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FOLIA UNIVERSITATIS AGRICULTURAE STETINENSIS
Folia Univ. Agric. Stetin. 2007, Agric., Aliment., Pisc., Zootech. 257 (3), 157–162
Brygida ŚLASKA, Grażyna JEŻEWSKA
GENETIC DIVERSITY IN THE RACCOON DOG
(NYCTEREUTES PROCYONOIDES GRAY, 1834)
AN ANALYSIS BASED ON MOLECULAR MARKERS∗
ANALIZA ZMIENNOŚCI GENETYCZNEJ JENOTÓW
(NYCTEREUTES PROCYONOIDES GRAY, 1834)
NA PODSTAWIE MARKERÓW MOLEKULARNYCH
Department of Biological Basis of Animal Production, Agricultural University of Lublin
ul. Akademicka 13, 20-049 Lublin, Poland
Abstract. In recent years, RAPD-PCR molecular markers, which are widely used in population
studies, have become a useful tool for population-genetic studies on many animal species. The
aim of this study was to analyse the genetic diversity of raccoon dogs using selected RAPD-PCR markers. Statistical analysis allowed testing selected primers in terms of their usefulness
for further genetic analyses. The markers that generate polymorphic DNA fragments include
OPA 09 and OPA 10. The results obtained have created a basis for further detailed genetic research aimed at understanding the genetic diversity at the level of the entire genome. It is
highly advisable to use OPA 09 or OPA 10 in studies on the within-population variability of raccoon dogs. An analysis of OPA 09 band patterns has revealed a sex-specific DNA fragment.
site.
Key words: raccoon dog, RAPD-PCR, genetic diversity.
Słowa kluczowe: jenot, RAPD-PCR, zmienność genetyczna.
INTRODUCTION
Genetic markers have been used in many research areas for decades; however, in the
early 1990s, when we observed an impressive progress in the techniques of molecular biology and cytogenetics, new groups of genetic markers were distinguished at the DNA
level. The technique named Random Amplification of Polymorphic DNA, or RAPD, developed by Williams et al. (1990) and Welsh and McClelland (1990) enabled simultaneous
detection of multiple polymorphic loci in the entire genome. RAPD is a variant of PCR,
a commonly used technique of in vitro amplification of DNA fragments. The method involves short arbitrary nucleotide sequences (arbitrary primers). The sensitivity of this
method is very high. RAPD-PCR markers are widely used in population studies. They are
usually applied in preliminary stages of a genome research project, especially when the
genetic diversity of a given species has not been fully explained. As soon as a genomic
DNA polymorphism is located using RAPD markers, detailed genetic analyses can by
started using other markers, e.g. AFLP.
∗
The research was financed by the Ministry of Science and Information Society during 2004–2006,
project No. 2 P06D 006 26.
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RAPD markers are widely used in the research on nuclear DNA variability between individuals and populations within a species, and the fact that the technique is simple and relatively inexpensive probably contributes to this success. The genome of raccoon dog is relatively poorly described and no reports have been published dealing with the studied RAPD
markers used in Nyctereutes procyonoides.
The aim of this study was to analyse the genetic diversity of raccoon dogs basing on selected RAPD-PCR markers.
MATERIAL AND METHODS
The analysis involved the breeding stock (F0 generation) of the three-generation raccoon
dogs population managed at a breeding farm located in south-eastern Poland, 208 animals
in total. DNA was extracted, subjected to quantitative and qualitative evaluation, and PCRamplified using the Taq PCR Core Kit (Qiagen). The reaction mix (30 µl) contained 80 ng
DNA, 3 µl PCR buffer, 4.2 µl Q solution, 200 µM of each nucleotide, 0.2 µM of arbitrary primer,
25 mM MgCl2, and 1 U Taq polymerase. The following primers were used for amplification:
OPA 02, OPA 07, OPA 09, OPA 10, OPA 12, and OPA 13 (Proligo Primers and Probes).
The amplification conditions were as follows: initial denaturation step (94°C, 5 min) followed by 46 cycles of denaturation (94°C, 1 min), annealing (36°C, 2 min), and extension
(72°C, 1 min), concluded by final extension step (72°C, 10 min) and cooling to 4°C. The
RAPD-PCR products were separated on a 2% agarose gel (with bromophenol blue loading
solution). The resulting gels were visualised under UV light (using a transilluminator) and
the images were stored by means of SCION IMAGE program (Syngen Biotech). We used
GeneRuler 50bp DNA Ladder (Fermentas) for the resulting DNA fragments sizing. The statistical analysis of the bands with respect to sex included the following parameters: mean
number of bands per specimen, number of loci, L (Lynch 1990), Nei's genetic diversity index, h (Nei 1973), Shannon's index, I (Lewontin 1972), and average heterozygosity,
H (Stephens et al. 1992).
