http://orcid.org/0000-0002-0464-3199
Figures
Abstract
Goat breeding has become an important sector in Eastern Europe, with Romania and Hungary being among the major producer countries. Given the limited number of research done up-to-date concerning genetic studies of indigenous goat breeds reared in Romania and Hungary, the current preliminary study aimed to analyze the variability of genes related to mastitis and gastrointestinal parasitism by using Kompetitive Allele Specific PCR (KASP™). We studied 52 single nucleotide polymorphisms (SNPs) belonging to 19 genes in indigenous breeds from both countries, namely Banat’s White (n = 36), Carpatina (n = 35) from Romania and Hungarian Milking (n = 79) and identified 16 polymorphic SNPs among 10 genes (PTX3, IL6, CLEC4E, IL8, IL1RN, IL15RA, TNFSF13, SOCS3, TNF and TLR3) in 150 animals. Furthermore, the diversity of the studied breeds was investigated. The PIC values ranged from 0.042 to 0.691. The mean values of observed and expected heterozygosity were 0.235 and 0.246 respectively. The highest observed heterozygosity was obtained for IL15RA g.10343904C>T in Banat’s White (0.464), IL15RA g.10354813C>T in Carpatina (0.577) and SOCS3 g.52626440T>G in Hungarian Milking (0.588). Pairwise FST values between the Romanian breeds and Romanian and Hungarian breeds were small (0.009 and 0.015), indicating the close relationship among the studied goat populations. From all the polymorphic SNPs identified, the Hungarian Milking breed showed the highest proportion of polymorphisms (100%), whereas the Carpatina breed had the lowest percentage (87.5%). The highest value of MAF was obtained for SOCS3 g.52626440T>G (0.46), IL15RA g.10343904C>T (0.47), IL15RA g.10344025C>T (0.45), and IL15RA g.10354813C>T (0.42). The 16 polymorphic SNPs identified in a panel of 150 unrelated individuals belonging to three Romanian and Hungarian indigenous goat breeds could be used in future genomic based breeding schemes as markers for genetic resistance to mastitis and gastrointestinal parasitism in goat breeds found in Eastern and Central Europe.
Citation: Ilie DE, Kusza S, Sauer M, Gavojdian D (2018) Genetic characterization of indigenous goat breeds in Romania and Hungary with a special focus on genetic resistance to mastitis and gastrointestinal parasitism based on 40 SNPs. PLoS ONE 13(5): e0197051. https://doi.org/10.1371/journal.pone.0197051
Editor: Juan J. Loor, University of Illinois, UNITED STATES
Received: September 19, 2017; Accepted: April 25, 2018; Published: May 9, 2018
Copyright: © 2018 Ilie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN--II--RU--TE--2014--4--0005, grant awarded to Dinu Gavojdian. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The goat farming sector in Romania has been rapidly developing during the last decade. Currently, Romania holds a national flock of 1.48 million goats, according to Eurostat reports [1]. The breed structure is being dominated by the indigenous unimproved Carpatina, which represents over 90% of the goats reared in Romania. Reports concerning the breed’s performance have shown modest production levels, with milk yields estimates of 220 to 350 kg/lactation, litter size of 130–160% and growth rates in kids ranging between 90 and 110 g/day [2,3]. The second indigenous goat breed found in Romania is the Banat’s White. The breed is currently listed as endangered and included in a conservation program, with a census of 1,002 purebred does reared in 5 farms. The Banat’s White has a milk production of 350–400 kg/lactation and is highly prolific, with an average litter size of 200–225% [4].
Nowadays the national Hungarian goat population is of 81,000 heads [1] and the main production is dairy [5]. Goats are being reared under extensive low-input production systems in over 7,000 farming units, with an average flock size of roughly 15 breeding does/farm [6]. The most frequent goat breed is the Hungarian Milking [7], with the adult body weight of 40 to 60 kg in does and 60 to 90 kg in adult bucks. Reports concerning the breed’s performance have shown modest production levels, with milk yields estimates of 200 to 300 kg/lactation and the litter size of 130–150% [8,9].
