Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:89-93
Canine-Derived Cosmid Probes Containing Microsatellites Can Be Used in Physical Mapping of Arctic Fox (Alopex lagopus) and Chinese Raccoon Dog (Nyctereutes procyonoides procyonoides) Genomes
From the Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University, PoznÄn, Poland (Rogalska-Niznik, Szczerbal, and Switonski); the Institute of Animal Genetics, Nutrition and Housing, University of Berne, Berne, Switzerland (Dolf and Schläpfer); and the Department of Animal Sciences, Swiss Federal Institute of Technology and Faculty of Veterinary Medicine, University of Zurich, Zurich, Switzerland (Schelling).
Address correspondence to Marek Switonski, Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University of PoznÄn, Wolynska 33, 60-637 PoznÄn, Poland, or e-mail: switonsk{at}jay.au.poznan.pl.
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Abstract |
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Rapid development of the canine marker genome map facilitates genome mapping of other Canidae species. In this study we present chromosomal localization of 18 canine-derived cosmid probes containing microsatellites in the arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes by the use of fluorescence in situ hybridization (FISH). The chromosome localizations in the arctic fox are in general agreement with data obtained from comparative genome maps of the dog and the fox. However, our studies showed that the order of the loci on some chromosomes was changed during karyotype evolution. Therefore, we suggest that small intrachromosomal rearrangements took place.
The arctic fox (Alopex lagopus) and the Chinese raccoon dog (Nyctereutes procyonoides procyonoides) belong to the Canidae family. In this family a wide range of chromosome diploid numbers is observed (Wayne et al. 1987) and, moreover, an extensive chromosome and karyotype polymorphism in some species has been described. The dog (Canis familiaris) has the highest diploid chromosome number (2n = 78), with all autosomes being acrocentric. In contrast, the red fox (Vulpes vulpes) has the lowest diploid chromosome number (2n = 34 + B) with a variable number (0–8) of supernumerary microchromosomes, described as B chromosomes (Mäkinen et al. 1985b). The arctic fox karyotype is characterized by centric fusion polymorphisms, and animals with 50, 49, and 48 chromosomes occur (Mäkinen et al. 1985a). In the karyotype of this species, 10 chromosome pairs have complete heterochromatic arms, for which size polymorphisms were found (Switonski and Gustavsson 1991). There are two subspecies of the raccoon dog: the Chinese raccoon dog (2n = 54 + B) (1–4) and the Japanese raccoon dog (2n = 38 + B) (2–5). Both subspecies have variable numbers of large B chromosomes (Mäkinen et al. 1986).
The canine marker genome map is very advanced and includes 1,800 genetic markers (Breen et al. 2001), but a recent report describes an extension of this map to 3,400 loci (Andre et al. 2002). It is known that a majority of the primer sequences of canine microsatellites amplify analogous sequences in the red fox (Fredholm and Wintero 1995; Zajac et al. 2000) and arctic fox (Klukowska et al. in press) genomes. It has also been shown that canine-derived cosmid probes can be successfully used in physical mapping of microsatellite loci in related Canidae species, such as the red fox (Yang et al. 2000) and the arctic fox (Rogalska-Niznik et al. 2000). The aim of the present study was to map a set of 18 microsatellite probes onto the arctic fox and Chinese raccoon dog using fluorescence in situ hybridization (FISH). This study adds new data to the comparative genome map of the dog, red fox, arctic fox, and raccoon dog obtained recently by comparative chromosome painting (Graphodatsky et al. 2001; Yang et al. 1999).
The red fox (known as a farm silver fox), the arctic fox (known as a farm blue fox), and the raccoon dog are fur animals bred in captivity. The fur industry is under pressure by both fashion and animal rights organizations. Therefore studies on housing environment related to improvement of fur animal welfare have become very important (Harri 2000). It is believed that selection on behavior traits may be useful. Therefore development of genome maps may facilitate identification of genes influencing not only fur quality but also behavior traits.
