A PRELIMINARY STUDY OF THE
INTERRELATION BETWEEN THE BODY WEIGHT OF RACCOON DOGS (Nyctereutes procyonoides) AND THEIR GENOTYPE ON THE BASIS OF
SELECTED MICROSATELLITE SEQUENCES*
Andrzej Jakubczak, Grażyna
Jeżewska-Witkowska,
Iwona Rozempolska-Rucińska, Brygida Ślaska
Department of Biological Basis of
Animal Production,
University of Life Sciences in Lublin, 13 Akademicka Street,
20-950 Lublin
* An
academic study financed from the research fund for 2008-2011, as research
project no. N N311 361435.
SUMMARY
INTRODUCTION
The history of fur
animal breeding dates back over a hundred years. Until recently animal farms
served as repositories for the fur industry, the breeders` chief aim being
maximum possible profitability. As science progressed the conception of
breeding has undergone changes. The drive to raise production profitability has
been linked with other aspects, not only those directly connected with proper
breeding. In the nineties research was undertaken on animal welfare as a factor
in the profitability of farm breeding [Śmielewska-Łoś, 2002].
The recent years have also seen a lot of attention devoted to aspects related
to the preservation of animal biodiversity. Research is underway to determine
the genetic variability of both wild and domestic animal varieties and
populations. Such monitoring is necesary as poor genetic diversity leads to a
drop in productivity and in animal well-being and to a deterioration of
reproduction related characteristics [Kania-Gierdzewicz, 2006; Szablewski i
Szwaczkowski, 2003]. The preservation of genetic variability can pose
particular problems in fur animal breeding since the farms mostly include more
or less restricted populations. In this case raccoon dogs whose population in
Poland is relatively sparse may be in particular danger. Along with the
development of molecular biology methods an intensive proliferation of research
on the identification of genes related to quantitative characteristics has
taken place. The elaboration of a marker genome map begins with the identification
of the greatest possible number of polymorphic genetic markers and with the
detection of the way those markers are conjugated with a given gene
[Świtoński i Cholewiński, 2000]. In the Canidae family the research concerns mostly dogs and foxes [Richman
et al., 2001], as well as raccoon dogs [Ślaska et al., 2007]. However, the
search for loci of quantitative characteristics should also involve other
fur-bearing animal species, which, in turn, could make for an increased
breeding development of characteristics crucially important for the breeder.
One characteristic that greatly affects breeding profitability is the size of
furs which is largely related to the body weight of animals [Filistowicz et
al., 1999].
In the present study an
attempt was made to analyse the genetic diversity of raccoon dogs bred at a
reproduction farm, on the basis of selected microsatellite sequences. The
population under analysis was divided into two groups of animals differing in
body weight. The division was assumed to identify possible differences in the
genetic structure of both the raccoon dog groups and, consequently, facilitate
the selection of the microsatellite loci which could be used for further
analysis as potential genetic body weight markers in the animals.
THE MATERIAL AND METHODS
The genetic material for
the analysis was sampled from the raccoon dogs bred at the reproduction farm.
Blood was drawn from posterior veins of 173 living animals into vacuum test
tubes containing an anticoagulant – EDTA.
A polymerase
chain reaction was conducted onboard the MJ Research PTC-225 thermocycler in
0,2 mL thin-walled test tubes. The starters used for the reaction were designed
according to data gathered from studies on the dog genome: CPH1, CPH3, CPH6,
CPH8, CPH11, CPH14, FH2004, FH2010, FH2016, FH2019, FH2054, FH2097, FH2140,
FH2141, FH2152, FH2164, FH2168, FH2320, ZuBeCa4 and ZuBeCa6. The standard
reaction mixture contained the following ingredients: ~150ng matrix DNA; 2.5μL 10xPCR
buffer for the PCR reaction, 2.0μL mixture of each of the four
nucleotides, 0.5μL of each starter per reaction, 1U DNA
polymerase and 1.5μL MgCl2. The standard reaction
comprised 35 successive cycles consisting of denaturation (95˚C for
30sec), the starter addition (58˚C for 30sec) and the elongation of the
newly synthetised DNA thread (72˚C for 60sec). The cyclic reaction was
preceded by a 10 minute preliminary denaturation at 95˚C. Additionally, in
order to complete the polymerase activity a final 10 minute elongation was
performed. For several PCR reaction starters it was necessary to modify the
standard reaction conditions in order to enhance the efficiency of the reaction
itself, as well as to obtain a highly specific product. The modifications
included: changes in the temperature of respective starter addition stages and
that of the final reactional elongation; in the number of cycles and in the
MgCl2 concentration. In order to optimise the starter addition
temperature a test PCR reaction was carried out using a gradient block to
determine the most appropriate temperatures for the respective starters
(Tab.1).