In the recent decades, several studies have attempted to investigate and identify genetic variants responsible for mastitis and gastrointestinal parasitism resistance in livestock. So far, studies on the genetic structure of goat breeds and genetic basis of immune cell involved in response to intramammary pathogens were not performed in Romanian and Hungarian indigenous goat breeds. Despite the management practices, mastitis and parasitic infections are among the main health constraints for the small ruminants sector worldwide, being responsible for causing heavy production losses and poor animal welfare [10–12]. Clinical mastitis is less frequent in dairy sheep and goats (5%) compared with dairy cattle, however, subclinical mastitis has a prevalence of up to 55% [13,14]. On the other hand, gastrointestinal nematode infections are the most prevalent parasitic diseases affecting sheep and goats productivity worldwide, especially under pasture-based production conditions. The condition can generate the reduction in skeletal growth, live-weight gain and in milk yield, causing significant economic losses, reduced animal performance and even leading to mortality in severe infestations [15,16].
Knowledge on the genetic structure of goat breeds, as well as new information about the genetic basis of immune cell involved in response to pathogens, will be beneficial to understand the role of genetic variants in resistance to mastitis and gastrointestinal parasitism. Furthermore, genetic improvement schemes and conservation plans are becoming more and more important in each country for all farm species, and are being designed based on both phenotypic and genomic data. For conservation plans, there is a need for detailed knowledge of the genetic make-up of the goat breeds in Central and Eastern Europe. The aim of the current pilot study was to analyze variability of 52 SNPs found on 19 genes related to mastitis and gastrointestinal parasitism in the indigenous goat breeds from Romania and Hungary through the use of a novel and fast method, called Kompetitive Allele Specific PCR.
Material and methods
Ethics approval
The research activities were performed in accordance with the European Union’s Directive for animal experimentation (Directive 2010/63/UE). The experimental design, sampling collection protocols and procedures were approved by the Institutional Ethics Committee of the Research and Development Station for Sheep and Goats Caransebes (Decision no. 39 from 05 November 2015).
Animals and DNA isolation
A total of 150 goats belonging to three Romanian and Hungarian indigenous breeds were included in the study. Hair follicles were sampled from 71 Romanian goats (Banat’s White n = 36 and Carpatina n = 35) and blood samples from 79 Hungarian goats (Hungarian Milking n = 79) were collected. The animals were selected following the criteria to be unrelated individuals and sampled from different farms (2–5 farms/breed unit) in order to reduce the genetic relationship among animals and to increase the breed representativeness. The collection sites and geographic coordinates of the studied breeds used in the study were given in S1 Table. Collection of samples was performed by authorized veterinarians and all samples were kept on 4°C until the further laboratory process. Genomic DNA was extracted following FAO/IAEA protocol [17] for the hair follicles and Zsolnai and Orbán protocol [18] for blood. DNA concentration was evaluated spectrophotometrically, with NanoDrop-2000 (Thermo Fisher Scientific Inc., MA, USA), and visually by standard agarose gel electrophoresis (1% agarose (w/v) in TBE). After extraction, all DNA samples were diluted for 50 ng and stored at -20°C until the further analysis.
Selection of SNPs
A total of 52 SNPs belonging to 19 genes (S2 Table) have been selected to be used in Kompetitive Allele Specific PCR assay based on studies [11,19–22] involving markers for genetic resistance to gastrointestinal infection and genes associated with resistance to mammary gland infection. SNPs data were collected from the caprine Single Nucleotide Database (dbSNP) of the National Center for Biotechnology Information (NCBI).
DNA genotyping and data analysis
Kompetitive Allele Specific PCR (KASP™, LGC Genomics, Teddington, Middlesex, UK) genotyping was performed for the bi-allelic discrimination of the selected SNPs (S2 Table). The data generated was viewed using SNP viewer software (version 1.99, Hoddesdon, UK). The raw allele calls received from LGC Genomics were analyzed with KlusterCaller software from LGC Genomics. Linkage analysis was performed by GENEPOP [23]. POPGENE 1.32 [24] was used to calculate the deviation from Hardy-Weinberg equilibrium (HWE), observed (Ho) and expected (He) heterozygosity values for each studied breed, Polymorphic Information Content (PIC) and pairwise FST values.
