Bull Vet Inst Pulawy 59, 321-326, 2015 DOI: 10.1515/bvip-2015-0047
Polymorphism of the PrP prion protein gene in Polish Merino and old-type Polish Merino from Brylewo flock Roman Niżnikowski1, Artur Oprządek2, Grzegorz Czub1, Marcin Świątek1, Dariusz Kubasik3, Paweł Brzoza3, Krzysztof Głowacz1, Magdalena Ślęzak1 1Division
of Sheep and Goat Breeding, Faculty of Animal Science, Warsaw University of Life Sciences, 02-786 Warszawa, Poland 2The Agricultural Property Agency, 00-215 Warszawa, Poland 3Breeding Center „GARZYN”, 64-120 Krzemieniewo, Poland
[email protected] Received: January 08, 2015
Accepted: June 17, 2015
Abstract The study was conducted on Polish Merino (143♀ and 39♂) and old-type Polish Merino sheep (176♀ and 61♂) in Brylewo flock (Wielkopolskie Province). The examined sheep were at the age of one year. Prion protein (PrP) genotype was determined in all animals. In both breeds four alleles (ARR, ARQ, AHQ, VRQ) were found. Highly significant effect of breed and insignificant impact of gender within breed was observed in regard to the frequency of occurrence of alleles and genotypes susceptible to classical scrapie in the sheep. Eight different PrP genotypes in Polish Merino and nine genotypes in old-type Polish Merino were identified. Very high frequency of ARR/ARR genotype in old-type Polish Merino and high frequency of ARR/ARQ genotype in Polish Merino were found, with relatively significant frequency of occurrence of the genotypes containing VRQ allele. In old-type Polish Merino, three animals (of both genders) had VRQ/VRQ genotype. Breeding work involving elimination of animals encoding valine at codon 136, and introduction of rams with ARR allele to the population increased the frequency of occurrence of ARR/ARR genotype and ARR allele in the population of old-type Polish Merino. To improve the distribution of the genotypes genetically resistant to scrapie in the flock of Polish Merino only rams with ARR/ARR genotype were left. This guarantees an increase in the frequency of occurrence of genotypes genetically resistant to scrapie in the offspring.
Keywords: sheep, PrP, alleles, genotypes, polymorphism.
Introduction The European Parliament has introduced regulations for prevention, control, and eradication of transmissible spongiform encephalopathies (1, 10). PrP prion protein gene was recognised as responsible for the appearance of scrapie in sheep. In this gene, series of polymorphisms at codons 136, 154, and 171 were identified, and these polymorphisms are considered to be responsible for genetic resistance to scrapie (3-6, 8). ARR allele guarantees the lowest sensitivity to scrapie, while allele encoding valine (VRQ) is regarded as responsible for the susceptibility to this disease (3, 4, 8, 9). In Poland, studies were performed to monitor the
presence of alleles determining susceptibility to scrapie in many breeds. (6, 7, 9, 13). The results showed the absence of alleles encoding valine in Polish Heath Sheep and various frequency of its presence in other breeds (6, 7, 9, 13). It is worthwhile to conduct breeding work which aims to eliminate VRQ allele from the sheep population, and at the same time increase the frequency of ARR allele, in order to improve the genetic resistance to this disease within individual herds and sheep breeds. That direction of research was adopted in this study conducted on two breeds: Polish Merino and old-type Polish Merino sheep. For comparative purposes, the selected breeds were sufficiently genetically distant from each other for
© 2015 R. Niżnikowski et al. This is an open access article distributed under the Creative Commons AttributionNonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/) Unauthenticated Download Date | 7/14/15 5:38 PM
322
R. Niżnikowski et al./Bull Vet Inst Pulawy/59 (2015) 321-326
many years before appearing in a herd-book. Hence, it was interesting to determine how the PrP genetics developed in these diverse breed populations.
