l KEYWORDS Dairy sheep; Greek breeds; Ovar- DRB1 exon2 alleles; genetic polymorphism; parasites PAGES 1 – 19 REFERENCES Vol. 3 No. 1 (2016) ARTICLE HISTORY Submitted: November 20, 2015 Revised: January 30, 2016 Accepted: February 02 2016 Published: February 08 2016 CORRESPONDING AUTHOR Panagiota Koutsouli, Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Greece. 75, Iera Odos, Votanikos, Athens 11855, Greece. e-mail: panagiota@aua.gr phone: +30 210 5294440 fax: +30 210 5294442 JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Article Allelic polymorphism of Ovar- DRB1 exon2 gene and parasite resistance in two dairy sheep breeds. Stavros Spetsarias1, Panagiota Koutsouli1*, Georgios Th eodorou1, Georgios Theodoropoulos2, Iosif Bizelis1. 1 Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens 11855, Greece. 2 Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens 11855, Greece. ABSTRACT. The Ovar-DRB1 gene locus is one of the most polymorphic genes of the Major Histocompatibility Complex (Ovar-MHC) and holds a functional role to antigen presentation. The aim of this study was: a) to describe the Ovar -DRB1 locus variability in two dairy Greek sheep breeds and b) to investigate associations between this variability with resistance to gastrointestinal parasitosis. Blood and faecal samples were collected from 231 and 201 animals of Arta and Kalarrytiko breeds, respectively. The identification of alleles was performed using the sequence–base method. Faecal egg counting (FEC) of the gastrointestinal parasites and measures of blood plasma pepsinogen levels were performed in order to evaluate parasitological parameters. From this study in the overall examined animals, thirty-nine Ovar-DRB1 alleles were identified, among them, ten new alleles, reported for the first time in the literature. In Arta breed a total of twenty-four alleles were found. Among the detected alleles, ten were breed specific and five were new. Regarding the Kalarrytiko breed, twenty-nine alleles were found, fifteen of them were unique and nine were new. The studied breeds differed in their allelic profile, with only 12 common from the total of 134 different recorded genotypes. A higher number of animals with high parasitic load and high plasma pepsinogen values were found in Kalarrytiko. Associations between Ovar-DRB1 alleles with FEC values were found with certain heterozygous genotypes to present significantly reduced FEC values. The large number of detected alleles with low frequencies and the fact that the majority of animals were heterozygous, make hard to find strong associations. S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 2 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 1 Introduction The Major Histocompatibility Complex of sheep (Ovar- MHC) is a gen e complex associated mainly with immunological properties. Οvar-DRB1 en codes the b1 structural core of an Ovar- MHC class II molecule, forming a large part of the binding region of the an tigenic peptide (PBR) of the molecule (Marsh and Bodmer, 1993). Compared to all other gen es of the Ovar- MHC, the Ovar-DRB1 gene locus exhibits the largest variability (Andersson and Rask, 1988). This variability is localized at specific positions of exon 2 (270 bp). Ovar-MHC class II molecules present an tigenic peptides deriv ed from extracellular proteins and parasites on th e T-cell receptor (TCR) for CD4+T-lym phocytes (Germain and Margulies, 1993; Fremont et al., 1996) which are expressed at higher concentrations on the surface of macrophages and B-lymphocytes (Outteridge et al., 1996). Since the polymorphic pattern of PBR amino acids determines its conformation and electric charge, strong hydrophobic bonds are created only with specific antigenic peptides (Th orsby, 1999). Thus, different Ovar -DRB 1 alleles favour the binding of differen t antigenic peptides in the PBR Ovar-MHC class II molecules (Rudensky et al., 1991). Consequen tly, the polym orphism of Ovar-DRB1 exon 2 locus is associated with the variability in resistance or susceptibility to diseases of a sheep population. Many studies have shown that there is a correlation between Ovar-DRB1 alleles and parasite resistance, particularly to nematodes (Outteridge et al., 1996; Paterson et al., 1998; Charon et al., 2002; Sayers et al., 2005; Hassan et al., 2011). This is also the case against other infectious agents, such as pathogenic bacteria and viruses (Larruskain et al., 2010). Gastroin tes tinal nematodes are th e most importan t source of infestation in sheep worldwide. Anthelmin thic treatm ent with pharmaceu tical compounds is a relatively expensive m ethod of combating th e infestation, which leads in many cases to the developm ent of anthelmin thic resistance (Roos, 1997). Additionally, a growing concern related to residues of pharmaceu tical compounds which pass down into the food chain, have driven a large number of farmers to adopt breeding prog rams which rely less on drug deliv ery. For these reasons, the selection of sheep breeds and individuals resistan t to parasitic infestation is promoted in many countries. Recen tly, Ovar- MHC loci hav e attracted the research interest as candidate D NA markers since several gen es from all classes of Ovar-MHC are involved in resistance or susceptibility to various sheep nematodes (Dukkipati et al., 2006). Generally, all research studies have mainly been carried out in breeds of meat and wool type. Since g enetic differences exis t between th e indigenous Greek and foreign sheep breeds (Rogdakis, 2002), there was g reat interest in exploring the Ovar-DRB1 genetic variability and the possible iden tification of new alleles in th e Greek breeds. For the pu rposes of this study, two dairy Greek breeds with differen t