RESULTS AND DISCUSSION
Available literature on the subject allows concluding that the markers we selected have never
been used in raccoon dogs before. As a result of the RAPD analysis, we obtained amplification
products of all the primers we used; however, OPA 13 was rejected due to both poor quality of
gel band pattern and its low efficiency. According to Pradeep et al. (2005), the marker was characterised by a high polymorphism in studies on the silkworm, Bombyx mori.
The amplified genetic material of all the primers used in our study, except for OPA 13,
was visible under UV light in the form of fluorescent bands (Fig. 1, 2).
Table 1 presents the characteristics of the analysed genetic markers of raccoon dogs. The
markers OPA 02, OPA 07, and OPA 12 produced between six (OPA 07) and nine (OPA 02)
bands; however, no polymorphism in these markers within the studied group of animals has been
observed (Table 1, Fig. 1), which may be a consequence of their origin from a single lot of 200
raccoon dogs imported from Finland in 1979 (Jeżewska et al. 1991).
Genetic diversity in the raccoon dog...
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Fig. 1. Electrophoretogram of RAPD-PCR analysis in raccoon dogs with use of OPA 07 primer (first
lane from the left – Gene Ruler 50 bp DNA Ladder)
Rys. 1. Elektroforegram analizy RAPD-PCR u jenota z wykorzystaniem startera OPA 07 (pierwsza
ścieżka od lewej – marker wielkości)
Fig. 2. Electrophoretogram of RAPD-PCR analysis in raccoon dogs with use of OPA 10 primer (first lane
from the right – Gene Ruler 50 bp DNA Ladder; second lane from the right – negative control)
Rys. 2. Elektroforegram analizy RAPD-PCR u jenota z wykorzystaniem startera OPA 10 (pierwsza
ścieżka od prawej – marker wielkości; druga ścieżka od prawej – kontrola negatywna)
Despite the fact that a small number of other imported raccoon dogs supplemented the
original stock in subsequent years, we may observe a low genetic diversity of the studied
group of animals. The markers (OPA 07 and OPA 12) that were used in the studies by
Pradeep et al. (2005) to distinguish two lineages of Bombyx mori showed high polymorphism, contrary to what we found in this study.
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Among the studied primers, only OPA 09 and OPA 10 produced polymorphic bands. In the
observed population, OPA 09 produced DNA fragments in six lengths, from ~400 to ~1050 bp,
whereas OPA 10 (Fig. 2) resulted in 14 bands, from ~180 to ~1200 bp. The polymorphic loci
comprised 66.67% and 35.71% for, respectively, OPA 09 and OPA 10. Average numbers of
bands ranged between 3.0 to 11.4 for, respectively, OPA 09 and OPA 10 (Table 1).
Table 1. Characteristics of selected RAPD-PCR markers in raccoon dogs
Tabela 1. Charakterystyka wybranych markerów RAPD-PCR u jenotów
Mnb
Primer
Starter
Nucleotide sequence
Sekwencja nuklotydów
5’→3’
Size ranges of DNA
fragments (bp)
Zakres wielkości
amplifikowanych
DNA (pz) *
Tnb
Poly
males
samce
females
samice
OPA 02
TGCCGAGCTG
170–1050
9
0
9.0
9.0
OPA 07
GAAACGGGTG
300–1000
6
0
6.0
6.0
OPA 09
GGGTAACGCC
400–1050
6
4
4.6
3.0
OPA 10
GTGATCGCAG
180–1200
14
5
11.3
11.4
OPA 12
TCGGCGATAG
270–1000
8
0
8.0
8.0
OPA 13
CAGCACCCAC
–
–
–
–
–
Tnb – total number of bands – całkowita liczba krążków, Poly – number of polymorphic bands – liczba polimorficznych krążków, Mnb – mean number of bands – średnia liczba krążków.
* Approximate values – wartości przybliżone.
Mamuris et al. (2002) used the RAPD technique to study genetic diversity among six European populations of hare, as well as to analyse the genetic structure within the Greek population. The number of loci as well as the number of polymorphic bands detected by the markers
OPA 02 and OPA 09 were higher in the population of hare compared with the studied population of raccoon dogs, whereas similar numbers of loci were found with use of OPA 10.
