Journal of Heredity 2003:94(6)
© 2003 The American Genetic Association 94:507-511
Brief Communication |
AFLP to Assess Genetic Variation in Laboratory Gerbils (Meriones unguiculatus)
From the Dipartimento di Biologia Evolutiva e Funzionale. Università degli Studi di Parma, Parco Area delle Scienze, 11/A, 43100 Parma, Italy, (Razzoli, Papa, Valsecchi, and Nonnis Marzano), and Department of Psychiatry, University of North Carolina, Taylor Hall #312, CB#7096, Chapel Hill, NC 27599 (Razzoli).
Address correspondence to M. Razzoli at the address above, or e-mail: razzoli{at}biol.unipr.it or razzoli{at}med.unc.edu.
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Abstract |
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The amplified fragment length polymorphism (AFLP) technique has been increasingly employed for characterizing inbred breeds of animals and detecting strain-specific polymorphisms. The majority of animals studies conducted in biomedical research are performed on rodent species, among which laboratory-reared Mongolian gerbils can be included. Despite the wide use of gerbils in scientific studies, their genetics has rarely been studied. Therefore we investigated the genetic differentiation of laboratory bred gerbils by means of AFLP markers. Six EcoRI/TaqI primer combinations were selected among 13 different combinations to assess the genetic polymorphisms in four stocks of animals: Charles River (CR), Harlan (Ha), Parma (Pr), and Crossbred (Cb). CR and Ha gerbils were purchased from commercial vendors, while Pr and Cb were derived from animals bred in our animal colony. A total of 228 fragments ranging between 70 and 650 bp were obtained. The mean percentage of polymorphic loci across primer combinations was 7.5%. Calculation of genetic distances through application of different algorithms (Nei's, BSI, and Jaccard's indexes) confirmed the poor genetic diversity between stocks. Nevertheless, a differentiation of the Pr and Cb stocks from the more homogeneous CR and Ha was revealed, in agreement with the different breeding derivation and management of the stocks.
Since the development of the polymerase chain reaction (PCR) (Saiki et al. 1988), many PCR-based fingerprinting methods have been developed to assess levels of genotypic variation in animal studies. Given the vast array of available molecular markers, the choice of the most suitable technique is one of the most critical issues in conducting research (Parker et al. 1998). Furthermore, the majority of laboratory animal strains originate from a few founder individuals or are the products of inadvertent artificial selection, which can account for their very low genetic variability. This seems to be the case for Mongolian gerbils (Meriones unguiculatus). Gerbils used in scientific studies all over the world originated from 20 pairs captured in eastern Mongolia and imported to Japan in 1935. Four of the descendent pairs were eventually imported into the United States in 1954, where the first American commercial colony was established at the Tumblebrook Farm (Rich 1968). Gerbils are extensively used in behavioral and biomedical research, and they are the animal model for studies of aging (Spangler et al. 1997), oncology (Fujioka et al. 2000), nutrition (Deming et al. 2000), reproduction (Heeb and Yahr 2001), stroke (Adachi et al. 2002), and epilepsy (Scotti et al. 1998). Despite this, there are only a few studies on the genetics of this species (Gray and Wong 1990; Okumura et al. 1995; Petrij et al. 2001; Shimizu et al. 1996). Recently low microsatellite variation was described in laboratory gerbils as compared to wild ones, as would be expected because of the historical derivation of laboratory strains (Neumann et al. 2001).
An increasing number of reports describe the amplified fragment length polymorphism (AFLP) analysis, originally proposed by Vos et al. (1995) and applied to plant genomes, as a useful tool for genetic mapping and fingerprinting in animals (Savelkoul et al. 1999). In particular, this technique, based on the selective amplification of restriction fragments ligated to adapters of known sequence, has the highest multiplex ratio, allowing for screening of the entire genome without the need for any a priori sequence knowledge (Mueller and Wolfenbarger 1999).
We carried out the present study in order to estimate the amount of genetic variability in captive gerbils as evidenced by AFLP markers. We chose to apply the AFLP technique to gerbils' genetic analysis for two main reasons. First, considering the paucity of data on the genetics of the species, it is important that the AFLP technique does not require any a priori sequence knowledge. Second, by allowing a simultaneous screening of several DNA regions randomly distributed throughout the genome, this technique can highlight extremely small genetic differences, as in the case of highly inbred strains.