management and productive characteristics were selected in order to describe th e variability of Ovar-DRB1 gene locus: i) Arta, a breed with high milk yield and litter size and ii) Kalarrytiko, a rare breed, with low milk yield bu t well adapted to harsh environmen tal conditions. Considering that associations have been documented between certain Ovar-DRB1 alleles and parasitological param eters, there was further in terest to investigate, whether similar associations among Ovar-DRB1 alleles and parasites could be found in order to use this knowledge as a criterion for selection prog rams to create animals resistan t to gastrointestinal parasites, avoiding the adminis tration of an tiparasitic drugs. S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 3 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 2 Materials and Methods 2.1 Animals and sample collection A total of 43 2 blood samples were taken randomly from two dairy Greek sheep breeds. Th e breeds reared at the region of Western Greece, in flocks w ith a mean size of approximately five hundred animals. The flock of Arta breed (sample size=23 1) was kept at 414 m altitude (Vassiliki, Etoloakarnania Prefectu re), grazing in private pastures throughou t th e year under a semi-extensive farming s ystem. The Arta ew es were fed supplemen tary concen trates according to th e requiremen ts of their productive s tage, milked mechanically twice per day and produ ced on average 276.08 ± 9.73 L of milk within a whole lactation. The flock of Κalarrytiko (sample size= 201) was kept at 838 m altitude (Small Gotista, Ioannina Prefecture) grazing in communal pastures under an extensive traditional farming system. During th e winter sufficien t herbage is not available and supplemen tary feed was given from October to March. Kalarrytiko ewes were milk ed by hand and produced on average 80.45 ± 3.02 L of milk. Blood samples were taken from each animal by jugular vein puncture, in order to use whole blood for genomic DNA isolation and plasma for evaluation of pepsinogen levels. Faecal samples were obtain ed from th e rectum of each animal to es timate parasitic burdens. 2.2 Genotyping of Ovar-DRB1 exon2 region 2.2.1 Identification of alleles by direct sequencing A touchdown PCR, using as substrate the g enomic DNA from each animal, was used for th e amplification of a 340 bp long DNA fragmen t con taining th e exon 2 of the Ovar-DRB1 locus. The total reaction volume was 25 μl and contained 110 -120 ng genomic DNA, 10 pmol/μl of th e DRB1-330 primers (forward prim er): ATTAGCCTCYCCCCAGGAGKC and DRB1-329 (revers e primer): CACCCCCGCGCTCACCTCGCCGC (Ballingall and Tassi, 2010) and 0.5 U Phusion® High - Fidelity DNA polymeras e (NEB). Initially, the reaction occurred in two hybridization cycles at the following temperatu res: 67°C, 66°C, 65°C, 64°C and con tinued with 30 cycles at 63°C. Touchdown PCR products w ere directly sequenced at Laboratories of CEMIA SA (Larissa), with the Genetic Analyzer ABI3730xl (Applied Bios ystems). The sequencing cycle kit BigDye Terminator v3.1 (Applied Biosystems ) was used for the reaction, according to manufacturer’s guidelines. Two suitable software packag es: “Gen eStudioTMProfessional sequence Analysis Software” and “Nu cleotide BLAST Basic Local Alignment Search Tool” w ere used for th e identification of alleles after sequencing. 2.2.2 Identification of novel alleles by cloning and sequencing Regarding the iden tification of candidate alleles (new alleles), cloning and sequencing was performed in th ree successive steps: At th e first step, for those animals presen ting new alleles, a simple PCR was performed with a differen t reverse prim er compared to th e respectiv e primer used in the Touchdown PCR. A 364 bp long DNA fragm ent containing the exon 2 locus of the Ovar-DRB1 gene was amplified. The total reaction volu me was 50 μl containing : a) 85-95 S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 4 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 ng genomic DNA, b) 10 pmol/μl of the forward primer DRB1-330, c) 10 pmol/μl of the revers e primer DRB1-313: ACACACTGCTCCACACTGG (Ballingall and Tassi, 2010) and d) 3,5 U Taq DNA polym erase (Fermen tas). The reaction was pe rformed at +62°C, for 37 cycles. At the s econ d step, due to th e fact that the PCR product consisted of the allelic pair of genotypes, cloning was performed, using a culture of susceptible bacterial cells, in order to initially seg regate them, and subsequ ently to find the candidate allele considered as new. Using th e ligation reaction, th e isolated 364 bp long DNA fragm ent con taining th e exon2, was inserted to th e plasmid pBluescript SK (2961 bp) and used for the transformation of susceptible Escherichia coli cells of the JM109 strain. The localization of cloned alleles of exon2 within a bacterial culture was performed with a Colony PCR. The products of Colon y PCR were incubated with 10U of th e restriction en zyme RsaI (NEB) in order to ascertain, from the dig es tion patterns in which of th e received transform ed bacterial colonies, clones of th e two alleles were contained. The third and final s tage of the process was th e selection of th e colonies containing the new alleles, from which the recom binant plasmids were isolated, in order to sequence th e inserts and to finally confirm the sequence of the alleged new allele as new indeed. The isolation of the plasmids was performed using standard reagent kitsNucleoSpin® Plasmid (Macherey - Nagel). 2.3 Parasitological analyses 2.3.1 Count of faecal parasite reproductive elements Faecal egg counts (EPG) w ere conducted for strongyle-type eggs cumulatively and for Nematodirus sp., Strongyloides sp., Trichuris sp., and Moniezia sp. eggs separately (Thienpon t et al., 1986). Faecal coun ts of coccidian oocysts were also enumerated. 