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Materials and Methods |
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Blood samples were collected from farm arctic foxes and Chinese raccoon dogs, and short-term lymphocyte cultures were established. Chromosome slides with metaphases were QFQ-banded prior to hybridization. International chromosome nomenclature for the arctic fox (Mäkinen et al. 1985a) and a nomenclature for the Chinese raccoon dog proposed by Pienkowska et al. (2002) were applied. Eighteen canine-derived cosmid probes containing microsatellites—ZuBeCa1, ZuBeCa2, ZuBeCa3, ZuBeCa5, ZuBeCa6, ZuBeCa7, ZuBeCa8, ZuBeCa9, ZuBeCa10, ZuBeCa12, ZuBeCa13, ZuBeCa16, ZuBeCa17, ZuBeCa20, ZuBeCa25, ZuBeCa28, ZuBeCa29, and ZuBeCa30 (Dolf et al. 2000; Ladon et al. 1998; Schelling et al. 1998a,b, 2000; Schläpfer et al. 1998, 1999; Switonski et al. 1998)—were used in the present study. Cosmid DNA (25 ng) was labeled with biotin-16-dUTP (Boehringer Mannheim) using Prime-a-Gene labeling system (Promega). The slides were denatured in 70% formamide/2x SSC for 2 min at 70°C, plunged immediately into ice-cold 70% ethanol, dehydrated, and air dried. Biotin-labeled cosmid probes were denatured by heat at 70°C for 10 min, preannealed for 15 min at 37°C, applied onto slides (12 ng DNA per 18 mm x 18 mm), and incubated overnight. Signal detection and amplification, using avidin-FITC and anti-avidin (Vector Laboratories), was applied. Staining was performed with propidium iodide (200 ng/ml). Image capturing and processing were performed with a fluorescence microscope (Nikon E600 Eclipse) equipped with a CCD camera (Hammatsu) driven by computer-aided software (Lucia).
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Results and Discussion |
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Eighteen canine-derived cosmid probes were successfully assigned to the chromosomes of the arctic fox and the Chinese raccoon dog by FISH. Localizations of two cosmid probes (ZuBeCa5 and ZuBeCa9) are shown in Figure 1. A summary of the chromosomal assignments in both investigated species, along with previous localizations of the same probes localized earlier onto chromosomes of the dog and the red fox by Yang et al. (2000), is presented in Table 1. We also show the localizations which follow the chromosome nomenclature of Breen et al. (1999), endorsed by the International Society of Animal Genetics (ISAG) conference (Minneapolis, MN; 2000). Moreover, comparative localization of these microsatellites in the dog, Chinese raccoon dog, and arctic fox chromosomes is shown in Figure 2.
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Comparative chromosome analysis of the dog, red fox, arctic fox, and Japanese raccoon dog by the use of reciprocal chromosome painting suggests that the ancestor of the Canidae family had a low chromosome diploid number, similar to the Japanese raccoon dog karyotype (Graphodatsky et al. 2001). Until now, three comparative chromosome maps existed: dog versus red fox (Yang et al. 1999), dog versus arctic fox (Graphodatsky et al. 2000), and dog versus Japanese raccoon dog (Graphodatsky et al. 2001). So far the Chinese raccoon dog has not been included in such studies. The Chinese and Japanese raccoon dog karyotypes were compared by banding techniques; however, the results were not concordant (Mäkinen et al. 1986; Ward et al. 1987). Correspondence between karyotypes of both subspecies can be predicted from localization of locus-specific probes (ZuBeCa markers) on chromosomes of the dog and the Chinese raccoon dog.
The presented assignments in the arctic fox genome are in agreement with the report on comparative chromosome maps of the dog, red fox, and arctic fox (Graphodatsky et al. 2000), in which 42 conserved autosome segments were identified. However, it should be emphasized that the chromosome painting approach may not detect intrachromosomal rearrangements. An interesting example concerns dog chromosome 9 (CFA9), Chinese raccoon dog 5 (NPP5), and arctic fox chromosome 12 (ALA12). ALA12q is painted by the CFA9 whole chromosome painting probe. Our study suggests that a fragment of NPP5q was inverted in ALA12q. The centromere position of canine CFA9 has been conserved according to the Chinese raccoon dog chromosome, but it is oriented toward the telomeric region of ALA12. Moreover, we found that three closely localized microsatellites on NPP5q (ZuBeCa12, ZuBeCa13, and ZuBeCa20) reside on two different fragments of CFA9 and ALA12. A rearranged order of the loci (ZuBeCa25 and ZuBeCa30) was also observed on CFA17 and ALA5p when compared with NPP13. This also was probably caused by an inversion.
The present study extends the physical genome map of the arctic fox to 21 loci, including those previously mapped by Rogalska-Niznik et al. (2000), and gives the first data about the physical map of the Chinese raccoon dog genome. Our data indicate that in the karyotype evolution of these species, intrachromosomal rearrangements also occurred. Further development of these maps will not only support studies on karyotype evolution in Canidae, but can also be used to identify genes important for selective breeding.
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Acknowledgments |
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This study was supported by the State Committee for Scientific Research, Poland (grant no. 6P06D 028 21) and by the Foundation for Polish Science (contract no. 13/2000). This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
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Footnotes |
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Corresponding Editor: Elaine Ostrander
Received July 15, 2002
Accepted September 27, 2002
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