The
length of the selected microsatellite sequences was identified with the ABI
3100 Avant Genetic Analyzer automatic sequencer that employs laser scanning of
fluorescent tagged DNA threads. For the analysis onboard the sequencer the
starters were divided into 6 multiplexes within which they differed in the
built-in fluorescent tag or were considerably divergent as to the size of the
final PCR product. The results were analysed using the sequencer related Gene
Mapper 3.5 software.
Table. 1. The PCR reaction conditions.
|
Locus |
MgCl2 concentration |
Biological stain |
Addition temperature |
|
CPH1 |
2.5 |
NED |
58˚ C |
|
CPH3 |
2.0 |
6-FAM |
58˚ C |
|
CPH6 |
2.0 |
6-FAM |
58˚ C |
|
CPH8 |
3.0 |
VIC |
58˚ C |
|
CPH11 |
3.0 |
VIC |
58˚ C |
|
CPH14 |
1.5 |
NED |
62˚ C |
|
FH2004 |
1.5 |
6-FAM |
58˚ C |
|
FH2010 |
1.5 |
VIC |
59˚ C |
|
FH2016 |
1.5 |
VIC |
58˚ C |
|
FH2019 |
1.5 |
6-FAM |
58˚ C |
|
FH2054 |
4.3 |
VIC |
58˚ C |
|
FH2097 |
1.5 |
6-FAM |
58˚ C |
|
FH2140 |
2.5 |
6-FAM |
58˚ C |
|
FH2141 |
1.5 |
NED |
58˚ C |
|
FH2152 |
1.5 |
VIC |
58˚ C |
|
FH2164 |
1.5 |
VIC |
58˚ C |
|
FH2168 |
2.0 |
NED |
58˚ C |
|
FH2320 |
2.0 |
6-FAM |
58˚ C |
|
ZuBeCa4 |
1.5 |
NED |
68˚ C |
|
ZuBeCa6 |
1.5 |
NED |
68˚ C |
All the animals under
analysis were weighed after developing adult coats. Considering the body weight
of the raccoon dogs the populations were divided into two groups. The first
group was comprised of raccoon dogs weighing up to 10kg (the L group), the
second of those heavier than 10kg (the C group). The low body weight group (L)
consisted of 83 animals whereas the C (high body weight) group comprised 90
animals.
In the study the allele
and genotype frequencies at the respective loci were calculated using the SAS
GENETIC [2000] statistical package.
THE RESULTS AND
DISCUSSION
In the raccoon dog
population under analysis 20 microsatellite loci were genotyped, out of which 2
were monomorphic (CPH1, ZuBeCa6) and one (FH2141) proved exceptionally
difficult to analyse on account of small differences in allele lengths.
Therefore, 17 microsatellite loci
were left for further analysis (Table 1). The number of alleles at the
respective loci ranged from 2 to 8
(FH2152 i FH2164). The lowest allele number was identified at the CPH6 and
FH2320 loci.
The allele number
identified for each locus often
varies from species to species or even from one canine variety to another
[Klukowska et al. 2001, 2003; Verardi et al., 2006; Altem et al., 2001; Puja et
al., 2005]. An example is provided by the CPH6 locus in raccoon dogs at which definitely fewer alleles were
identified as compared with the dog, red fox and arctic fox populations. In the
case of the above species between 7 and 11 alleles were identified [Klukowska
et al. 2003]. A similar situation was valid for the CPH3 locus at which 12 alleles were identified in dogs [Klukowska et al.