2.3.2 Estimation of pepsinogen levels in blood plasma Plasma pepsinogen in each animal was determined by th e method of Hirschowitz (1955) as modified by Korot’ko & Islyamova (1963). Pepsinogen is the inactive p recursor of pepsin, on e of th e main proteolytic enzymes of digestiv e system. Pepsin acts as peptidase, catalysing th e hydrolysis of peptide bonds from the aromatic amin o acids (tyrosine, phen ylalanine, tryptophan). In th e presence of parasites larvae th e intense inflammation of the gastric mucosa of the abomasum, affects the function of the gastric glands and HCl production is decreased. As a consequen ce pepsinogen could n ot be converted to pepsin and inserts in blood circulation, increasing the plasma peps inogen levels. In brief, blood plasma samples were collected from the supernatant after centrifugation (3000 rpm, 30 min) and six standard solutions of tyrosine were prepared. A s taining solution of F olin -ciocalteau was added in each sample. The optical density of blank, standard tyrosine solu tions and samples were m easured at 560 nm in a spectrophotometer. The concen tration of samples in pepsinog en was given as values of mI.U. tyrosine. S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 5 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 2.4 Statistical analysis The genotypic frequencies (observed and expected estimations) and the allelic frequencies were estimated with GENEPOP (Rousset, 2008). The abov e software was also used to m easure the observed and expected heterozygosity and hom ozyg osity. The appropriate samplin g function z was used to test for significant differences betw een ratios of heterozygosity. Th e null hypothesis was Η0: pA–pΚ=0 while the alternative one was Η1: pA ≠ pΚ. Sample sizes were large en ough (>100) and th e null hypoth esis was rejected wh en z > z 0,05 or z < - z 0,05. Associations between genotypes and parasite categories were perform ed at first, with a χ 2 contingency Pearson test but then th e simulated Monte Carlo method was preferred becaus e in many cases the number of observations was fewer than 5. Th e EPG values or F EC (Faecal Egg Counts) and pepsinogen concen tration values (mI.U. tyrosine) declined from normality . Their analysis was done using the Bartlett’s tes t for checking homogen eity of the sample an d the non pa rametric tes t of Kruskal-Wallis. Associations betw een Ovar-DRB1 genotypes and FEC values of parasites were investigated using non parametric tests (Mann-Whitney or Kruskal-Wallis depending from the number of levels for th e specific alleles ) considering th e fixed effect of genotype for th e specific i allele with three lev els (firs t level: animals without the specific allele, second level: animals with on e copy of th e i allele and third lev el: animals with two copies of the i allele). The same analysis was perform ed in order to reveal associations between Ovar-DRB1 genotypes and pepsinogen levels. The above analyses were carried ou t a) on th e total examin ed animals for alleles which are common betw een the s tudied breeds and b) on a within breed basis for alleles which are unique for each breed. 33 Results 3.1 Genotyping analysis and polymorphism of Ovar-DRB1 exon2 gene 3.1.1 Genotypic frequencies One hundred and thirty four gen otypes were recorded in the total of 432 examined animals. Eighty-one (81) and sixty-five (65 ) differen t gen otypes w ere found among the 23 1 an d 201 individuals from Arta and Kalarrytiko breeds, respectively. Twelv e common g enotypes were found between breeds which are presented in supplementary Table A. Among th e common genotypes only th ree were homozygous for alleles: DRB1*0402, DRB1*0901 and DRB1*2101. Homozygous genotypes among the examined animals were quite few, only eleven (11) and ten (10) in Arta and Kalarrytiko, respectively. A different set of genotypes with relative high frequency was observ ed in each breed. Th e most common genotypes and in descending order for Arta breed w ere: DRB1*0101-DRB1*0102, DRB1*0102-DRB1*1001, DRB1*0102-DRB1*2101, DRB1*0101-DRB1*0901, DRB1*0102-DRB1*0901, DRB1*0102-DRB1*0402. On the other hand in Kalarrytiko breed the genotypes with higher frequ ency and in descending order w ere: DRB1*0402-DRB1*0702, DRB1*0702-DRB1*0702, DRB1*0102-DRB1*0402, DRB1*0702-DRB1*1602. Am ong th e recorded genotypes th e S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 6 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 percentag e of new alleles was 23 % and 54 % for Arta and Kalarrytiko breed, respectively. A percentag e of 52 % and 49 % of Arta and Kalarrytiko genotypes, respectiv ely, w ere represen ted by only one individual. Half of the g enotypes in both breeds had extrem ely low genotypic frequ encies below 0.01. Taking into accoun t the above results it is evident that each breed is characteris ed by its specific g enotypic profile. 