Sheep genotyping for PrP gene polymorphisms in rare Greek breeds

Loukia V. Ekateriniadou, Cynthia H. Panagiotidis, Anastasios Terzis, Konstantoula

Ploumi, Alexandros Triantafyllidis, Panagiotis Deligiannidis, Costas Triantaphyllidis,

Theodoros Sklaviadis

L. V. Ekateriniadou, PhD,

National Agricultural Research Foundation - Veterinary Research Institute,

570 08 Ionia, Thessaloniki, Greece

C. H. Panagiotidis, PhD,

A. Terzis, BA,

T. Sklaviadis, PhD

Prion Disease Research Group,

School of Pharmacy, Aristotle University of Thessaloniki

54 124 Thessaloniki, Greece

A. Triantafyllidis, PhD,

P. Deligiannidis, MS,

C. Triantaphyllidis, PhD,

School of Biology, Aristotle University of Thessaloniki

54 124 Thessaloniki, Greece

K. Ploumi, DVM,

National Agricultural Research Foundation, Agricultural Research Station of

Chalkidiki, 63200 Agios Mamas, Greece

1

Summary Current data indicate that at least 1-1.5% of Greek sheep flocks are affected by

scrapie (Greek Ministry of Rural Development and Food). The aim of the present

work was to determine the PrP genotype profile of rare Greek sheep breeds, in order

to find breeding animals resistant to scrapie. Thus, the PRNP genes of 388 sheep

from 13 rare breeds were analysed for polymorphisms at codons 136, 154, 171 and a

total of five haplotypes and eleven different genotypes were found. Genotypes of low

risk Group R1 (ARR/ARR) and Group R2 (ARR/ARQ, ARR/ARH, and ARR/AHQ)

appeared at high frequencies, i.e. 14.43% and 44.4%, respectively. As the frequency

of the VRQ haplotype, known to be associated with a high risk for scrapie, is very

low (0.4%), these breeds cannot be considered valine breeds. The highest frequency

of scrapie resistance genotypes was observed in the Kimi and Kalaritiko breeds. A

pilot-breeding programme to increase the frequency of scrapie resistant genotypes

will be established soon in Greece. The high frequency of scrapie-resistant genotypes

in the country’s rare breeds (a total of 58.83%) and the availability of recently

genotyped low-risk Class R1 and R2 individuals should greatly facilitate and expedite

such a breeding programme.

KEY WORDS: scrapie resistance, rare Greek sheep breeds, allelic (haplotype) frequency,

PCR, RFLP.

2

Introduction

Scrapie is a slowly progressive infectious disease of sheep and goats that causes

degeneration of the central nervous system. The main constituent of the infectious

agent is an aberrant isoform (PrPSc) of the normal cellular (PrPC) prion protein (PrP),

which is a cell–surface glycoprotein (Madec and others 1997, Prusiner 1998). In

sheep, polymorphisms at codons 136, 154 and 171 of the host gene that encodes PrP

are known to be closely linked to susceptibility to natural and experimental scrapie

(Laplanche and others 1993, Westaway and others 1994, Belt and others 1995, Smits

1995, Bossers and others 1996, Hunter and others 1996, Dawson and others 1998,

Baylis and others 2002).

Breed differences in both the frequencies of different PrP alleles and the correlation of

the alleles with the disease are known; however, some clear genetic rules have

emerged. According to the British National Scrapie Plan (Dawson 2003), a

classification system of five risk groups (R1-R5) has been established with R1

indicating a very low risk of scrapie and R5 indicating genotypes associated with the

highest susceptibility.

The PrP genotypes of a majority of the North-Central European, as well as some of

the Asian sheep breeds, have been studied and the polymorphisms characteristic of

each breed are known (Hunter and Cairns 1998, Bossers and others 1999,

Thorgeirsdottir and others 1999, Tranulis and others 1999, Vaccari and others 2001,

Siros and others 2002, Gombojav and others 2003). Breeds can be characterized as

either “simple” or “complex” types (Ikeda and others 1995, Hosie and Dawson 1996,

O’ Doherty and others 2001), based on plurality of their PrP allele variants and

resultant geneotypes.

