RACCOON DOG (NYCTEREUTES PROCYONOIDES GRAY) PARENTAGE TESTING BASED ON STR MARKERS

Brygida Ślaska¹, Grzegorz Zięba¹, Grażyna Jeżewska-Witkowska^2
1 Department of Biological Bases of Animal Production, University of Agriculture, Lublin, Poland
² Department of Biological Basis of Animal Production, University of Life Sciences in Lublin, Poland

ABSTRACT

We presented a practical and efficient parentage analysis test for raccoon dogs. The method makes use of 15 polymorphic microsatellite markers. Parentage control was performed for 3 generations of raccoon dog (207 animals altogether) by amplification of microsatellites. Exclusion probabilities ranging from 0.959 assuming one known parent to 0.998 assuming both known parents, were attained. It is possible to use the set of 15 markers in this study for routine parentage testing, but further research should be done to examine whether the set are practically effective. Microsatellite markers are useful tools for parentage testing in raccoon dogs.

Key words: Canidae, microsatellites, parentage testing, raccoon dog.

INTRODUCTION

The raccoon dog (Nyctereutes procyonoides Gray, 1834), is one of the farm fur bearing animals, belonging to the order of carnivores (Carnivora), family Canidae. Profitability of fur animal farming, including raccoon dogs, depends on a high level of reproduction-related traits, mainly fertility and fecundity, which in turn are conditioned by both genetic and environmental factors [13]. Each breeder tends to create conditions that would enable obtaining the highest number of offspring, bearing in mind the quality of the breeding material. Selection response in the farms of fur bearing animals can result from the selection conducted, on the basis of correct pedigree data. For economic reasons, it was important to reduce the number of males needed for breeding, and polygamous mating trials were performed in the 1970s [15], and since then the polygamous mating have been used in the fur industry. Due to a relatively short period of farming, the literature lacks any reports related to control of the males' fecundity in raccoon dog farms. Taking into consideration this fact, the infertile male could cause losses, such as lack of offspring from the unfertilized females. It is especially important for the raccoon dog which is seasonally monoestrous, so a breeder could lose the possibility of obtaining cubs for a year. To minimize losses which can occur in the case of infertile males, and increase fertility in the raccoon dog farms, a female, as a rule, is mating not only one, but different males, twice or three times during one estrus, which lasted from 2 to 6 days [16]. In the current situation it is not always possible to establish, which male is a biological father of the obtained offspring. It is particularly important for breeding work in farms, and selection of animals for the breeding stock. Inappropriate selection could lead to both a decrease in breeding value and appearance of inbreeding depression with all its negative results, such as a decreased fertility of animals, in the foreground [10]. Therefore, it is important to determine the biological father of the offspring, coming from a female, which was mating different males, twice or three times during one estrus. If we can establish an efficient parentage testing system for raccoon dog, this problem will be solved.

STR (Short Tandem Repeat) sequences have been used for parentage testing in many species, but the application of microsatellites to parentage testing in dogs, which belong to Canidae, like the raccoon dog, is relatively recent [2,4,6,8,9]. So far, the parentage testing based on STR markers in raccoon dog has not been presented in literature.

The aim of the present study was to evaluate usefulness of 15 microsatellite markers for parentage testing in the raccoon dog.

MATERIAL AND METHODS

In general, 3 generations of raccoon dog (altogether 207 animals) maintained at a Polish breeding farm in the years 2002-2004, were under observation. Blood samples were collected from animals using sterile test-glasses (Medlab) with K₂EDTA as an anticoagulant. Genomic DNA was isolated using QIAamp DNA Blood Mini Kit (QIAGEN). 15 microsatellites (FH3300; C01.246; REN112I02; REN288J16; PEZ17; REN144A06; FH2097; AHT103; C03304; ACE; FH3596; REN198P23; REN230G12; REN01N09; BAC_382-K19), were used in the present study. All primers as well as conditions of PCR (Polymerase Chain Reaction) amplification of microsatellite sequences come from domestic dog (Canis familiaris) literature data as modified by Slaska et al. [14]. The allele identification within the sequences of microsatellite loci was conducted by capillary electrophoresis performed in a sequencer ABI PRISM^TM 3100-Avant Genetic Analyzer using: 3100-AVANT ABI PRISM DATA COLLECTION and GENE MAPPER SOFTWARE 3.5.

The Windows-based computer program, CERVUS [11] was used to calculate allele frequencies, run simulations and perform parentage analysis using data from all types of codominant markers. The exclusion probability (PE) for raccoon dog in the case where only one parent was available for genotyping (PE₁: Equation 1) in the case where both parents could be genotyped (PE₂: Equation 2) as well as the Combined PE were calculated according to Jamieson and Taylor [7]. The calculations consider alleles at each marker with frequencies p_i...p_n.