3.1.2 Allelic frequencies A set of thirty-nine (39) alleles for both breeds w ere identified. The allelic frequencies for each breed and for th e total number of animals are presented in Table 1. Among the alleles found, a total of 29 are already known and registered in the Database for Ovar-MHC (IPD-MH C Database EMBL-EBI), while the remaining ten (10) are reported for the first time. Table 1 illustrates the number of identified alleles in Arta (24 alleles) and in Kalarrytiko (29 alleles ) flocks, the number of shared and the number of unique alleles for each breed. Th e unique alleles for Arta were ten out of 24 (DRB1*0104, DRB1*0302, DRB1*0308, DRB1*1001, DRB1*1003, DRB1*1501, DRB1*1901, DRB1*2001, DRB1*2201 and DRB1*12). Also, in Arta breed a subset of five alleles from the total of 24 (DRB1*1606, DRB1*1607, DRB1*1608, DRB1*2502 an d DRB1*12) represented new alleles, reported first time in literature. The following a lleles: DRB1*0102, DRB1* 0101 and DRB1*0402, were detected in descending order with frequencies of 0.232, 0.139 and 0.095, respectively. For the above alleles th eir cumulative inciden ce was equal to 0.465. In Kalarrytiko a set of fifteen alleles, from the total of 29 detected, were breed specific (DRB1*0403, DRB1*0702, DRB1*0803, DRB1*1102, DRB1*1301, DRB1*1302, DRB1*1502, DRB1*160202, DRB1*1604, DRB1*1605, DRB1*2003, DRB1*2401, DRB1*11, DRB1*26 and DRB1*30). A subset of 9 alleles, from the total of 29 detected, were new (DRB1*160202, DRB1*1606, DRB1*1607, DRB1*1608, DRB1*2003, DRB1*2502, DRB1*11, DRB1*26 and DRB1*30). In Kalarrytiko th e most frequent alleles were: DRB1*0702, DRB1*0402 and DRB1*1606 with frequ encies 0.211, 0.192 and 0.107 respectively, and cumulati ve incidence equal to 0.510. 3.1.3 Frequencies of homozygosity and heterozygosity - Deviations from Hardy-Weinberg equilibrium The percentag e of 84.49% in the overall examined animals (n=432) were classified as heterozygotes and the remaining 15.51% as homozygotes. Estimates of observ ed homozygosity and heterozygosity (mean ± S.E.M.) within th e Arta breed was pA=0.125±0.022 an d qA=0.875±0.022, respectively. For the Kalarrytiko breed the relativ e es timat es were pK=0.189±0.028 and qK=0.811±0.028. The above estimates were not differen tiated betw een breeds (z = -1.82>-1.96, Ρ>0.05). The unbiased estimation of expected heterozyg osity according to Nei (1978) was HexpA=0.890 and HexpΚ=0.886 in Arta and Kalarrytik o breed, respectively and the abov e estimations w ere n ot differen t between them (P>0.05). Observed and expected estimations of heterozygosity differed significan tly in Kalarrytiko breed reflecting deviation from Hardy- Weinberg equilibrium. More specifically, the observed h eterozygote animals (qΚ=0.811) were fewer than expected (HexpΚ= 0.886) (P<0.05). S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 7 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 Table 1. Frequencies of Ovar-DRB1 alleles in Arta breed (n =231), in Kalarrytik o breed (n=201) and in the total examin ed animals (n=432). Alleles Allelic frequency Arta breed Kalarrytiko breed Total DRB1*0101 0.1385 0.0174 0.0822 DRB1*0102 0.2316 0.0647 0.1528 DRB1*0104 0.0043 - 0.0023 DRB1*0302 0.0022 - 0.0012 DRB1*0304 0.0498 0.0050 0.0289 DRB1*0308 0.0043 - 0.0023 DRB1*0311 0.0152 0.0522 0.0324 DRB1*0402 0.0952 0.1915 0.1400 DRB1*0403 - 0.0025 0.0012 DRB1*0601 0.0498 0.0025 0.0278 DRB1*0702 - 0.2114 0.0984 DRB1*0801 0.0238 0.0025 0.0127 DRB1*0803 - 0.0199 0.0104 DRB1*0901 0.0844 0.0398 0.0637 DRB1*1001 0.0887 - 0.0475 DRB1*1003 0.0130 - 0.0069 DRB1*1102 - 0.0025 0.0012 DRB1*1301 - 0.0025 0.0012 DRB1*1302 - 0.0025 0.0012 DRB1*1501 0.0065 - 0.0035 DRB1*1601 0.0130 0.0025 0.0081 DRB1*1602 - 0.0697 0.0336 DRB1*160202# - 0.0373 0.0035 DRB1*1604 - 0.0075 0.0046 DRB1*1605 - 0.0100 0.0069 DRB1*1606# 0.0022 0.1070 0.0081 DRB1*1607# 0.0022 0.0697 0.0012 DRB1*1608# 0.0152 0.0075 0.0463 DRB1*1901 0.0130 - 0.0023 DRB1*2001 0.0152 - 0.0023 DRB1*2003# - 0.0025 0.0035 DRB1*2101 0.0801 0.0075 0.0174 DRB1*2201 0.0043 - 0.0509 DRB1*2401 - 0.0050 0.0336 DRB1*2502# 0.0043 0.0025 0.0116 DRB1*11# - 0.0473 0.0220 DRB1*12# 0.0433 - 0.0231 DRB1*26# - 0.0025 0.0012 DRB1*30# - 0.0050 0.0023 #: New alleles -: No allele found S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 8 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 3.2 Parasitological analysis 3.2.1 Faecal examination Faecal samples were collected from 157 Kalarrytiko and 224 Arta sheep to obtain the faecal egg count of gastrointes tinal parasites. In th e total examined animals (n=381), a percen tage of 33% (126/381) was positively infected. Specifically, eggs from various parasites were detected in 55.41% (87/157) and 17.41% (39/224) animals from Kalarrytiko and Arta breed, respectiv ely (Table 2). A g reater variety of parasite reproductive elements was found in Kalarrytik o: eggs of strongyles, Nematodirus spp, Strongyloides spp., Trichuris spp., Moniezia spp. and oocytes of coccidia while in Arta breed w ere mainly detected, eggs of strong yles, Strongyloides spp., Capillaria spp. and coccidia. The above findings on FEC and pepsinogen values between th e studied breeds could be considered as the possible effect of th e farming system com bined with the differen t pasture locations used by the flocks. Table 2. Prevalence (%) and faecal egg counts (FEC) (epg) of gas troin testinal parasites and pepsinogen levels (m I.U. tyrosine) in sheep of the Arta and Kalarrytiko breeds. Infection Arta (N=224) Kalarrytiko (N=15 7) n Prevalence % FEC Median (Qr) Pepsinogen Median (Qr) n Prevalence % FEC Median (Qr) Pepsinogen Median (Qr) Helminths* 25 11.16 100 (200) 319.26 (356.46) 52 33.12 500 (1625) 228 (374) Strongyles 13 5.80 100 (200) 405.20 (255.06) 46 29.30 500 (1175) 223 (345.5) Strongyloides spp. 10 4.46 200 (200) 220.96 (517.99) 7 4.46 200 (1000) 138 (371) Nematodirus spp. 0 - - - 1 0.64 N/A N/A Trichuris spp. 1 0.46 N/A N/A 1 0.64 N/A N/A Capillaria spp. 1 0.45 N/A N/A 0 - - - Moniezia spp. 0 - - - 7 4.46 300 (400) 212 (374) Coccidia 17 7.59 200 (100) 317.13 (442.95) 57 36.31 200 (350) 381.5 (571.5) *: Infection with all the investigated parasites except coccidian -: No parasite found N/A: Not applicable 3.2.2 Pepsinogen levels The percentage of animals with pathological values of tyrosine was higher in Kalarrytik o breed (Table 2). More specifically, 56.1% for Kalarrytiko (88 animals from the total of 157 examined) and 43.6% for Arta (89 animals from the total of 224 examin ed) exhibited >375 m S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 