Material and Methods The study was conducted on ewes and rams up to the age of one year, selected to the herd: Polish Merino (143♀ and 39♂) and old-type Polish Merino sheep (176♀ and 61♂) originating from the Brylewo flock (Wielkopolskie Province) (Table 1.). The sheepfold in Brylewo appeared in the herd-book under the number 001, established in 1919 for Merino sheep kept there to the present day. First of all, rams from Brylewo flock as well as some imported rams were used for tupping. The last time imported rams were used was in 2009. During each year of the study, blood was collected from the jugular vein into tubes containing EDTA. DNA was isolated from blood leukocytes. In order to obtain high quality DNA suitable for multiple use, blood was purified from the heme compounds, which were erythrocyte lysis products. DNA was isolated by chromatography on mini-columns of silicate (A&A Biotechnology, Poland), and subsequently served as a template DNA for amplification of polymorphic gene allele fragment. Sample genotyping was performed with KASPar® system (www.kbioscience.co.uk), which uses a single nucleotide polymorphism (SNP) based on primers listed in Table 2. A high reliability of
SNP method compared to the sequencing method was proved by Green et al (2). Based on the reading of genotyped DNA samples within the ewes and rams, distribution of alleles and genotype frequency within individual years of research was determined. Every year, each sheep with alleles encoding valine was removed from the flock. In the case of rams, only ARR allele carriers were left in the herd for further breeding. The obtained material was evaluated in terms of the frequency of alleles and genotypes occurrence between the breeds, as well as within the breeds, and between genders and years of study. SPSS v.21 software was used for statistical calculations (11). The impact of breed and within breeds, sex, and year of study on the frequency of occurrence of alleles and genotypes were assessed using χ2 test. The effect of breed in the case of allele values was read from 3 degrees of freedom, and for genotypes – 8 degrees of freedom. Within Polish Merino sheep, gender was rated with 3 degrees of freedom for alleles, and 7 degrees for genotypes as well as for the year of study – 9 degrees for alleles and 21 degrees for genotypes. Within old type of Polish Merino sheep, gender was rated with 3 degrees of freedom for alleles, and 8 degrees for genotypes as well as for the year of study – 9 degrees for alleles and 24 degrees for genotypes. For all data, separately for alleles and genotypes, the impact of breed on its frequencies was rated with one degree of freedom. The results are presented in Tables 3-6.
Table 1. Experimental material used in the study Breed Polish Merino
Old-type Polish Merino
♀
♂
♀
♂
2009
72
14
36
14
2010
19
18
48
19
2011
26
3
36
11
2012
26
4
56
17
143
39
176
61
Year of study
Total Total
182
237
Table 2. The primers and SNP of the prion protein gene Locus
PRNP prion protein
Primers 3’-5’
CACAGTCAGTGGAACA AGCC/ CTTTGCCAGGTTGGGG
SNP
Changes
Localization
AY909542:g.385A>G
A/G
exon 3
AY909542:g.386G>T
G/T
exon 3
AY909542:g.479C>T;
C/T
exon 3
AY909542:g.534G>A
G/A
exon 3
Unauthenticated Download Date | 7/14/15 5:38 PM
R. Niżnikowski et al./Bull Vet Inst Pulawy/59 (2015) 321-326
323
Table 3. Frequency of PrP allele occurrence in tested sheep breeds Alleles Total ARR ♀ Polish Merino ♂ ♀ Old-type Polish Merino ♂ ♀ Total ♂ Total
ARQ
AHQ
VRQ
n
159
89
16
22
286
%
55.6
31.1
5.6
7.7
100.0
n
42
23
4
9
78
%
53.8
29.6
5.1
11.5
100.0
n
250
64
5
33
352
%
71.0
18.2
1.4
9.4
100.0
n
88
20
3
11
122
%
72.1
16.4
2.5
9.0
100.0
n
409
153
21
55
638
%
64.1
24.0
3.3
8.6
100.
n
130
43
7
20
200
%
65.0
21.5
3.5
10.0
100.0
n
539
196
28
75
838
%
64.4
23.4
3.3
8.9
100.0
Table 4. Frequency of PrP allele occurrence within breeds depending on the year of study Alleles Total ARR
ARQ
AHQ
VRQ
n
103
44
6
19
172
%
59.9
25.6
3.5
11.0
100.0
n
37
26
5
6
74
%
50.0
35.1
6.8
8.1
100.0
n
28
23
5
2
58
%
48.3
39.7
8.6
3.4
100.0
n
33
19
4
4
60
%
55.0
31.6
6.7
6.7
100.0
n
201
112
20
31
364
%
55.2
30.8XX
5.5XX
8.5
100
n
71
18
1
10
100
%
71.0
18.0
1.0
10.0
100.0
n
82
20
4
28
134
%
61.2
14.9
3.0
20.9
100.0
n
77
12