3

In Greece, where the sheep population stands at about 9.5 million, there are twenty-

six native breeds (Rogdakis 2002), nine of which represent significant populations. As

of 2004, scrapie has been diagnosed in 70 Greek flocks, with all the scrapie-affected

sheep being crossbred individuals. As scrapie has not yet been found in any of the rare

Greek breeds, it is of great interest to determine if their seeming resistance can be

correlated to any of the known scrapie resistance-associated genotypes.

The Chios breed, a well-known Greek breed of sheep and one of the world’s best milk

producers, is found both in Northern Greece (Chios Mainland) and on the Aegean

island of Chios (Chios island). The Chios Mainland breed is the result of

crossbreeding between local sheep of the Chios Island breed and breeds from

Anatolia.

In addition to the Chios breed, Northern Greece is also home to the breeds of Thrace,

Katsika, Kozani, Florina and Sarakatsaniko. The Florina breed is raised in the

lowlands of Northern Greece and in some regions of Former Yugoslavian Republic of

Macedonia. The mountainous breeds of Thrace and Sarakatsaniko have many

similarities and probably share a similar origin (Kivircik). Finally, the Katsika breed

originated from the Zackel breed, as did the Kozani breed. It is worth noting that the

latter breed is particularly well-adapted to the hostile environmental conditions of

Greece.

Central Greece has four main native breeds of sheep, the Kalaritiko, Pilio,

Karamaniko and Argos breeds. The mountainous Kalaritiko breed originated from an

ancient local breed and the Italian breed, Comisana. The lowland Karamaniko breed

and another mountainous breed, that of Pilio, both originated from the breed of

Zackel. Argos sheep are a lowland breed of Central Greece.

4

In addition to the previously mentioned breed of Chios island, the Skopelos, Kimi and

Zakynthos breeds are also located mainly on islands. The breed of Skopelos, one of

the best known old Greek breeds, is found on the Sporades islands of Skopelos and

Skiathos. It is a crossbreed between local sheep and breeds from Anatolia. Kimi

sheep live in the mountains of Evia, an island just northeast of Athens and the

Zakynthos breed, having many similarities to the Italian breed Berkamasca, is found

on the Ionian island of Zakynthos. These insular island breeds share not only

exceptional breed characteristics such as high milk yield and excellent prolificacy, but

also the unfortunate designation of being in serious danger of extinction (Boyazoglu,

1991).

In response to European Union regulations requiring member countries to institute

scrapie resistance breeding schemes, a pilot-breeding programme to increase the

frequency of scrapie resistant genotypes will be established soon in Greece. To

facilitate and expedite such a breeding programme, the present study to determine the

PrP genotype profile of rare Greek sheep breeds and to identify “low-risk”

individuals suitable for such a programme was undertaken.

Materials and Methods

Sheep

Blood samples were obtained from a total of 388 healthy sheep, from 47 farms and 13

rare Greek breeds raised in three geographic areas of Greece: Northern Greece

[Thrace (THR), Chios Mainland (CMD), Katsika (KAT), Kozani (KOZ), Florina

(FLO) and Sarakatsaniko (SAR) a total of 176 sheep], Central Greece [Karamaniko

(KAR), Argos (ARG), Kalaritiko (KAL) and Pilio (PIL) a total of 117 sheep] and

some Greek islands (Chios (CIL), Kimi (KIM), Skopelos (SKO) and Zakynthos

5

(ZAK) a total of 95 sheep]. All these breeds have been characterized by the Greek

Ministry of Agriculture as rare Greek breeds.

DNA extraction

DNA was isolated from EDTA-treated blood by a standard phenol-chloroform

extraction protocol (Sambrook and others, 1989).

DNA amplification and Genotype analysis

Two pairs of primers (V1 and V2) designed by Yuzbasiyan-Gurkan and others (1999)

were used for the amplification of the PrP gene as described by Yuzbasiyan-Gurkan

and others (1999).

Detection of polymorphisms at codons 136 and 154. Polymorphisms at codons 136

and 154 were detected by RFLP analysis as described by Hunter and others (1993)

with one modification: for the DNA amplification, the V2 primers of Yuzbasiyan-

Gurkan and others (1999) were used. One hundred of the samples were checked by

both methods to verify our modified protocol.