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Materials and Methods |
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Animals
Fourteen male and 19 female adult (6 months of age) Mongolian gerbils were employed. Animals could be assigned to four different stocks (Charles River [CR], Harlan [Ha], Parma [Pr], and Crossbred [Cb]) according to their origin and/or to the breeding scheme to which they were subjected. Animals included in the CR stock (n = 8) were purchased from the vendor, Charles River Italia S.p.A. (strain: Crl: (MON)BR; Charles River Italia S.p.A., Como, Italy) in 2001. Charles River purchased the entire colony from Tumblebrook Farms, Inc. (Wilmington, MA) in 1996 and the Crl: (MON)BR strain was rederived from those gerbils. Animals included in the Harlan (Ha) stock (n = 7) were purchased from the vender Harlan Italy (strain: Hsd:MON; Harlan Italy, S. Pietro al Natisone, Italy) in 2000. Harlan Italy imports gerbils directly from Harlan Sprague Dawley (Madison, WI), where the breeding of gerbils originated by founder specimens from the University of Missouri. The breeding of the Parma (Pr) stock started in our animal facility in 1990 from 80 gerbils purchased from Tumblebrook Farm Inc. (USA). In 1996, 80 gerbils were purchased from Charles River and crossbred with those already present in the Parma colony. As a general rule, pairings in the colony were carried out in order to maintain the highest level of possible outbreeding. Seven animals belonging to the 10th generation of the Pr stock were used in this study. Animals of the Cb stock (n = 11) represent the second generation of a breeding scheme started in 2000 in our animal facility which consisted of crossbreeding Pr animals with gerbils purchased in 2000 from Charles River Italia S.p.A. (strain: Crl: (MON)BR; Charles River Italia S.p.A., Como, Italy), with minor involvement of the Ha stock purchased in 2000. All animals were maintained at the animal colony of the Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma, Parma, Italy.
AFLP Procedure
Genomic DNA was extracted and purified from ethanol-fixed muscle tissue of gerbils by means of the Aquapure genomic DNA kit (Bio-Rad Laboratories, Hercules, CA). Five hundred nanograms of genomic DNA were digested with EcoRI and TaqI restriction endonucleases (Amersham Pharmacia Biotech, Milwaukee, WI) at two different times because of different optimal digestion temperatures. Restriction was first carried out in a 25 µl volume containing TaqI endonuclease (5 U), "One-Phor-All-Buffer PLUS" (Amersham Pharmacia Biotech), 50 ng/µl of bovine serum albumin (BSA), 5 mM dithiothreitol (DTT). After incubation for 1.5 h at 65°C, 15 µl of a solution containing EcoRI (5 U) and the same DTT, BSA, and buffer composition was added to reach a final 40 µl volume. Then the digestion was continued at 37°C for 1.5 h.
Ligation of synthetic adapters (see Table 1) to the restriction fragments was carried out for 16 h at 16°C after adding 10 µl of a solution containing 1 U of T4 DNA ligase (USB, Cleveland, Ohio), EcoRI adapters (5 pmol), TaqI adapters (50 pmol), 1 mM adenosine triphosphate (ATP), 5 mM DTT, 50 ng/µl BSA in "one phor all buffer plus" to the 40 µl restriction solution. After ligation, the final 50 µl reaction volume was diluted four times in sterile apirogen water. Preamplification with primers carrying one selective nucleotide was performed in 50 µl volumes containing 15 µl of diluted ligate, equal quantities of EcoRI and TaqI primers (75 ng), 0.2 mM each of the four dNTPs, 1 U of Taq polymerase (Roche Molecular Biochemical, Mannheim, Germany), 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 50 mM KCl. The PCR was carried out at the following amplification conditions: denaturation for 30 s at 94°C, annealing for 1 min at 56°C, extension for 1 min at 72°C (30 cycles), followed by 7 min at 72°C to complete partial amplifications. Preamplified template was diluted 30-fold with sterile apirogen water and 5 µl of diluted product were further used for selective amplification in 20 µl of PCR mix containing EcoRI-labeled (Cy5) primer (10 ng), unlabeled TaqI primer (30 ng), 1 U of Taq polymerase (Roche Molecular Biochemical, Mannheim, Germany), 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, and 0.2 mM each of the four dNTPs. The selective amplification PCR profile was the following: 3 min at 94°C, 11 cycles of touchdown PCR starting from 94°C for 30 s, 65°C for 30 s (0.7°C decrease at each cycle), 72°C for 2 min, and an additional 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min. Final extension of partial amplifications was reached at 60°C for 30 min.
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A 1.7 µl volume of PCR product and 0.3 µl of DNA internal size standards (CEQ DNA Size Standard kit–600, Beckman-Coulter, Fullerton, CA) were added to 40 µl of deionized formamide (J. T. Baker, Phillipsburg, NJ). Samples where then loaded into the CEQ 2000 DNA Analysis System (Beckman-Coulter, Fullerton, CA).
Data Analysis
Original fragment data were examined with the Genographer software (version 1.6.0; J. J. Benham, Montana State University, 2001) for export to standard graphical formats. Further analyses of AFLPs were accomplished with the TFPGA software. Since AFLP analysis produces dominant markers, the assumption of the Hardy-Weinberg genotypic proportion must be made to calculate allele frequencies for statistical elaboration. Allele frequencies were estimated on the basis of Lynch and Milligan's (1994) Taylor expansion estimate and were rounded to exactly match observed samples, leading to the calculation of Nei's (1978) unbiased identity values. Genetic similarities for the four stocks were calculated using two pairwise similarity indices: the band sharing index (BSI) (Nei 1987) and Jaccard's coefficient (Ajmone-Marsan et al. 2002).