1
4
94
%
81.9
12.7
1.1
4.3
100.0
n
108
34
2
2
146
%
74.0
23.2
1.4
1.4
100.0
n
338
84
8
44
474
%
71.3XX
17.7
1.7
9.3NS
100.0
n
539
196
28
75
838
%
64.4
23.4
3.3
8.9
100.0
2009
2010 Polish Merino 2011
2012
Total within breed
2009
2010 Old-type Polish Merino 2011
2012
Total within breed
Total Effect of year within breed: Polish Merino – no statistical significance; Old-type Polish Merino – XX – P ≤ 0.01; NS – no statistical significance
Unauthenticated Download Date | 7/14/15 5:38 PM
324
R. Niżnikowski et al./Bull Vet Inst Pulawy/59 (2015) 321-326
Table 5. Frequency of PrP genotype occurrence in tested sheep breeds Genotypes ARR/ ARR
♀ Polish Merino ♂
♀
Oldtype Polish Merino
♂
♀ Total gender ♂
ARR/ ARQ
ARR/ AHQ
ARQ/ ARQ
ARQ/ AHQ
VRQ/ ARR
VRQ/ ARQ
VRQ/ AHQ
VRQ/ VRQ
Total
n
36
64
9
7
5
14
6
2
0
143
%
25.2
44.7
6.3
4.9
3.5
9.8
4.2
1.4
0.0
100.0
n
9
13
3
4
1
8
1
0
0
39
%
23.1
33.3
7.6
10.3
2.6
20.5
2.6
0.0
0.0
100.0
n
90
47
2
4
2
21
7
1
2
176
%
51.1
26.8
1.1
2.3
1.1
11.9
4.0
0.6
1.1
100.0
n
30
17
3
1
0
8
1
0
1
61
%
49.3
27.9
4.9
1.6
0.0
13.1
1.6
0.0
1.6
100.0
n
126
111
11
11
7
35
13
3
2
319
%
39.5
34.9
3.4
3.4
2.2
11.0
4.1
0.9
0.6
100.0
n
39
30
6
5
1
16
2
0
1
100
%
39.0
30.0
6.0
5.0
1.0
16.0
2.0
0.0
1.0
100.0
n
165
141
17
16
8
51
15
3
3
419
%
39.4
33.7
4.1
3.8
1.9
12.1
3.6
0.7
0.7
100.0
Total
Table 6. Frequency of occurrence of PrP genotypes within breeds depending on the year of the study Genotypes ARR/ AHQ
ARQ/ ARQ
ARQ/ AHQ
VRQ/ ARR
VRQ/ ARQ
VRQ/ AHQ
VRQ/ VRQ
Total
ARR/ ARR
ARR/ ARQ
n
27
31
3
4
2
15
3
1
0
86
%
31.4
36.0
3.5
4.7
2.3
17.4
3.5
1.2
0.0
100.0
n
6
18
3
2
2
4
2
0
0
37
%
16.2
48.7
8.1
5.4
5.4
10.8
5.4
0.0
0.0
100.0
2009
2010 Polish Merino
n
4
16
3
2
2
1
1
0
0
29
%
13.8
55.2
10.4
6.9
6.9
3.4
3.4
0.0
0.0
100.0
n
8
12
3
3
0
2
1
1
0
30
%
26.7
40.0
10.0
10.0
0.0
6.7
3.3
3.3
0.0
100.0
n
45
77
12
11
6
22
7
2
0
182
0
100
2011
2012 Total within breed
%
24.7
42.3
XX
6.6
X
6.0
X
3.2
NS
12.2
NS
3.9
NS
1.1
NS
n
24
13
1
2
0
9
1
0
0
50
%
48.0
26.0
2.0
4.0
0.0
18.0
2.0
0.0
0.0
100.0
n
28
9
2
2
1
15
6
1
3
67
%
41.8
13.4
3.0
3.0
1.5
22.3
9.0
1.5
4.5
100.0
n
30
12
1
0
0
4
0
0
0
47
%
63.9
25.5
2.1
0.0
0.0
8.5
0.0
0.0
0.0
100.0
2009
Oldtype Polish Merino
2010
2011 n
38
30
1
1
1
1
1
0
0
73
%
52.0
41.0
1.4
1.4
1.4
1.4
1.4
0.0
0.0
100.0
n
120
64
5
5
2
29
8
1
3
237
2012 Total within breed
%
50.6
XX
1.2
NS
27.0
2.1
2.1
0.8
12.2
3.3
0.4
n
165
141
17
16
8
51
15
3
3
100 419
%
39.4
33.7
4.0
3.8
1.9
12.2
3.6
0.7
0.7
100.0
Total Effect of year within breed: Polish Merino – no statistical significance; Old-type Polish Merino P ≤ 0.01. Differences between breeds include genotypes: XX – P ≤ 0.01; X – P ≤ 0.05; NS - no statistical significance
Unauthenticated Download Date | 7/14/15 5:38 PM
R. Niżnikowski et al./Bull Vet Inst Pulawy/59 (2015) 321-326
Results The distribution of alleles in assessed sheep breeds within gender are presented in Table 3. Highly significant differences were demonstrated in regard to alleles distribution between breeds, but no statistical significance was noted between the genders within a specific breed. Four alleles (ARR, ARQ, AHQ, and VRQ) were found in both breeds. Moreover, large diversity in the frequency of alleles in the breeds was identified. Polish Merino was characterised by a lower frequency of ARR and VRQ alleles and higher frequency of ARQ and AHQ alleles compared to oldtype Polish Merino. Frequencies of alleles within breeds depending on the year are presented in Table 4. The impact of year on the frequency of alleles distribution was highly significant in Polish Merino, but no significant effect was observed in old-type Polish Merino. Different levels of ARR allele frequency were observed in both breeds in subsequent years. Lower frequency of VRQ allele was observed in 2011-2012 compared to 2009-2010. Frequency distribution of scrapie genotypes is presented in Table 5. Gender within breed did not significantly affect the frequency of genotype, while the differences in this regard between both breeds were highly significant. In Polish Merino 8 genotypes were found: ARR/ARR, ARR/ARQ, ARR/AHQ, ARQ/ARQ, ARQ/AHQ, VRQ/ARR, VRQ/ARQ, and VRQ/AHQ. In old-type Polish Merino in addition to the aforementioned genotypes, VRQ/VRQ genotype was also