Detection of polymorphisms at codon 171. The three common codon 171 alleles were

detected using two sets of primers (V1 and V2) by a series of two digestions. After

the DNA amplification with the V1 primers or the V2 primers, the PCR products

were treated with the enzyme BslI to detect the R allele and with the enzyme AccI to

detect the H allele, respectively. In both tests, the allele refractory to digestion was the

Q allele (Yuzbasiyan-Gurkan and others 1999).

Twenty-two samples (two from each genotype found) were confirmed with a capillary

electrophoresis method (Zslonai and others 2003).

6

Statistical Analysis

Genetic heterogeneity among the entire set of samples, pairwise differentiation among

population samples and deviations from Hardy-Weinberg equilibrium for each locus

were tested using probability tests or, where possible, exact tests. The program

GENEPOP 3.3 (Raymond and Rousset 1995) was used to perform the above analyses.

RESULTS

PrP allelic (haplotype) variants

Dimorphisms A/V and R/H in codons 136 and 154, as well as the trimorphism Q/R/H

in codon 171, of the gene encoding PrP were determined for 388 individual healthy

animals. Samples were obtained from 47 farms and 13 different breeds located in

three different geographical regions (Northern Greece, Central Greece and Greek

Islands). Five different PrP haplotype were observed, resulting in eleven separate

genotypes. The haplotype frequency distribution for each breed is shown in Table 1.

In general, most of the breeds examined possess three to four different haplotypes.

Two haplotypes, ARQ and ARR, are present at high mean frequencies overall (50.5%

and 36.7% respectively) in the population tested. The breeds with the highest

frequencies (>45%) of the scrapie-resistance associated ARR haplotype are Kimi,

Kalaratiko and Katsika.

With regard to the AHQ haplotype, it is the dominant haplotype (44.7%) in one breed

examined, that of Skopelos. In all other breeds tested, however, the AHQ haplotype

frequency is lower than 15%, and it is absent in the CIL, KAL and ARG breeds (in

the KOZ breed the AHQ allele is also absent but possibly due to the low sample size).

These latter breeds, i.e. CIL, KAL and ARG all have a significant (>10%) frequency

of the ARH polymorphism. Most importantly, perhaps, is the finding that the VRQ

7

haplotypes is present in only two breeds, FLO and SKO, and at a low frequency

(5.5%) in those.

The breeds of KAR, PIL, ZAK and THR have many similarities. As noted above,

none of them have the ARH haplotype but all do have a significant AHQ component.

Moreover, the ARR, ARQ and AHQ haplotype frequency profiles are very similar for

these three breeds. Consistent with these similarities, no pairwise differentiation test

among these breeds was statistically significant (p> 0.05).

Another similarity in breed frequency profiles was observed with the ARR, ARH and

ARQ haplotypes in the CMD and SAR breeds of Northern Greece, with the pairwise

test between these breeds revealing no statistical significance (p> 0.05). It is

interesting to note that the pairwise test between the CMD breed and the CIL breed,

from which it originated, is highly significant (p<0.001), indicating a substantial

genetic drift of the CMD breed.

PrP genotypes

As mentioned above, the present study of thirteen rare Greek sheep breeds detected

five PrP haplotype variants giving rise to eleven different genotypes. Table 2 shows

the obtained total results per breed, per geographical area along with the frequency of

each genotype. All breeds were in Hardy Weinberg equilibrium (P>0.05) except for

the Florina breed (P<0.001), due possibly to the complete absence of ARR/ARQ

heterozygotes in the population tested despite the high frequencies of the

corresponding homozygotes. Four genotypes (ARR/VRQ, AHQ/ARH, AHQ/AHQ

and AHQ/VRQ) were detected for the first time in Greece (Table 2). No VRQ/ VRQ

sheep were found in any of the breeds tested and, in fact, genotypes with valine at

codon 136 appeared only in the breeds of FLO and SKO.