9 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 I.U. tyrosine. However, in Kalarrytiko breed animals with values up to 500 m I.U. tyrosin e showed a very sharp increase in high levels up to 6.315 m I.U, while for Arta breed the relativ e values showed a lower increase up to the value of 2.243 m I.U. tyrosine. The Spearman correlation coefficient betw een F EC and pepsinog en values was nearly zero. 3.3 Associations between genotypes, parasitic load, and pepsinogen levels Investigation of possible associations between genotypes and F EC valu es was carried ou t for the genotypes of common alleles with relatively high frequ ency in the total examined animals or within each breed. We found few significan t associations betw een certain genotypes and FEC values which are presented in Table 3. Animals heterozygous for alleles DRB1*0101, DRB1*0102 and DRB*03 11 had significan tly lower FEC valu es in comparison with th e remaining genotypes. In the case of DRB1*0311 allele, within the group “heterozyg otes”, animals heterozygous for alleles DRB1*0302, DRB1*0304 and DRB1* 0308, were also included, because th e above three alleles have a tigh t nucleotide resem blance with DRB1* 03 11 (th ey belong to the same allelic family). On th e opposite direction the h eterozyg ous animals for DRB1*0402 allele had high er F EC values in com parison with th e homozygotes and th e remaining genotypes. Aditionally, animals carrying one or tw o copies of DRB1*0702 allele (with th e highest frequency in Kalarrytiko breed), had the higher m edian FEC numbers in comparison with the remaining genotypes. Table 3. Associations between Ovar-DRB1 genotypes and faecal egg counts (F EC) (epg ). Allele Ovar-DRB1 genotypes n FEC Median (Interquartile Range) P DRB1*0102 No copy of the allele 265 0 (200) * Heterozygote 106 0 (25) Homozygote 7 0 (200) DRB1*0101 No copy of the allele 316 0 (200) ** Heterozygote 57 0 (0) Homozygote 5 0 DRB1*0402 No copy of the allele 281 0 (100) *** Heterozygote 85 0 (350) Homozygote 12 50 (175) DRB1*0311 No copy of the allele 331 0 (200) ** Heterozygote 47 0 (0) Homozygote 0 N/A DRB1*0702 No copy of the allele 319 0 (100) *** Heterozygote 48 100 (300) Homozygote 10 100 (42 5) *: Significant association p<0.05 **: Signifant association p<0.01 ***: Significant association p<0.001 S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 10 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 Pepsinogen levels did not differ significantly among the various parasites in the studied breeds (Table 2). Fu rth ermore, no significant association was found betw een genotypes an d pepsinogen levels with one exception for the h omozygote animals for DRB1*0402 allele in th e total population which tend to have lower m edians values of pepsinogen levels in comparison with the remaining genotypes (P<0,07). 4 Discussion The studied breeds differ considerably in their morphological and productive traits . Arta is a breed of high milk production, kept in lowland of Western Greece in large flocks. Kalarrytik o is a rare b reed of m ountain type of medium productivity with a small population size in comparison to Arta breed (Rogdakis, 2002) and this fact could explain the significant deficit of heterozygote individuals found in Kalarrytiko. However, the fact that a greater number of alleles was found in Kalarrytiko as well as, more n ew alleles were identified in the above breed, confirms the necessity to preserve the small and rare breeds because in their genetic pool, a considerable amount of gen etic variation, can be found. The large number of alleles and th e high levels of heterozygosity found in both breeds are expected findings, due to the fact that Ovar-DRB1 gene locus is the most polymorphic among th e Οvar-MHC loci (Andersson and Rask, 1988). Generally, relying on the number of detected alleles, th e results of the present study could be compared with relative studies using the sequencing method for the detection of Ovar - DRB1 genetic variability. Schwaiger et al. (1993, 1994) identified 47 alleles in flocks from Perendale, Coopw orth, Landrace, Merino and Texel sheep. Paterson (1998) iden tified 5 alleles in a population of Soay feral sheep, while Gruszczynska (1999) found 36 and 28 alleles in tw o flocks of Merino sheep. From th e same scientific team (Gruszczynska et al., 2005) 36 and 30 alleles were found respectively, in two flocks of Polish Heath sheep. Additionally, Charon et al. (2002) in another flock of Polish Heath breed found 20 Ovar-DRB1 alleles, while Konnai et al. (2003) detected 28 alleles in Suffolk, 14 in Cheviot and 9 in Corriedale sheep breeds. Sayers et al. (2005) identified 8 alleles in Texel and 7 in Suffolk breeds. Finally, in the study of Balingall and Tassi (2010) 38 new alleles were iden tified in a sample of 214 animals from 15 differen t breeds. Considering all the above information, as well as the number of alleles found from th e presen t study, it is justified to argu e that the Greek sheep breeds contain considerable am oun t of genetic variation in Ovar-DRB1 locus. There is substantial evidence that genetic factors affect the resistan ce of animals in gastrointestinal parasite numbers (Bishop et al., 2004; Sayers et al., 2005), although many other factors as the type, ag e and sex of the parasite, th e intensity of infection, th e age, die t and frequency of th e host defecation, act on the coun ted number of reprodu ctive elemen ts of gastrointestinal parasites. Also, the FEC values are affected by time of year and w eath er conditions (Abbott et al., 2009). Possibly, the large numbers of gastroin tes tinal parasit es in Kalarrytiko animals occu rred due to a relatively large number of fertile and adult parasites, which