Statistical description of the selected genetic markers are presented in Table 2. The
mean values of Nei's genetic diversity (h) ranged between 0.183 and 0.389. The highest
value of this index was scored for OPA 10 for females, whereas Shannon’s index exceeded
0.5 for both sexes considered jointly for both OPA 09 and OPA 10 (Table 2). The unbiased
estimate of loci number (L) ranged from 1.335 (females, OPA 09) to 1.692 (females, OPA 10).
The average number of loci for both sexes for OPA 09 and OPA 10 remained
at a similar level (Table 2).
The primers we have selected exhibit high polymorphism and are widely used in studies
on other species of animals or plants (Ur-Rahman et al., 1997; Horng et al., 2000; Mamuris
et al., 2002; Pradeep et al. 2005). In our study, the average heterozygosity (H) was 0.344
and 0.395 for OPA 10 and OPA 09, respectively, with the values being lower compared
with those reported by other authors for other animal species.
An analysis of the band patterns has revealed DNA fragments characteristic for sex.
A band of ~450 bp (OPA 09) was scored for males only. This may be a consequence
Genetic diversity in the raccoon dog...
161
of sex-specific DNA sequences present in the Y chromosome. Antoniou and Skidmore
(1995) have demonstrated that the Y-chromosome-specific bands can be amplified by
RAPD-PCR with the OPA 06 primer. However, those results have not been confirmed by
Horng and Huang (2000), who, using OPA 06 and 100 other primers, did not score bands
that are sex-specific in the bovine genome.
Table 2. Statistical characteristics of selected RAPD-PCR markers in raccoon dogs with respect to sex
Tabela 2. Charakterystyka statystyczna wybranych markerów RAPD-PCR u jenotów, z uwzględnieniem płci
Primer
Starter
OPA 09
Sex
Płeć
h
I
L
male
samice
0.287
0.421
1.520
female
samce
0.213
0.335
1.335
0.385
0.571
1.650
male
samice
0.183
0.260
1.341
female
samce
0.389
0.569
1.692
0.368
0.540
1.655
Both sexes – Łącznie
OPA 10
Statistical indices – Wskaźniki statystyczne
Both sexes – Łącznie
h – Nei's genetic diversity index – wskaźnik różnorodności genetycznej Neia, I – Shannon’s index –
indeks Shannona, L – number of loci – liczba loci.
The results of the RAPD analysis using OPA 09 and OPA 10 primers, which generate
polymorphic DNA fragments, indicates that they can be applied in the studies on genetic
variability and polymorphism in raccoon dogs. Their use is particularly advisable in the
studies on the variability with the populations.
CONCLUSIONS
1. The RAPD-PCR analysis of raccoon dogs resulted in amplification products of the following
primers: OPA 02, OPA 07, OPA 09, OPA10, and OPA12. All the mentioned markers have
been positively evaluated in terms of the quality of the gel band pattern and efficiency.
2. Statistical analysis of the selected primers allowed determining their usefulness
in further genetic analyses. The markers generating polymorphic DNA fragments
in raccoon dogs include OPA 09 and OPA 10.
3. The results obtained give grounds for detailed genetic analyses aimed at an evaluation
of the genetic variability at the level of the entire genome. Application of OPA 09 or OPA
10 is highly advisable for studies on the diversity within the populations of raccoon dogs.
4. The analysis of the bands has revealed sex-specific DNA fragments. A band of approx.
450 bp (OPA 09) was present only in males.
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Streszczenie. W ostatnich latach użytecznym narzędziem w badaniach populacyjno-genetycznych różnych gatunków zwierząt stały się markery molekularne RAPD-PCR, które są szeroko
wykorzystywane w badaniach populacyjnych. W pracy przeprowadzono analizę zmienności
genetycznej jenotów na podstawie wybranych markerów RAPD-PCR. Analiza statystyczna wybranych do badań starterów pozwoliła na określenie ich przydatności do dalszych analiz genetycznych. Do markerów generujących polimorficzne fragmenty DNA zaliczyć należy OPA 09
i OPA 10. Uzyskane wyniki stanowią podstawę do szczegółowych analiz genetycznych, mających na celu ocenę zróżnicowania genetycznego na poziomie całego genomu. Użycie OPA 09
lub OPA 10 jest w dużym stopniu celowe w badaniach zmienności wewnątrzpopulacyjnej jenotów. Analiza wzorów prążkowych OPA 09 wykazała istnienie fragmentu DNA charakterystycznego dla płci.