8

In general, breeds of Northern Greece have a large variety of PrP genotypes (Table 2),

while those of Central Greece display a lesser variety. For purposes of scrapie-

resistance breeding, the most interesting breed of Northern Greece is that is that of

KAT which has a high frequency of ARR/ARR genotype (25.4%), a very good

proportion (46.1%) of genotypes with at least one ARR allele and a low frequency of

genotypes belonging to risk Group R3. Similarly, in Central Greece, the KAL breed

shows a high frequency (90.4%) of scrapie resistant genotypes (risk Group R1 and

R2). Of the Greek island breeds sampled, the best, in terms of its overall PrP

genotype profile, seems to be KIM, with a 40.0% frequency of ARR/ARR and

53.35% frequency of ARR/ARQ and ARR/AHQ genotypes (Table 2).

Table 2 also displays the general distribution of PrP genotypes in the 388 samples.

More than half of the individuals sampled (58.83%) are genotypes of the risk Groups

R1 and R2. Polymorphisms with valine in codon 136 appeared only at a frequency of

0.77% for the overall population tested. The comparison of the frequency of the

various genotypes in the three different regions is shown also in Table 2.

DISCUSSION

The results of the present study reveal the frequencies of the haplotypes, as well as

those of the genotypes, for the three scrapie susceptibility-associated PrP codons (136,

154 and 171) in 13 different rare Greek breeds, from three different geographical

areas. It is the first report of PrP genotype frequencies in these breeds. The analysis

revealed five different PrP allelic variants resulting in eleven different genotypes. The

VRQ haplotype, which is linked to the highest risk for scrapie, was present only in

two breeds (FLO and SKO, Table 1) and with an average frequency of 0.4% for the

total population tested. This is far lower than the VRQ frequencies found in other

9

European breeds (Belt and others 1995, Hunter and others 1996, Bossers and others

1999, Tranulis and others 1999). From these results it seems that Greek rare breeds

belong mainly to the group of non-valine breeds.

Of the 13 breeds examined, the lowest number of genotype variants per breed was

four, and that was seen with only four breeds (THR, KAR, PIL and KIM). All other

breeds have five or more (5 to 9) genotypes, indicating that they are rather complex.

With regard to phylogenetic evolution, the ARQ haplotype is the oldest PrP haplotype

in the ovis species (Thorgeisdottir and others 1999). High frequencies of this

haplotype were found in the breeds of KOZ, CMD, SAR and THR (75%, 67.8%,

67.2% and 64.6%, respectively). Despite the high frequency of the ARQ/ARQ

genotype in the breeds of CMD, THR, and KOZ (50.8%, 50%, and 50%, respectively)

no scrapie cases have been reported in purebred flocks of these breeds. In Greece, the

only cases of sheep affected by scrapie are those of crossbred flocks, mainly from

crosses with the breed of CMD (unpublished data).

The AHQ haplotype was present in the majority of breeds tested in this study. It was

not found, however, in the breeds of KOZ, ARG, KAL and CIL. This haplotype

frequency is highest, at 44.7%, in the breed of SKO. Interestingly, while the CMD

breed carries the AHQ allele (6.9%), the CIL breed, from which it originated, does

not.

It is difficult to hypothesize why there are such differences in the PrP haplotype

frequencies among the Greek breeds. Apart from possible selective advantages of

special alleles that have already been pointed out, one must bear in mind the effect of

the small populations of many of these breeds. Genetic drift phenomena are quite

possible, a fact that has also been suggested based on previous examination of these

Greek breeds by isozyme analyses (Rogdakis 2002, Koutsouli and Rogdakis 2002)

10

and Restriction Fragment Length Polymorphism analysis of mitochondrial DNA

(Katana 2002).

Importantly, four genotypes (ARR/VRQ, AHQ/ARH, AHQ/AHQ and AHQ/VRQ)

were detected for the first time in Greece (Table 2). Moreover, sheep with the

VRQ/VRQ genotype were not found in the present study of purebred breeds.

In accordance with the Greek Surveillance Programme for TSE’s Control (2004),

scrapie positive farms in Greece will be checked and all the animals that do not carry

at least one ARR allele will be stamped out. ARR/ARR rams, and ewes carrying at

least one ARR allele, would be kept, or introduced from an available breeding

population (once established), to such scrapie positive farms. It is worth noting, in this

regard, that in a recent survey of more than two hundred crossbred sheep from 16

scrapie-affected flocks across Greece (Billinis and others 2004), the frequency of

individuals carrying at least one ARR allele was reported to be less than 15 percent.

