The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:71-74
Brief Communication |
Low Microsatellite Variation in Laboratory Gerbils
From the Institute of Zoology, Martin-Luther-University Halle-Wittenberg, Domplatz 4, D-06108 Halle (Saale), Germany (Neumann and Gattermann), Institute of Animal Breeding and Husbandry with Veterinary Clinic, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany (Maak and von Lengerken), and Federal Institute of Neurobiology, Magdeburg, Germany (Stuermer).
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Abstract |
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The Mongolian gerbil has become a model organism of increasing importance for the understanding of aging, epilepsy, the process of domestication or sociobiological questions. We report the development and characterization of the first nine polymorphic dinucleotide repeat loci in this species. Average observed heterozygosity and allele number of laboratory animals measured 0.136 (SE = ±0.065) and 1.78 (SE = ±0.278) compared to 0.761 (SE = ±0.025) and 9.2 (SE = ±0.57) found for a reference group of wild gerbils. The extreme low genetic variation observed in laboratory animals is caused by several severe population size bottlenecks due to the initial founder event and the later establishment of subpopulations. Reduced levels of allelic polymorphism in experimental animals hamper genetic mapping or parental studies. Therefore experiments relying on kinship analyses have to be carried out on wild animals. Estimates of genetic identity and parental exclusion were calculated as Pid = 2.8 x 10-12 and Pex > 0.999 in wild gerbils. Laboratory gerbil strains show the expected high degree of genetic similarity. However, significant allele frequency differences (P < .001) between American and European gerbils at some microsatellite loci may still allow discrimination between breeding lines.
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Introduction |
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The Mongolian gerbil (Meriones unguiculatus Milne Edwards, 1867) is a common rodent inhabiting the open steppe and semidesert regions of Mongolia and China. Its history as a laboratory animal started in 1935 when 20 pairs were captured in Mandschuria and brought to Japan for breeding. From there, 11 pairs of gerbils were imported into the United States in 1954, of which five females and four males at Tumblebrook Farm Ltd. formed the breeding stock for laboratories in the United States and Europe (Marston 1972; Schwentker 1963). Since then the Mongolian gerbil has become an extensively used experimental animal, for example, in neuroscience, auditory research, and physiology. The species serves as an important genetic model for aging (Spangler et al. 1997) and limbic epilipsy (Scotti et al. 1998). However, despite being an established model organism, the genetics of the Mongolian gerbil has hardly been explored (Gray and Wong 1990).
A number of behavioral and reproduction studies focus on the social organization and the cooperative breeding system of M. unguiculatus (e.g., Elwood 1979; Weinandy and Gattermann 1999), but most data were obtained from captive populations of laboratory animals and only a few field studies have been carried out (e.g., Ågren et al. 1989). Studies under laboratory and seminatural conditions (Ågren 1976, 1984) have shown that gerbils live in family groups and defend their territories. Within families there is usually a single reproducing pair suppressing reproduction of subordinated group members. Despite the formation of stable pair bonds, females often seek extrapair mating opportunities outside their territories to prevent inbreeding (Ågren 1990; Ågren et al. 1989).
The ongoing reproductive isolation of experimental gerbil strains has led to a number of anatomical, physiological, and behavioral pecularities not found in wild animals. Brain size reduction, seizures, and different learning performance are considered as evidence for a new case of domestication in a rodent species (Stuermer et al. 1997).
The low number of founder individuals in the Tumblebrook population has led to a substantial degree of inbreeding among captive gerbils. Previous data describing the genetic variability in laboratory bred animals are restricted to isozyme studies. Electrophoretic analysis of lactate dehydrogenase and alkaline phosphatase revealed no variation between different color morphs (Shimizu et al. 1996). A comparative survey of four gerbil strains kept in Japan found them indistinguishable at 23 protein loci except for liver acid phosphatase Acp2 (Okumura et al. 1995). These data support the view of a low inter- and intrastrain diversity among experimental animals.
Because of the obvious lack of genetic data and the need to evaluate the prognostics of genetic mapping experiments or parental studies, we have developed and characterized a set of microsatellite loci. Based on the data derived from nine loci we have estimated the genetic diversity of laboratory and wild gerbils.