act within the organism to enhance the immun e animal hypersensitivity reaction causing an increase of the perm eability of abomasum mucosa, resulting i n th e estimation of very hig h values mI.U. tyrosine (Yakoob et al, 1983b). As already m entioned, the environmen tal factors S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 11 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 (climate and habitat) acting on th e studied breeds were differen t and do not allow a furth er comparison betw een FEC valu es and the tyrosine values estimated for both breeds. Pepsinogen levels in plasma were determin ed in order to assess the extent of damage in the gastric mucosa of animals by gastroin testinal parasites. The endoparasitic infection is associated with pathophysiological disorders such as the increase of plasma pepsinogen lev els, the albumin catabolism and plasma protein losses in the gastrointestinal tract (Yakoob et al, 1983a). For this reason, the estimation of pepsinogen concentrations in the blood plasma has been proposed as an auxiliary diagnostic technique of endoparasitic infections (parasitic gastroen teritis) in ruminants (Anderson et al., 1965). According to Stear et al. (1995a, b) th e plasma pepsinogen concentration expresses the hos t response to infection by nem atodes, while FEC values reflect th e behaviour of pes ts in th e hos t. In particular, th e above researchers found that in adult ewes infected naturally by gastroin tes tinal nematodes, the increase of plasma pepsinogen con centration was merely an expression of the immunological hos t response to infection and had no relation to the parasitic load. The identification of resistan t and susceptible sheep to nematodes is more effective when the estimation of FEC values is don e in parallel with the estimation of plasma pepsinogen levels (Stear et al., 1995a, b). In some regions where Ostertagia spp is detected, serum plasm a analysis is a useful diagnostic tool. In g eneral, pepsinogen levels > 375 mI.U. tyrosin e correspond to clinical symptoms. However, interpretation problems may arise in immunized animals that are infected by gastroin testinal parasites. I n these animals, there are no clinical signs but, as mentioned, plasma pepsinogen lev els may be elevated because of immunological hypersensitivity reaction that occu rs in the abomasum mucosa. Most studies on th e concen tration of pepsinogen in the blood plasma have been conducted in sheep experimen tally infected with nematodes (Yakoob et al., 1983b; Fox et al., 1988; Mostofa et al., 1990; Lawton et al., 1996) while there are few studies in sheep infected with nematodes in a natural way (Yak oob et al., 1983a). Besides th e determination of plasma pepsinogen levels when the genus Haemonc hus spp dominates, hematocrit m easuring provides a rapid estimation of th e degree of anaemia which is the characteristic clinical sign of n ematodosis caused by the above parasite. Also, in some countries the serological diagnosis (ELISA) is used mainly for the species Ostertagia spp and Cooperia spp. However, so far there is insufficien t information on th e association betw een serological titres and parasitic load. Generally, from this study neither significant differences of pepsinogen levels were foun d betw een heterozygote and homozygote Ovar-DRB1genotypes, nor an y relationship betw een the various genotypes with high or low pepsinogen levels. However, there was a trend for homozygous animals carrying DRB1*0402 allele to have lower median values of mI.U. tyrosin e in comparison with the other genotypes. Genetic factors play an importan t role in resistance to nematodes (Bisset et al., 1992). As the es timated coefficient of heritability for F EC values rang es from 0.2 to 0.4 in various sheep breeds which have previously been infected with parasites (Stear et al., 1997a, b), this suggests that a sheep s election prog ram of genetic resistance to nematodes can b e effectively implemen ted (Stear et al., 2001). According to research studies of Behnke et al. (2003, 2006) MHC polymorphism of class II genes and mainly of TNF gen e regions determin es the resistance or th e host susceptibility to diseases caused by gastroi n tes tinal parasites. In the study of Davies et al. (2006) in Scottish Blackface sheep, it was shown that specific QTLs in chromosom es 2, 3, 14, and 20 are related with the resistance to infection of Τeladorsagia S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 12 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 circumcincta. The analysis of ch romosom e 20 revealed that the Ovar-MHC region is significantly correlated with resistan ce to gastroin tes tinal nematodes. Furth ermore, QTLs related to th e specific activity of th e IgA immunoglobulins against nematodes are found on ch romosomes 3 and 20. Associations of MHC DRB1 alleles with resistance to n ematodes have been found not only in sheep (Outteridge et al., 1996; Sayers et al., 2005) bu t also in cattle (Stear et al., 1988 & 1990 ; Gasbarre et al., 1993) and mice (Froeschke and Somm er, 2005). Early studies in Sco ttis h Blackface lam bs, based on th e hybridization of oligonucleotides in exon 2 of Ovar-DRB1 gene, have shown that, lambs infected natu rally with Osterdagia circumcincta, had FEC values associated with Ovar-DRB1 alleles (Buitcamp et al., 1994; Schwaiger et al., 1995; Stear et al., 1996). In addition, the significan t effect of Ovar-DRB1 alleles on th e dras tic reduction of F EC values, was detected by substitution of the common allele I, with G2 allele, identical with th e allele Ovar-DRB1*0203, which confers resistance to nematode parasites. The association of G2 allele with low F EC values in Scottish Blackface sheep was con firm