To support our Surveillance Programme for TSEs, a pilot-breeding programme for

increasing the frequency of the scrapie resistant genotypes will begin soon, based on

ARR/ARR rams and ewes carrying at least one ARR allele. Our current aim is to

identify and to provide “scrapie resistant” animals for breeding. The high frequency

of scrapie-resistant genotypes (58.83%) in Greek breeds presents a strong possibility

for selecting low-risk, recently PrP-genotyped animals for reproduction.

In parallel studies aimed at developing potential targets for scrapie therapy, all

haplotypes described in this work are currently being cloned and expressed to

generate a new battery of molecules to study the molecular mechanisms of self-

aggregation due to the differential conformations of the PrP variants.

11

ACKNOWLEDGEMENTS The authors thank the staff at CISA – INIA, Madrid, Spain who performed the

capillary electrophoresis experiments. The authors also thank the European Union for

partial funding of this study through QLRT-2001-00959 Small ruminant TSE

epidemiology pathology and diagnostic tests (SRTSENETWORK).

REFERENCES

1. BAYLIS, M., GOLDMANN, W., HOUSTON, F., CAIRNS, D., CHONG, A.,

ROSS, A., SMITH, A., HUNTER, N. & MCLEAN, A. R. (2002). Scrapie

epidemic in a fully PrP-genotyped sheep flock, Journal of General Virology

83, 2907-2914.

2. BELT, P. B.G.M., MUILEMAN, I. H., SCHREUDER, B. E.C., RUIJTER,

J.B.-D., GIELKENS, A. L. J. & SMITS, M. A. (1995). Identification of five

allelic variants of the sheep PrP gene and their association with natural

scrapie, Journal of General Virology 76, 509-517.

3. BILLINIS C, PSYCHAS V, LEONTIDES L, SPYROU V, ARGYROUDIS S,

VLEMMAS I, LEONTIDES S, SKLAVIADIS T, PAPADOPOULOS O.

(2004). Prion protein gene polymorphisms in healthy and scrapie-affected

sheep in Greece, Journal of General Virology 85, 547-54.

4. BOSSERS, A., SCHREUDER B. E.C., MUILEMAN, I.H., BELT P. B.G.M.

& SMITS M.A. (1996). PrP genotype contributes to determining survival

times of sheep with natural scrapie, Journal of General Virology 77, 2669-

2673.

5. BOSSERS, A., HARDERS, F. I. & SMITS, M. A. (1999). PrP genotype

frequencies of the most dominant sheep breed in a country free from scrapie,

Archives of Virology 144, 829-834.

6. BOYAZOGLU J.G. (1991). Milk breeds of sheep. In Genetic Resources of

Pig, Sheep and Goat. Ed. K. Maijala, Elsevier Science Publishers B.V.,

Amsterdam. Chapt. 18, pp 327-336.

7. DAWSON, M. (2003). The National Scrapie Plan: progress to date and future

developments. State Veterinary Journal 13, 40-46.

12

8. DAWSON, M., HOINVILLE, L. J., HOSIE B. D. & HUNTER, N. (1998).

Guidance on the use of PrP genotyping as an aid to the control of clinical

scrapie, Veterinary Research 149, 623-625.

9. DEPARTMENT FOR ENVIRONMENT, FOOD AND RURAL AFFAIRS ON

BEHALF OF THE GB AGRICULTURE DEPARTMENT (2001). National

Scrapie Plan for Great Britain, Crown Copyright 2001, PB5742.

10. GOMBOJAV, A., ISHIGURO, N., HOTIUCHI, M., SERJMYADAG, D.,

BYAMBAA, B. & SHINAGAWA, M. (2003). Amino acid polymorphisms of

PrP gene in Mongolian Sheep, Journal of Veterinary Medicinal Science 65,

75-81.

11. HOSIE, B. D. & DAWSON, M. (1996). Scrapie genotyping for Suffolk sheep.

Veterinary Research 138, 215-216.

12. HUNTER N., GOLDMANN W., BENSON G., FOSTER J. D. & HOPE J.

(1993). Swaledale sheep affected by natural scrapie differ significantly in PrP

genotype frequencies from healthy sheep and those selected for reduced

incidence of scrapie, Journal of General Virology 74, 1025-1031.