Equation 1

Equation 2

The Combined PE was calculated as:

combined PE₁=1-(1-PE₁ for marker 1)(1-PE₁ for marker 2)..(1-PE₁ for marker n),

combined PE₂=1-(1-PE₂ for marker 1)(1-PE₂ for marker 2)..(1-PE₂ for marker n).

RESULTS

Values of the exclusion probability in the case where only one parent was available for genotyping (PE₁), the exclusion probability in the case where both parents could be genotyped (PE₂), and the Combined PE (parentage testing) were presented in Tables 1 and 2. STR genetic markers were used in our investigations.

The exclusion power in raccoon dog for each marker, depending on the availability of parental data, was different and ranged from 0.048 (REN230G12) to 0.371 (REN198P23) for paternity exclusion (PE₁) (Table 1), and from 0.137 (REN230G12) to 0.551 (REN198P23) for paternity exclusion (PE₂) (Table 2). As the number of markers increased, the combined PE₁ (Table 1) as well as PE₂ went up (Table 2). Finally, the combined PE for 15 markers reached 0.959 (Table 1) and 0.998 (Table 2), in case when data of only one of parents were available as well as of both parents, respectively.

DISCUSSION

So far, the parentage testing based on STR markers in raccoon dog has not been presented in literature. Thus, the present study as first presents STR markers for parentage control in this species.

Microsatellite repeat sequences are well dispersed in the genome, highly polymorphic, and have been shown to be powerful tools in genome mapping of dog [12]. They have been used for parentage testing in many species, but the application of microsatellites to parentage testing in dogs which belong to Canidae, like raccoon dog, is relatively recent. There are a few reports on microsatellite-based parentage testing [2,4,6,8,9]. The previous studies of microsatellite loci showed very high DNA sequence conservatism inside the Canidae family [3]. Taking this fact into consideration, the received results can be compared only to those in Canis familiaris, because some panels based on microsatellite markers have already been studied in the dog, but not in the raccoon dog.

Fredholm and Wintero [4] performed parentage control of 12 different dog breeds by the amplification of microsatellites. Maternity indices were calculated for 12 loci, and the probability of maternity was estimated to be 99.99%. Koskinen and Bredbcka [9] using ten polymorphic microsatellite markers examined dogs from four different breeds. According to them, assuming one known parent, exclusion probabilities ranged from 0.9934 (Golden Retriever) to 0.9993% (Finnish Hound). Ichikawa et al. [6] examined an accurate method for parentage testing in dogs - microsatellite DNA repeat length polymorphisms. They selected twenty microsatellites and examined their application for parentage testing in Beagles and Labrador Retrievers. The final combined PEs in Beagles and Labrador Retrievers were 0.999994 and 0.999920, respectively. Their results suggest that the examined twenty markers can be applied for routine parentage testing in dogs. Klukowska et al. [8] studied nine microsatellite polymorphism in six dog breeds. The exclusion probability (PE) for the studied breeds in the case where both parents could be genotyped varied from 0.9946 (Boxer) to 0.9996 (Dachshund). In the case where only one parent was available for genotyping the PE varied from 0.6677 (Bouvier des Flandres) to 0.7090 (boxer). According to DeNise et al. [2] The American Kennel Club (AKC) authorized a study to determine the power to exclude non-parents and identify individuals using DNA genotypes of 17 microsatellite markers in two panels. 9561 samples represented 108 breeds. The primary panel of 10 markers exceeded 99% power of exclusion for canine parentage verification of 61% of the breeds. In combination with the secondary panel of seven markers, 100% of the tested breeds exceeded 99% power of exclusion. The results of their analysis indicated that, on average, the primary panel meets the AKC’s needs for routine parentage testing, but that a combination of 10–15 genetic markers from the two panels could yield a universal canine panel with enhanced processing efficiency and reliability.

A higher value of exclusion probability in dog compared with the raccoon dog, was caused by a higher number of high polymorphic microsatellite markers description in dog, because of comparatively well known Canis familiaris genome. According to Breen et al. [1] the integrated marker FISH/RH map of the dog includes 4249 genetic markers. Hitte et al. [5] presented a recent, detailed comparative RH map of the dog genome containing about 10 000 canine markers. However, the knowledge about the raccoon dog genome is at a preliminary stage. In spite of this, it is possible to use the set of 15 markers in this study for routine parentage testing, but further research should be carried out to examine whether the set is practically effective. In conclusion, microsatellite markers are useful tools for parentage testing in raccoon dogs. The results of the present study provide new information about the raccoon dog.

CONCLUSIONS

1. On the basis of STR markers, exclusion probabilities ranging from 0.959 assuming one known parent to 0.998 assuming both known parents.

2. It is possible to use the set of 15 markers in this study for routine parentage testing in raccoon dogs.

ACKNOWLEDGMENTS

This study was carried out within a research project financed by the Ministry of Scientific Research and Information Technology in years 2004-2006, grant no. 2 P06D 006 26.

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Accepted for print: 6.08.2007

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