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Materials and Methods |
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The isolation procedure for microsatellites followed standard protocols with modifications (e.g., Neumann and Wetton 1996). DNA was extracted from six male gerbils (three wild and three laboratory) and doubly digested with restriction enzymes AluI and HaeIII. A genomic library was established in XL-1 Blue MRF' (Stratagene) transformed with size fractionated (0.3–0.8 kb) DNA fragments ligated into the SmaI site of pUC18 (Pharmacia). Approximately 1500 recombinant clones were transferred to microtiter plates, replica plated onto nylon membranes, and screened with a cocktail of DIG-labeled oligonucleotides (CA)15, (TC)15, (AGG)5, and (GGAA)4. Hybridized colonies were visualized using the CSPD system (Roche). Thirty positive clones were sequenced containing 18x d(CA), 5x d(CT), 2x d(GGAA), 1x d(GGA), and 4x mixed d(CA/ CT) motifs. Primers were designed for 13 loci using OLIGO 5.0 (MedProbe, Norway). Polymerase chain reaction (PCR) was performed using the Ready-To-Go-system (Pharmacia). Thirty picomoles of each primer (one was labeled with Cy5 Amidite as fluorescent dye) and 0.01–0.1 µg of genomic DNA were added to a total volume of 25 µl. After an initial denaturation step of 180 s at 94°C the amplification proceeded for 35 cycles as follows; 60 s at 94°C, 60 s annealing at primer specific temperature (Table 1) and 120 s at 72°C (Thermocycler UNO II, Biometra). PCR products were electrophoresed through 6% denaturing polyacrylamide gels on an automated sequencer A.L.F. express II (Pharmacia). The amplification of nine dinucleotide repeat loci Mungµ1–Mungµ9 produced unambiguous allele patterns.
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Inheritance and linkage of microsatellites were examined in seven gerbil families consisting of laboratory and second generation wild animals (2 laboratory x laboratory, 3 laboratory x wild, and 2 wild x wild). Linkage analysis was carried out using the sequential LOD score method (Morton 1955) compiled over all informative families.
To estimate the level of genetic polymorphism, 45 laboratory and 40 wild gerbils were analyzed. DNA was prepared from ethanol fixed liver tissues and frozen ear samples using a commercial kit system (E.Z.N.A. Tissue DNA Kit II, peqlab Biotechnologie GmbH). The laboratory animals comprise five different strains. Sixteen gerbils came directly from Tumblebrook Farm Inc./USA in 1995. A further 17 animals were supplied by the Leibniz Institute of Neurobiology in Magdeburg/Germany and are crossbred animals from the Charles River corporation (strain: CRW/Mon) and the Technical University of Darmstadt/Germany. Five animals belonged to a second strain kept in Magdeburg which originated from Molegart-breed/France. A single individual came from a laboratory at the University of Cologne/Germany and six animals represent the laboratory strain Zoh: CRW bred at the Institute of Zoology Halle/ Germany. The latter population derived from three pairs supplied by Charles River Wiga (Sulzbach/Germany) stock label CRW/ (Mon)BR in 1992.
Wild gerbils were captured during a joint expedition of the Leibniz Insitute of Neurobiology Magdeburg/Germany and the State University of Mongolia to Central Mongolia in 1995. Animals were trapped at six different locations about 130–140 km southwest and 100 km west of Ulaanbaatar.
All laboratory and wild animals were pooled into two separate groups defined as "laboratory" and "wild" because of the small number of individuals representing a particular strain or population. Expected heterozygosity (HETexp) was calculated from Hardy–Weinberg assumptions for each locus 
. Deviations from Hardy–Weinberg equilibrium (HWE) were tested by the chi-squared test to detect potential genetic heterogeneity within the pooled samples. A test of genic differentiation combined over all polymorphic loci (Fisher exact test) was performed between American (Tumblebrook) and European laboratory gerbils using the computer program GENPOP, version 3.1d (Raymond and Rousset 1995). Based on the observed allele frequencies (f) we calculated the probabilities of profile identity (two random individuals share the same alleles = Pid) and parental exclusion (the probability of detecting an incorrectly assigned parent = Pex) combined over all loci, according to Gundel and Reetz (1981) and Bruford et al. (1992).
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Results |
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Primer sequences and characteristics of nine microsatellites in the Mongolian gerbil are presented in Table 1. Sequential LOD score analyses produced no evidence of close linkage (z < -2 for all
< 0.05 and z < 0 for all < 0.3) between these markers. The two pure laboratory pedigrees provided no information because all parents proved homozygous at each locus. Allele segregation among all offspring followed Mendelian inheritance. No measurable frequencies of null alleles were detected. A genetic variability comparison of laboratory and wild gerbils documented a dramatic reduction of allele numbers and observed heterozygosity in the domestic animals (Table 2). Four loci (Mungµ1, 2, 3, 9) were monomorphic and allele numbers at the five remaining loci ranged from 2 (Mungµ4, 5, 8) to 3 (Mungµ6, 7) in our sample, giving a mean allele number of 1.78 (SE = ±0.278). This differed significantly (P < .001, Mann–Whitney) from the high diversity of these loci in wild gerbils exhibiting an average allele number of 9.2 (SE = ±0.57). The mean observed heterozygosity in laboratory animals was 0.136 (SE = ±0.065) compared with 0.761 (SE = ±0.025) in wild gerbils (P < .001, Mann–Whitney). Significant differences in allele distribution were detected between animals from Tumblebrook Farm (USA) and European laboratories (P < .001). Private alleles restricted to the American or European strains were found at loci Mungµ6 and 7 in rather low frequencies. Alleles C (f = 0.011) and F (f = 0.011) at locus Mungµ6 were confined to Europe and allele E (f = 0.022) at locus Mungµ7 to Tumblebrook Farm (USA).