13. HUNTER, N., FOSTER, J. D., GOLDMANN, W., STEAR, M. J., HOPE, J.

& BOSTOCK, C. (1996). Natural scrapie in a closed flock of Cheviot occurs

only in specific PrP genotypes, Archives of Virology 141, 809-824.

14. HUNTER, N. & CAIRNS, D. (1998). Scrapie-free Merino and Poll Dorset

sheep from Australia and New Zealand have normal frequencies of scrapie-

susceptible PrP genotypes, Journal of General Virology 79, 2079-2082.

15. IKEDA, T., HORIUCHI, M., ISHIGURO, N., MURAMATSU, Y., KAI-

UWE, G. D. & SHINAGAWA, M. (1995). Amino acid polymorphisms of PrP

with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan,

Journal of General Virology 76, 2577-2581.

16. KATANA, E. (2002). Analysis of genetic structure of rare breeds of Greek

sheep. M.Sc. Thesis, Aristotle University of Thessaloniki, pp86, (in Greek).

17. KOUTSOULI, P. & ROGDAKIS E. (2002). Genetic structure of Greek sheep

breeds: III. Genetic variation within and between breeds. Animal Science

Review 30, 29-44, (in Greek).

18. LAPLANCHE, J. L., CHATELAIN, J., WESTAWAY, D., THOMAS, S.,

DUSSAUCY, M. M., BRUGERE-PICOUX, J. & LAUNAY, J. M. (1993).

13

PrP polymorphisms associated with natural scrapie discovered by denaturing

gradient gel electrophoresis, Genomics 15, 30-37.

19. MADEC, J. Y., VANIER, A., DORIER, A., BERNILLON, J., BELLI, P. &

BARON, T. (1997). Biochemical properties of protease resistant prion protein

PrPsc in natural sheep scrapie, Archives of Virology 142, 1603-1612.

20. O’DOHERTY, E., AHERNE, M., ENNIS, S., WEAVERS, E., ROCHE, J. F.

& SWEENEY, T. (2001). Prion protein gene polymorphisms in pedigree sheep

in Ireland, Research in Veterinary Science 70, 51-56.

21. PRUSINER, S. B. (1998). “Prions”, Proceedings of the National Academy of

Sciences, USA 95, 13363-13383.

22. RAYMOND, M. & ROUSSET, F. (1995). GENEPOP (version 3.3.):

population genetics software for exact tests and ecumenicism, Journal of

Heredity 86, 248-249.

23. ROGDAKIS, E. (2002). Greek native sheep breeds. Description, Phylogeny,

Genetic improvement and Management. Agrotypos Publisher. Co, Athens pp

208, (in Greek).

24. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1989). Molecular

Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor

Labroratory Press.

25. SIROS, W., KRAUS, M., SCHMOLL, F., ACHMANN, R. &

BAUMGARTNER, W. (2002). PrP genotyping of Austrian Sheep Breeds,

Journal of Veterinary Medicine A 49, 415-418.

26. SMITS, M. A. (1995). Identification of five allelic variants of the sheep PrP

gene and their association with natural scrapie, Journal of General Virology

76, 509-517.

27. “SURVEILLANCE PROGRAMMED FOR TSE’S CONTROL 2004”,

Department of Infectious and Parasitic Diseases, Directory of Animal Health,

General Veterinary Directory, Greek Ministry of Rural Development and

Food.

28. THORGEIRSDOTTIR, S., SIGUDARSON, S., MAR THORISSON, H.,

GEORGSSON, G. & PALSDOTTIR, A. (1999). PrP gene polymorphism and

natural scrapie in Icelandic sheep, Journal of General Virology 80, 2527-2534.

14

29. TRANULIS, M. A., OSLAND, A., BRATBERG, B. & ULVUND, M. J.

(1999). Prion protein gene polymorphisms in sheep with natural scrapie and

healthy controls in Norway, Journal of General Virology 80, 1073-1077.

30. VACCARI, G., PETRAROLI, R., AGRIMI, U., ELENI, C., PERFETTI, M.

G., DI BARI, M. A., MORELLI, L., LIGIOS, C., BUSANI, L., NONNO, R.

& DI GUARDO G. (2001). Prp genotypes in Sarda breed sheep and its

relevance to scrapie. Archives of Virology 146, 2029-2037.