2003], while only 5 in the analysed raccoon dog population. Verardi et al.
[2006] identified 14 alleles at the said locus
in dogs and grey wolves. Klukowska et al. [2001] recorded over 10 alleles at
the CPH8, FH2004 and FH2140 loci whereas only 3 to 7 alleles were identified in
the analysed group of raccoon dogs.
When analysing the
results attention should be paid to the allele distribution in the racoon dog
population depending on the body weight of the animals. The C and L group
frequencies are similar in the majority of cases, although in some situations
greater numbers of one or two alleles in these groups can be noted. At the
CPH11 locus the frequency of the A
allele was approximately twenty times higher in the animals with lower body
weights. Similar results were obtained for the A allele at the CPH8 locus, for the B allele at FH2097 and
for D at the FH2164 locus. In the
group of animals with high body weights a definite preponderance of the A
allele was identified at the FH2016 locus
and of the B allele at the FH2019 locus.
Moreover, 8
group-specific alleles were identified for the C and L groups in the material
under analysis. Alleles characteristic of the animals with lower body weights
were identified at the CPH3, FH2010, FH2019, FH2140, FH2164 and FH2004 loci. In the group of raccoon dogs with
high body weights the H allele was identified at the FH2152 locus and the F allele at the ZuBeCa4 locus.
The presence of
group-specific alleles in the animals with low and high body weights could
prompt further studies to find genetic markers of this characteristic. At the
same time, considering very low frequency of the specific alleles, the research
should be continued with particular attention paid to animals clearly differing
in body weight. Genetic map elaboration
on the basis of reference families where the parental generation animals
originate from varieties (or lines) divided by a maximum possible genetic
distance is impracticable in the case of fur-bearing animals. Within a
respective species it is impossible to single out lines clearly differing with
regard to useful characteristics, while divergent selection in order to obtain
maximum heterozygosity in the F1 generation is not economically viable and
hence not practiced. Therefore, a solution of sorts can be the use for such
analyses of individual animals that belong to one population but clearly differ
in the scope of useful characteristics.
When discussing the
results attention should be paid to the allele frequency at the respective loci. Despite the observed polymorphism,
in most cases a clear difference in the allele frequency at a given locus can be determined. Only at the
FH2164 and CPH8 loci no definite
preponderance of one or two alleles was observed. In the remaining cases the
frequency of a given allele amounted to between 40 and 80%. A particularly high
frequency was observed for the B allele at locus
FH2320. 2 alleles were present at this locus,
yet the proportion of the animals with the A allele genotype amounted to
between only 15 and 19 for both groups. This was similar in the case of the
FH2010 locus. The B allele
constituted 80% of the total of the 4 identified alleles, both in the C and L
groups.
This tendency can
unfortunately lead to a future elimination of rare alleles from the population
and, as a result, to a rise in homozygosity. An example of this can be the
FH2010 locus at which over 70% of
animals were characterised by the homozygotic BB distribution.
The loci at which group-specific genotypes of the analysed raccoon dogs
were observed are juxtaposed in Table 2. Out of the 17 analysed loci 13 had genotypes characteristic of
the L or C groups. The greatest number of specific genotypes of the low body
weight animals was observed at loci
FH2152 (6 genotypes) and ZuBeCa4 (7). In the C group of raccoon dogs the number
of such genotypes ranged from 1 to 3. At 5 out of the total analysed loci characteristic genotypes were
present only in one of the animal groups.
The presence of specific
genotypes in the groups of animals differing in body weight did not only stem
from the presence or absence of a characteristic allele. At the CPH8, FH2016,
FH2054, FH2097 and FH2168 loci C or L
group-specific alleles were not observed previously, though chracteristic
genotypes were identified for these loci.