found. In old-type Polish Merino, ARR/ARR genotype had the highest frequency, while in Polish Merino ARR/ARQ genotype had a higher frequency. These were the most common genotypes in both breeds, which should be considered as a positive result. Unfortunately, a relatively high frequency of genotypes containing VRQ in both breeds were observed. In old-type Polish Merino, three sheep with VRQ/VRQ genotype were found. Frequency of scrapie genotypes depending on the year within the breed are presented in Table 6. The impact of year was highly significant only in old-type Polish Merino. In that breed, an increase in the frequency of the ARR/ARR genotype and the genotypes with ARR allele, as well as the progressive reduction of VRQ allele were observed. In 2012, just after the study ended, all animals with VRQ allele were eliminated.
Discussion Old-type Polish Merino requires breeding work to eliminate the VRQ allele carriers, even though ARR allele frequency was high. However, in the case of both breeds, the frequency of the VRQ allele occurrence was much higher than it was previously demonstrated in national and foreign research in relation to the Merino sheep and other breeds (3-9, 13). On the other hand, the
325
high frequency of ARR allele in old-type Polish Merino is in agreement with the results obtained by other authors (8, 13), who performed their studies on Berrichon du cher. However, very high and unprecedented in other reports frequency of VRQ allele distribution. which was observed in 2010 in the oldtype Polish Merino required further investigation (7, 8, 9, 13). Herd-book analysis showed that in 2009, a number of purchased rams, which were genotyped later, had been used for tupping. The analysis found that three of them were VRQ heterozygous. Immediately after, these rams were eliminated from the herd, but they had managed to affect their offspring, which were obtained and assessed in 2010 in old-type Polish Merino. That is why interventions in breeding work, involving the elimination of the VRQ allele carriers from sheep population, is necessary. Despite that incident, the breeding programme used in the oldtype Polish Merino which led to a reduction of the frequency of VRQ allele occurrence, should be implemented into breeding programme of Polish Merino, held in the same flock. The beneficial effect of the applied breeding programme, which eliminated genotypes encoding valine, was considered as reasonable in practically all previous reports (3-9, 13). Animals were eliminated, but their sole occurrence indicates the need to refine the assumptions of the breeding programme. The range of genetic diversity in both breeds was similar to the results of other studies (3-8, 9). The results obtained indicate the need for stricter criteria for breeding work by qualifying for breeding only ARR/ARR genotype rams and eliminating VRQ allele carriers. Suggestions about reducing the conditions encoding valine at codon 136, appearing in a number of sources, were recognised through breeding work as possible to achieve (3, 6, 7, 9). The applied old-type Polish Merino`s breeding programme proved to be useful and worth recommending in breeding work to eliminate genotypes described as susceptible to classical scrapie from the sheep population. This enables to fulfil the goals consistent with the EU legislation (1, 10, 11). In the case of Polish Merino from Brylewo flock, there is a need to refine the criteria for breeding by introducing into the flock only breeder rams with ARR/ARR, and by annual introduction of ewes which are noncarriers of the genotype encoding valine at codon 136. This guarantees ARR allele in further offspring. Such action should result in a time-efficient creation of the sheep population with alleles genetically resistant to classical scrapie, which is worthwhile to recommend for in the breeding work carried out in other sheep breeds. In conclusion, the results of the present study demonstrated as follows: a highly significant impact of breed and insignificant impact of gender within breed on the frequencies of distribution of alleles and genotypes of classical scrapie in Polish Merino and oldtype Polish Merino sheep; the presence of four alleles (ARR, ARQ, AHQ, and VRQ) in both breeds, eight