31. WESTAWAY, D., ZULIANI, V., COOPER, C. M., DA COSTA, M.,

NEUMANN, S., JENNY, A. L., DETWILER, L. & PRUSINER, S. B. (1994).

Homogygosity for prion protein alleles encoding glutamine -171 renders sheep

susceptible to natural scrapie, Genes & Development 8, 959-969.

32. YUZBASIYAN-GURKAN, V., KREHBIEL, J. D., CAO, Y. & VENTA, P. J.

(1999). Development and usefulness of new polymerase chain reaction-based

tests for detection of different alleles at codons 136 and 171 of the ovine prion

protein gene, American Journal of Veterinary Research 60, 884-887.

33. ZSLONAI, A., ANTON, I., KUHN, C. & FESUS, L. (2003). Detection of

single-nucleotide polymorphisms coding for three ovine prion protein variants

by primer extension assay and capillary electrophoresis, Electrophoresis 24,

634-638.

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Table 1. Frequency distribution of PrP haplotypes (n – number of chromosomes)*

Breed Abr n PrP haplotype ARR ARH ARQ AHQ VRQ

Kozani KOZ 8 25.0% - 75.0% - -

Katsika KAT 78 48.7% 15.4% 28.2% 7.7% -

Thrace THR 48 31.2% - 64.6% 4.2% -

Florina FLO 30 40.0% - 43.3% 13.35% 3.35%

Chios, Mainland CMD 130 23.8% 1.5% 67.8% 6.9% -

Sarakatsaniko SAR 58 19.0% 6.9% 67.2% 6.9% -

Karamaniko KAR 24 37.5% - 58.3% 4.2% -

Argos ARG 62 30.6% 11.3% 58.1% - -

Kalaritiko KAL 84 54.8% 13.1% 32.1% - -

Pilio PIL 64 42.2% - 54.7% 3.1% -

Chios Island CIL 78 35.9% 11.5% 52.6% - -

Kimi KIM 30 66.7% - 23.3% 10.0% -

Skopelos SKO 38 26.3% - 23.7% 44.7% 5.3%

Zakynthos ZAK 44 38.7% - 54.5% 6.8% -

All breeds 776 36.7% 5.8% 50.5% 6.6% 0.4%

* The highest ARR haplotype frequencies, the lowest ARQ haplotype frequencies and the unusual high frequency of the AHQ haplotype are shown in bold.

16

Table 2. Comparison of the genotypes frequencies (%) of the thirteen different breeds in the three regions (North Greece, Central Greece, Islands)

GENOTYPES NORTHERN GREECE CENTRAL GREECE ISLANDS THR CMD KAT KOZ FLO SAR KAR KAL ARG PIL CIL KIM SKO ZAK

ARR/ARR 12.5 10.8 25.6 33.3 8.35 19.0 9.7 21.9 5.15 40.0 10.5 9.1

R1 Total 12.5 10.8 25.6 33.3 8.35 19.0 9.7 21.9 5.15 40.0 10.5 9.1 ARR/ARQ 29.2 23.2 20.5 50.0 34.6 50.0 50.0 25.8 40.6 43.6 46.6 15.8 50.0

ARR/ARH 1.5 23.1 3.4 21.4 16.1 17.9

ARR/AHQ 8.3 1.5 2.6 6.7 8.35 6.7 15.8 9.1

R2 Total 37.5 26.2 46.2 50.0 6.7 38.0 58.35 71.4 41.9 40.6 61.5 53.3 31.6 59.1ARQ/ARQ 50.0 50.8 12.8 50.0 33.3 37.9 33.3 4.8 41.9 31.3 28.2 5.3 27.3

ARQ/ARH 1.5 5.1 10.3 4.8 6.5 5.15

ARQ/AHQ 9.2 5.1 20.0 13.8 6.2 21.05 4.5

AHQ/ARH 2.6

AHQ/AHQ 1.5 2.6 6.7 21.05

R3 Total 50.0 63.0 28.2 50.0 53.3 62.0 33.3 9.6 48.4 37.5 33.35 6.7 47.4 831.ARR/VRQ 6.7

R4 Total .7 6AHQ/VRQ 10.5

R5 Total 10.5

17

18