ed by th e study of Stear et al. (2005). There was also, an association of G2 allele with reduced number of adult parasites of Τ. circumcincta species, although no association was found with the length size of female parasites. A possible explanation is that the MHC effect on FEC values functions th rough th e control of th e number of adult parasites and not th e control of th eir fertility. In the study of Hassan et al., (2011) the G2 allele, renamed as Ovar-DRB1*1101, gave increased resistance against the nematode T. circumcincta in Suffolk lambs. Eviden ce was also, presen ted that th e resistance of animals, due to Ovar-DRB1*1101 allele, was more acquired and less innate an d furthermore it was depended from the excretion of adult parasites th rough the multiplication of mast cells of th e mucosa. In the study of Sayers et al. (2005) the allele Ovar-DRB1*0203 was associated with reduced FEC values in Suffolk breed. The above strong association seems to be due, not only to an antibody production that protects the animals carrying G2 allele, but also to the genetic linkage of G2 allele with an allele of resistance mounted on another locus position. This probably occu rs because the loci close to on e another on the same chrom osome, tend to stay together during th e reduction and th erefore th ey are linked gen etically (McCririe et al., 1997). Additionally, th e parasite Trichostrongylus colubriformis in sheep appears capable to n egatively regulate many genes of immunologic importance, especially Ovar-DRB1 and DRA, of th e migratory cells in lymph (Knight et al., 2010). Several genetic mechanisms have been proposed to explain the MHC allelic va riability. New alleles appear through point mutations, genetic recombination or genetic transmutation s (Janeway and Travers, 1996). The simultaneous antag onistic evolution (coevolution) betw een host and parasites is another proposed mechanism for maintenance of genetic variability both in host and parasitic species (Paterson et al., 1998; Cutrera et al., 2011). The h ypothesis that th e high levels of MHC gen etic polym orphism of hos t species, driven by th e parasites themselves, is the result of the action of balancing selection, interprets th e redu ction of homozygous and the increase of heterozygous frequencies in a particular population (H edrick and Kim, 1998). The balan cing selection maintains g enetic polym orphism through th ree different genetic mechanisms: a) the heterozygote advantag e (overdominance) b) the advantag e of rare alleles and c) the variance in th e levels of selective pressure (Charbonn el and Pemberton, 2005). In the case of overdominance, selection will not fix or eliminate one or th e other allele, but will make a stable balance of heterozygotes, in which both alleles will be present in the population, S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 13 HAF © 2013 Vol. III, No. 1 ISSN: 2283-3927 at frequ encies determin ed solely by the selection rates against the two homozygote genotypes. In this case, the deg ree of MHC heterozygosity increases the rang e of parasites which are recognized by the immun e system and thus increases th e relativ e Darwinian fitn ess of MHC heterozygote genotypes compared to that of the homozygote (Pa terson et al., 1998). In the case of the second m echanism (advantag e of th e rare allele) (Hamilton, 1980; Takahata and Nei, 1990), the Darwinian fitness of th e allele is reduced when th e frequency of allele in the population increases. Genotypes with rare MHC alleles presen t a s trong selectiv e advantage as fewer pathogens hav e been expos ed and adapted to them (Clark e and Kirby, 1996). The MHC alleles are favou red at low frequencies, while an increase of their freq uencies could only cause a shift in the gen etic composition of parasitic population. Thus , according to the case of rare allele, the interaction of host-parasite is considered as a dynamic procedure. The action of parasites decreases the Darwinian fitness of the most common MHC alleles of the host with which in teracts (Pa terson et al., 1998). The third proposed mechanism is related with variances on the rate of selective pressure. Variations in type or size of the parasite population in th e area, lead to constant chang es in the intensity of selection and thus to maintenance of the polym orphism. This phenomenon is dependen t on the presen ce of th e same pathogen (Hedrick, 2002; Charbonnel and Pemberton, 2005). The possibility that an in teraction between various genotypes, as this is reflected by th e different breeds and th e farming system could not be excluded although this was im possible to be investigated in th e present study. Genetic selection schemes applied in Australian conditions by selecting Merino sheep with low faecal egg – counts gave good selection response and have not resulted in any n egative correlation response of th e economically importan t production traits. Sheep resource flocks helmin th resistant (Ryling ton Merino) hav e been created providing significant contribution in understanding th e breeding for diseas e resistance (Karlsson & Greeff, 2012). From the oth er hand th e div ersity of environmen tal conditions in this continent, highlights the need of various strategies and makes difficult to give universal recommendations in order to con trol gastro-intestinal parasites (Lars en, 2014). 