Genetic Variation in 28 Dog Breed Populations With 100 Microsatellite Markers J. Hered., January 1, 2003; 94(1): 81 - 87. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||