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Two loci Mungµ8 and 9 showed deviation from HWE in wild gerbils (P < .05) due to heterozygosity deficiencies. An excess of observed homozygotes was also responsible for the violation of the HWE in laboratory animals at locus Mungµ8 (P < .05). Probability estimates of genetic identity were calculated as Pid = 0.056 and Pex = 0.467 in laboratory and Pid = 2.8 x 10-12 and Pex > 0.999 in wild gerbils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the genetic variation in laboratory bred and wild Mongolian gerbils we have isolated a set of nine dinucleotide repeat loci. Their polymorphic nature and Mendelian inheritance was confirmed. All markers may be considered as genetically independent because no evidence for linkage was obtained. Pronounced differences in microsatellite variation between two pooled groups of laboratory and wild gerbils correspond with previous biochemical data and reflect the genetic history of the captive gerbil population. The low degree of polymorphism is a consequence of the initial founder event and following bottlenecks due to the establishment of subpopulations at different breeding locations. The genetic diversity of the investigated laboratory gerbil strains is below those observed in inbred mouse or rat strains (Love et al. 1990; Serikawa et al. 1992). In contrast, the data of wild gerbils fall in the range of variation found in other outbred wild Cricetidae populations (e.g., Cricetus cricetus; Neumann K, unpublished data).
All alleles present in the studied laboratory strains could be traced back to alleles in wild gerbils and have probably originated from early founder animals. Indications for this are also the high frequencies of most of these alleles in the wild gerbil sample. However, the increase in single low-frequency alleles because of mutations cannot be excluded. Small length differences by one repeat, for example, at locus Mungµ7, are in agreement with common stepwise mutations in microsatellites.
Observed deviations from HWE at two loci Mungµ8, 9 (P < .05) in wild and one locus Mungµ8 (P < .05) in laboratory gerbils are most likely explained by genetic differences between sampled populations or strains, since a general trend for increased proportions of homozygotes is observed at almost all loci.
Laboratory gerbil strains show the expected high degree of genetic similarity. Four microsatellite loci were homozygous for the same alleles in all tested domestic animals. This is in concordance with the breeding and distribution history of gerbils in North America and Europe and proves their common origin. Significant allele frequency differences (P < .001) at the remaining five loci between animals from Tumblebrook Farm (USA) and European gerbils may still allow the discrimination between breeding stocks. The detection of private alleles in the two laboratory gerbil pools could be a result of the small sample sizes but may equally account for true genetic strain diversity. Unfortunately our material did not contain laboratory gerbils from Japan. Their genetic composition could be more heterogeneous due to the larger number of unrelated founders (but see Okumura et al. 1995).
Mapping and linkage studies of genetically important loci in Mongolian gerbils are possible but require a larger set of informative markers than in most other experimental animals. However, the low level of genetic variation among strains and the existence of obvious phenotypic differences, for example, seizure-sensitive and seizure-resistant strains may ease the identification of affected genetic loci.
High combined probability estimates of genetic identity and low parental exclusion strongly interfere with kinship analyses in laboratory gerbils. Therefore genetic studies concerning mating system and kinship have to be carried out on wild animals. The microsatellite markers reported here provide a powerful system to accomplish this aim.
Laboratory Mongolian gerbils are an example for a thriving captive rodent population despite its apparent lack of substantial genetic variation. The existence of some distinguishing features such as an increased susceptibility to cerebral infarctions in domestic versus wild animals may be caused by genetic alterations, for example, the fixation of rare alleles and could therefore present inbreeding effects.
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Acknowledgments |
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We thank M. Strobel for technical support and Dr. K. Seidelmann for useful discussions and help with the statistics. K. Williams is acknowledged for checking the English. The authors like to thank three anonymous reviewers for their useful comments on the manuscript.
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Footnotes |
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Address correspondence to Karsten Neumann at the address above or e-mail: neumann{at}zoologie.uni-halle.de.
Corresponding Editor: Robert Wayne
Received January 10, 2000
Accepted October 31, 2000
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References |
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