Unauthenticated Download Date | 7/14/15 5:38 PM
326
R. Niżnikowski et al./Bull Vet Inst Pulawy/59 (2015) 321-326
genotypes of PrP prion protein in Polish Merino, and nine genotypes in old-type Polish Merino; very high frequency of ARR/ARR genotype in old-type Polish Merino and high frequency of ARR/ARQ genotype in Polish Merino with a relatively significant frequency of VRQ allele, and three sheep of old-type Polish Merino with VRQ/VRQ genotype. Breeding work involving the elimination of vectors encoding valine at codon 136 and the introduction of rams with ARR allele to population was suggested in order to increase the frequency of occurrence of ARR/ARR genotype and ARR allele in the population of old-type Polish Merino. In the case of Polish Merino sheep, a significant improvement of the frequency of genotypes which are known to be genetically resistant to scrapie can be achieved by refining the criteria of selection for breeding by only leaving breeders with ARR/ARR. This will guarantee an increase of the frequency of occurrence of genotypes genetically resistant to scrapie in future offspring. Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article. Animal Rights Statement: The authors declare that the experiments on animals were conducted in accordance with local Ethical Committee laws and regulations as regards care and use of laboratory animals.
2.
4.
5.
6.
7.
8.
9.
10.
11.
References 1.
3.
Commission decision of 13 February 2003 laying down minimum requirements for the establishment of breeding programmes for resistance to transmissible spongiform encephalopathies in sheep (notified under document number C (2003) 498) Green B.T., Heaton M.P., Clawson M.L., Laegreid W.W.: Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep. Mamm Genome 2006, 17, 1121–1129.
12. 13.
Kaal L.M.T.E., Windig J.J.: Rare sheep breeds and breeding for scrapie resistance in the Netherlands. Book of Abstracts of the 56th Annual Meeting of the European Association for Animal Production. Wageningen Academic Publishers, Wageningen, 2005, p. 375. Van Kaam J.B.C.H.M., Finocchiaro R., Vitale M., Portolano B., Vitale F., Caracappa S.: PrP allele frequencies in non –infected Valle del Belice and infected cross –bred flocks. Book of Abstracts of the 56th Annual Meeting of the European Association for Animal Production. Wageningen Academic Publishers, Wageningen 2005, p. 376. Lühken G., Buschmann A., Groschup M.H., Erhardt G.: Prion protein alleleA136H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Merinoland sheep. Arch Virol 2004, 149, 1571–1580. Niżnikowski R., Lühken G., Strzelec E., Lipsky S., Popielarczyk D., Erhardt G., consortium ECONOGENE: PRNP genepolymorphism in 136, 154 and 171 codons in Polish sheep breeds. Med Weter 2006, 62, 938–941. Niżnikowski R., Głowacz K., Czub G., Ślęzak M., Świątek M.: Polymorphism of the PrP prion protein gene in national sheep breeds with mixed wool, Polish Merino and European mouflon (Ovis aries musimon). Nauka Przyr Technol 2013, 7, 59. Palhiere I., Brochard M.I., Moazami-Goudarzi K., Laloe D., Amigues Y., Bed’hom B., Neuts E., Leymarie C., Pantano T., Cribiu E.P., Bibe B. Verrier E.: Impact of strong selection for the PrP major gene on genetic ariability of four French sheep breeds (Open Access publication). Genet Sel Evol 2008, 40, 663–680. Rejduch B., Knapik J., Piestrzyńska-Kajtoch A., KozubskaSobocińska A., Krupiński J.: Frequency of genotypes in the PrP prion protein gene locus in the Polish sheep population. Acta Vet Hung 2009, 57, 30–49. Regulation (EC) No 260/2003of 12 February 2003 amending Regulation (EC) No 999/2001 of the European Parliament and of the Council as regards the eradication of transmissible spongiform encephalopathies in ovine and caprine animals and rules for the trade in live ovine and caprine animals and bovine embryos. Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. SPSS 21.0 for Windows, IBM Ltd Wiśniewska E., Mroczkowski S.: Different breeding strategies for scrapie resistance depending on breed –specific PrP allele and genotype frequencies in the Polish sheep. Züchtungskunde, 2009, 81, 180–189.
Unauthenticated Download Date | 7/14/15 5:38 PM