Differences between stocks in average heterozygosity, percent of polymorphic loci, BSI, and Jaccard's index values were analyzed by means of a one-way analysis of variance (ANOVA) (four levels: CR, Ha, Pr, Cb), followed by Tuckey HSD post hoc test for unequal sample sizes. Data were subjected to angular transformation to satisfy ANOVA assumptions. Differences were considered statistically significant when P <.05.
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Results and Discussion |
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Originally developed for the analysis of plant genomes (Vos et al. 1995), the AFLP technique is a recent acquisition in investigations of animal genomes, for which EcoRI and TaqI are considered the most suitable primer combinations for vertebrates (Ajmone-Marsan et al. 1997). We tested 13 possible EcoRI/TaqI combinations, among which we selected six primer combinations for subsequent analysis on the basis of the average number of polymorphic bands and their resolution (Table 1). The great majority of the fragments migrated between 70 and 600 bp. Depending on the primer pair, the number of distinguishable bands selected for the analysis ranged between 24 and 50 (Table 2). The mean percentage of polymorphism across primer combinations was 7.5%.
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We defined the majority of the polymorphisms as dominant, on the presence versus the absence of a specific band. We could also detect heterozygous patterns for several markers after taking into account the signal intensity of adjacent monomorphic bands. Pooling the values of individual animals from the four stocks, the overall unbiased heterozygosity was 0.0329 and the percentage of polymorphic loci (95% criterion) was 9.9174% (Table 3). No significant differences in these parameters comparing the four stocks were found. As for the genetic identity between the four stocks, the Nei's unbiased identity index values ranged from 0.9888 (Ha versus Pr) to 0.9963 (CR versus Cb) (Table 4). A pronounced degree of genetic similarity across the four stocks of animals was also detected when employing BSI and Jaccard's similarity indexes, with values ranging from 0.9797 to 0.9906 (BSI) and 0.9604 to 0.9807 (Jaccard's index) (Table 5).
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In spite of the overall similarity and the low genetic variability across different laboratory stocks, significant differences in BSI values (F3,21 = 15.62, P <.0001) between stocks were found. In particular, both the CR and Ha stocks showed BSI values significantly higher than either the Cb (Cb versus CR and Cb versus Ha: P <.001) or Pr stocks (Pr versus CR and Pr versus Ha: P <.0001). These results suggest a higher level of genetic similarity within the CR and Ha stocks rather than the Cb and Pr stocks. This probably can be explained by the breeding history of the Pr stock. It was founded in 1990, starting from animals purchased from Tumblebrook Farm, Inc. In 1996, 80 gerbils were purchased from Charles River Italia and crossbred with those already present in the Parma colony. The contribution of Ha gerbils to the breeding scheme can be considered minimal, consisting of the crossbreeding of eight Ha females with animals from the Pr stock in 2000.
Recently Neumann et al. (2001) developed the first polymorphic dinucleotide repeat loci in Mongolian gerbils by means of the microsatellite technique. Despite the extremely low levels of genetic variation found in laboratory gerbils as compared to both wild gerbils and to other inbred strains of rodents, Neumann et al. (2001) claim the possibility of a distinction between breeding laboratory lines of gerbils. Due to the different technique employed and to the different gerbil lines analyzed, a direct comparison between Neuman et al. (2001) and our data cannot be done. In particular, due to the small number of individuals representing the different laboratory populations in their study, Neumann et al. pooled all the "laboratory-bred gerbils" in a single group, not allowing referral of the results to the different lines of laboratory gerbils.
Both studies highlight the extremely low amount of genetic variability present in laboratory gerbils as a result of their breeding history. Despite the analytical strengths of microsatellite markers, such as codominance and hypervariability, it must be taken into account that the rapid evolution to which microsatellites are subjected may lead to marker coalescence, which in turn may cause an underestimation of the actual genetic differences. Therefore the AFLP technique seems more suitable for detecting genetically important loci on gerbils, since it generates hundreds of informative genetic markers. Because of its unparalleled sensitivity to minor genetic differences, high reliability, and user-friendliness, the AFLP technique can be considered a key molecular tool for some time to come.
In conclusion, the present study can be added to the recently acquired AFLP characterization of mammals, particularly of inbred strains (Ajmone-Marsan et al. 1997, 2002; Kim et al. 2001; Nijman et al. 1999; Otsen et al. 1996; Ovilo et al. 2000; Prochazka et al. 2001). More importantly, this study represents the first screening of genetic variability at the genomic level in different lines of laboratory gerbils. Therefore it may constitute a starting point for further research involving the use of AFLP markers to relate the genetics of different laboratory strains of gerbils to their phenotypic differences, since AFLPs are based on mutations causing phenotypic variation by modulating gene expression and function.
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Acknowledgments |
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The authors wish to thank Dr. Stefano Allesina and Dr. Stefano Leonardi for consulting on data analysis, Valeria Vascelli for her assistance in animal care, and Tearrah Wilkins for editing the language. This study was supported by grants from FIL 2001 and FIL 2002 to F. Nonnis Marzano and P. Valsecchi.
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Footnotes |
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Corresponding Editor: Roger H. Reeves
Accepted July 31, 2003
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