This fact does not also seem to be related to a definite difference of allele
frequency in both the raccoon dog groups. An example can be the CPH8 locus at which the DG and FG genotypes
were identified exclusively in the L group while the D, E and G allele
frequencies were similar. On the other hand, the frequency of the B allele at
the FH2054 locus was higher in the
high body weight group of the raccoon dogs, which seemed to suggest that the
presence of specific genotypes in this group could be linked to the B allele.
However, the FH2054 locus genotypes
which were specific to the C group animals did not have the B allele but only
the A, D and C alleles.
The incidence of animals
with genotypes defined as specific was very low in both groups. Such animals constituted
between 1 and 6% of the low body weight raccoon dogs and between 1 and 3% of
those with high body weights.
CONCLUSION
Although at this stage
of research it is impossible to determine the reasons for the presence of
characteristic alleles and genotypes in raccoon dogs that differ in body
weight, further possible analyses searching for genetic markers of this
characteristic should involve those loci
at which such specificity has been observed. Simultaneously, it seems necessary
to monitor the genetic variability of the raccoon dog population since a clear
preponderance of one of the alleles at the majority of the analysed loci can be symptomatic of a tendency
towards a rising homozygosity of the animals.
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Table 2.
Characteristic genotypes of the analysed groups of raccoon dogs.
|
LOCUS |
The
number of specific genotypes |
GROUP C |
GROUP L |
|||
|
GROUP C |
GROUP L |
The genotype |
The frequency |
The genotype |
The frequency |
|
|
CPH3 |
3 |
0 |
A/E |
0,0135 |
|
|
|
B/C |
0,0135 |
|||||
|
E/E |
0,0676 |
|||||
|
CPH_8 |
2 |
4 |
A/C |
0,0270 |
A/F |
0,0112 |
|
E/F |
0,0135 |
C/C |
0,0225 |
|||
|
|
D/G |
0,0112 |
||||
|
F/G |
0,0225 |
|||||
|
FH2010 |
2 |
1 |
B/D |
0,0143 |
A/C |
0,0230 |
|
C/D |
0,0429 |
|||||
|
FH2016 |
2 |
1 |
A/E |
0,0615 |
C/C |
0,0132 |
|
E/E |
0,0308 |
|||||
|
FH2019 |
3 |
0 |
A/D |
0,0145 |
|
|
|
B/F |
0,0145 |
|||||
|
C/D |
0,1594 |
|||||
|
FH2054 |
2 |
0 |
A/C |
0,0145 |
|
|
|
A/D |
0,0580 |
|||||
|
FH2097 |
2 |
1 |
A/B |
0,0145 |
D/E |
0,0120 |
|
A/E |
0,0145 |
|||||
|
FH2140 |
2 |
2 |
A/B |
0,0222 |
A/C |
0,0250 |
|
A/E |
0,0222 |
C/E |
0,0125 |
|||
|
FH2152 |
1 |
6 |
A/B |
0,0213 |
B/C |
0,0123 |
|
B/D |
0,0247 |
|||||
|
C/E |
0,0370 |
|||||
|
C/H |
0,0123 |
|||||
|
D/F |
0,0123 |
|||||
|
D/H |
0,0247 |
|||||
|
FH2164 |
0 |
1 |
|
|
A/D |
0,0341 |
|
FH2168 |
3 |
1 |
B/E |
0,0149 |
C/F 0,0118 |
|
|
D/D |
0,0149 |
|||||
|
D/E |
0,0149 |
|||||
|
E/E |
0,0597 |
|||||
|
ZuBeCa4 |
3 |
7 |
B/B |
0,0317 |
A/F |
0,0278 |
|
B/E |
0,0159 |
A/F |
0,0278 |
|||
|
D/E |
0,0635 |
C/E |
0,0278 |
|||
|
|
C/F |
0,0139 |
||||
|
D/D |
0,0139 |
|||||
|
E/E |
0,0278 |
|||||
|
E/F |
0,0139 |
|||||
|
FH2004 |
1 |
0 |
B/C |
0,0143 |
|
|