5 Conclusions Our results suggest the considerable amount of g enetic variability in th e Ovar-DRB1 locus for the two Greek dairy sheep breeds, which is underlined due to th e high number of detected alleles as well as the finding of new alleles from this study. In the Kalarrytiko breed, a rare breed with small population size, a higher number of n ew alleles were iden tified than in th e Arta breed. This finding suggests that rare Greek sheep breeds poten tially are an importan t gene pool of new alleles. Ou r results also indicated that the majority of detected alleles hav e low frequen cies and subsequently the majority of animals w ere in heterozyg ote state for both breeds. Although certain genotypes of Ovar-DRB1 alleles were associated with lower median FEC values, the abov e findings make more complicated th e selection of desirable g enotypes resistant to gastrointes tinal parasites. Considering the central role that th e major histocompatibility complex (MHC) plays in the immune sys tem, our s tudy contributes to th e accumulated knowledge on MHC effects on parasite resistance, howev er furth er s tudies in th e future will be required with other genes involved in MHC region in order to cl arify th e genetic background of parasite resistan ce. S. Spetsa ria s et al. - Int. J. of Health, Animal science a nd Food safety 3 (2016) 1 - 19 14 Appendix: Supplementary table Table A. Observed (obs) and ex pected (exp) g enotypes for Ovar-DRB1 all eles detected in the studied breeds Genotypes obs (exp) Genotypes obs (exp) Genotypes obs (exp) Arta DRB1*0101-DRB1*0101 6 (4.373) DRB1*0901-DRB1*0304 1 (1.946) DRB1*1901-DRB1*0101 1 (0.833) DRB1* 0102-DRB1*0101 14 (14.855) DRB1* 0901-DRB1*0311 # 2 (0.592) DRB1*1901-DRB1*0102 1 (1.393) DRB1*0102-DRB1*0102 8 (12.301) DRB1*0901-DRB1*0402 # 4 (3.722) DRB1*1901-DRB1*0402 2 (0.573) DRB1*0304-DRB1*010 3 (3.193) DRB1*0901-DRB1*0801 2 (0.931) DRB1*1901-DRB1*0601 1 (0.299) DRB1* 0304-DRB1*0102 # 7 (5.338) DRB1*0901-DRB1*0901# 2 (1.607) DRB1*2001-DRB1*0402 3 (0668) DRB1*0304-DRB1*0304 2 (0.549) DRB1*1001-DRB1*0101 7 (5.69 2) DRB1*2001-DRB1*0901 3 (0.592) DRB1*0308 -DRB1*0101 1 (0.278) DRB1*1001-DRB1*0102 12 (9.516) DRB1*2101-DRB1*0101 1 (5.137) DRB1*0311-DRB1*0102 1 (1.625) DRB1*1001-DRB1*0302 1 (0.089) DRB1*2101-DRB1*0102 12 (8.588) DRB1*0402-DRB1*0101 # 3 (6.109) DRB1*1001-DRB1*0304 1 (2.046) DRB1*2101-DRB1*0311 1 (0.562) DRB1* 0402-DRB1*0102 # 9 (10.213) DRB1*1001-DRB1*0311 1 (0.623) DRB1*2101-DRB1*0402 1 (0.532) DRB1*0402-DRB1* 0104 1(0.191) DRB1*1001-DRB1*0402 3 (3.913) DRB1*2101-DRB1*0801 1 (0.883) DRB1*0402-DRB1*0304 2 (2.195) DRB1*1001-DRB1*0601 2 (2.046) DRB1*2101-DRB1*0901 2 (3.130) DRB1* 0402-DRB1* 0308 1 (0.191) DRB1*1001-DRB1*0901 1 (3.469) DRB1*2101-DRB1*1001 8 (3.291) DRB1*0402-DRB1* 0311 # 1 (0.668) DRB1*1001-DRB1*1001 1 (1.779) DRB1*2101-DRB1*1901 1 (0.482) DRB1* 0402-DRB1* 0402 # 4 (2.052) DRB1*1003-DRB1*0101 1 (0.833) DRB1*2101-DRB1*2101# 2 (1.445) DRB1*0601-DRB1* 0101 3 (3.193) DRB1*1003-DRB1*0102 5 (1.393) DRB1*2201-DRB1*0102 2 (0.464) DRB1* 0601-DRB1*0102 8 (5.338) DRB1*1501-DRB1*0101 1 (0.417) DRB1*2502-DRB1*2502 1 (0.002) DRB1*0601-DRB1* 0304 1 (1.148) DRB1*1501-DRB1*0402 1 (0.286) DRB1*12-DRB1*0101 1 (2.777) DRB1*0601-DRB1*0402 3 (2.195) DRB1*1501-DRB1*0801 1 (0.072) DRB1*12-DRB1*0102 5 (4.642) DRB1*0601-DRB1* 0601 1 (0.549) DRB1*1601-DRB1*0101 4 (0.833) DRB1*12-DRB1*0104 1 (0.087) DRB1*0801-DRB1*0102 2 (2.553) DRB1*1601-DRB1*0402 # 1 (0.573) DRB1*12-DRB1*0304 1 (0.998) DRB1*0801-DRB1*0311 1 (0.167) DRB1*1601-DRB1*0601 1 (0.299) DRB1*12-DRB1*0601 1 (0.998) DRB1*0801-DRB1* 0402 # 1 (1.050) DRB1*1607-DRB1*0101 1 (0.139) DRB1*12-DRB1*0901 1 (1.692) DRB1*0801-DRB1*0601 1 (0.549) DRB1*1608-DRB1*0101 1 (0.972) DRB1*12-DRB1*1001 3 (1.779) DRB1*0801-DRB1*0801 1 (0.119) DRB1*1608-DRB1*0102 4 (1.625) DRB1*12-DRB1*2101 4 (1.605) DRB1*0901-DRB1*0101 10 (5.414) DRB1*1608-DRB1*0304 1 (0.349) DRB1*12-DRB1*1606 1 (0.043) DRB1*0901-DRB1*0102 # 9 (9.052) DRB1*1608-DRB1*2001 1 (0.106) DRB1*12-DRB1*12 1 (0.412) Kalarrytik o DRB1*0304-DRB1*0102 # 1 (0.130) DRB1*1601-DRB1*0402 # 1 (0.192) DRB1*1607-DRB1*0102 1 (1.816) DRB1*0402-DRB1*0101 # 1 (1.344) DRB1*1602-DRB1*0102 2 (1.816) DRB1*1607-DRB1*0311 4 (1.466) DRB1*0402-DRB1*0102 # 11 (4.993) DRB1*1602-DRB1*0702 11 (5.935) DRB1*1607-DRB1*0402 3 (5.377) DRB1*0402-DRB1*0311 # 7 (4.032) DRB1*160202-DRB1*0402 2 (2.880) DRB1*1607-DRB1*0702 5 (5.9 35) DRB1*0402-DRB1* 0402 # 8 (7.297) DRB1*160202-DRB1*0702 1 (3.180) DRB1*1607-DRB1*1602 2 (1.955) DRB1*0702-DRB1*0101 4 (1.484) DRB1*160202-DRB1*1102 1 (0.037) DRB1*1607-DRB1*1606 4 (3.003) DRB1*0702-DRB1*0102 2 (5.511) DRB1*160202-DRB1*160202 5 (0.262) DRB1*1607-DRB1*1607 4 (0.943) DRB1*0702-DRB1*0402 13 (16.322) DRB1*1604-DRB1*0102 1 (0.195) DRB1*1608-DRB1*0402 1 (0.576) DRB1*0702-DRB1*0601 1 (0.212) DRB1*1604-DRB1*0702 2 (0.636) DRB1*1608-DRB1*0702 2 (0.636) DRB1*0702-DRB1*0702 11 (8.903) DRB1*1605-DRB1*1605 2 (0.015) DRB1*2101-DRB1*2101 # 1 (0.008) DRB1*0801-DRB1*0402 # 1 (0.192) DRB1*1606-DRB1*0101 2 (0.751) DRB1*2401-DRB1*0402 1 (0.384) DRB1*0803 -DRB1*0311 1 (0.419) DRB1*1606-DRB1*0102 5 (2.788) DRB1*2502-DRB1*0402 1 (0.192) DRB1*0803 -DRB1*0402 3 (1.536) DRB1*1606-DRB1*0304 1 (0.215) DRB1*11-DRB1*0402 3 (3.64 8) DRB1*0803 -DRB1*0702 1 (1.696) DRB1*1606-DRB1*0311 4 (2.252) DRB1* 11-DRB1*0901 1 (0.758) DRB1*0803 -DRB1*0803 1 (0.070) DRB1*1606-DRB1*0402 3 (8.257) DRB1* 11-DRB1*2003 1 (0.047) DRB1*0901-DRB1*0102 # 1 (1.037) DRB1*1606-DRB1*0403 1 (0.107) DRB1*/11-DRB1*2101 1 (0.142) DRB1*0901-DRB1*0311 # 1 (0.838) DRB1*1606-DRB1*0702 9 (9.115) DRB1* 11-DRB1*160202 1 (0.711) DRB1*0901-DRB1*0402 # 5 (3.072) DRB1*1606-DRB1*0803 1 (0.858) DRB1* 11-DRB1*1606 1 (2.037) DRB1*0901-DRB1*0702 1 (3.392) DRB1*1606-DRB1*0901 2 (1.716) DRB1*11-DRB1*/11 3 (0.426) DRB1*0901-DRB1*09 01# 1 (0.299) DRB1*1606-DRB1*1602 5 (3.003) DRB1*26-DRB1*1607 1 (0.070) DRB1*1301-DRB1*0702 1 (0.212) DRB1*1606-DRB1*2401 1 (0.215) DRB1*30-DRB1*0702 2 (0.424) DRB1*1302-DRB1*0702 1 (0.212) DRB1*1606-DRB1*1606 2 (2.252) Symbol #: the common genotypes between the two breeds S. 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