Results
In this study, 52 SNPs across 19 genes were studied using the KASP genotyping assays. Details of SNPs including SNP ID, gene name, chromosome location, genomic location, functional domain of the gene and alleles at each locus are presented in S2 Table. Out of the 52 SNPs, 16 (30.77%) were found to be polymorphic, 24 (46.15%) monomorphic and 12 (23.08%) failed in our studied goat breeds (S3 Table). However, a total of 40 SNPs (monomorphic and polymorphic) were used from a total of 6,000 genotypes assayed. A total of 24 alleles and 42 genotypes were found at 16 polymorphic SNPs in the 150 studied goats. The polymorphic SNPs were found in the following 10 genes: PTX3, IL6, CLEC4E, IL8, IL1RN, IL15RA, TNFSF13, SOCS3, TNF and TLR3. From polymorphic SNPs identified, four were located in chromosome 13, two were located in each of chromosomes 5, 6, 11 and 19 and one in each of chromosomes 1, 4, 23, and 27 respectively. The monomorphic SNPs were excluded from further analysis.
For the polymorphic SNPs, the assays produced 2,142 identified allele calls and 258 unidentified allele calls with an allele call rate of 89.25% and a mean of unidentified allele calls of 10.75%. All polymorphic SNPs revealed more than 84% identified allele calls/SNP. Among the polymorphic SNPs, twelve SNPs (75%,) were located within the coding region of the gene (CLEC4E g.93527308C>T, IL8 g.86041868A>G, IL8 g.86040123G>A, IL1RN g.46358256A>G, IL1RN g.46353777G>T, IL15RA g.10343904C>T, IL15RA g.10344025C>T, IL15RA g.10354726G>A, IL15RA g.10354813C>T, TNFSF13 g.26523480A>G, TNF g.26141981T>A and TLR3 g.14987931C>G), two were located in introns (PTX3 g.108076746C>T and SOCS3 g.52626440T>G), one in the 3′UTRs (IL6 g.29257937T>C) and one was an upstream variant (CLEC4E g.93538087T>C). However, when the experiment was designed, non-synonymous SNPs were mainly chosen in order to increase the probability that there would be a change in the characteristic of the proteins encoded for the investigated genes. Furthermore, for polymorphic SNPs located on the same chromosome (BTA13) and the same gene (IL15RA) a linkage analysis was performed and shown that all four SNPs are independent.
The genetic indices of Ho, He, PIC and FST values for different breeds under study were calculated and shown in Table 1. PIC values also revealed that rs635583012 / SOCS3 g.52626440T>G (0.640–0.693), rs661943224 / IL15RA g.10343904C>T (0.659–0.693), rs648293427 / IL15RA g.10344025C>T (0.632–0.693), IL15RA g.10354813C>T (0.640–0.691), rs661914424 / TLR3 g.14987931C>G (0.583–0.664) and IL1RN g.46353777G>T (0.467–0.417) are the most polymorphic markers.
Expected and observed heterozygosity values detected in two Romanian and one Hungarian breeds were similarly low (<0.600). The mean global observed and expected heterozygosity was 0.235 and 0.246 respectively. The highest observed heterozygosity was obtained for rs661943224 / IL15RA g.10343904C>T in Banat’s White (0.464), rs635969404 / IL15RA g.10354813C>T in Carpatina (0.577) and rs635583012 / SOCS3 g.52626440T>G in Hungarian Milking (0.588) indicating high levels of within-population diversity.
Pairwise FST values between the Romanian breeds and Romanian and Hungarian breeds were small ranging within the range of 0.0–0.024 and 0.0–0.040 respectively, indicating the close relationship among the studied breeds (Table 1). Thereby, low genetic differentiation between breeds (FST) were obtained, of 0.9% and 1.5%, respectively. With the Romanian breeds showing less differentiation. Moderate genetic differentiation was observed only for one SNP (rs661943224 / IL15RA g.10343904C>T) in Romanian breeds/Hungarian Milking (0.040).
The Hardy-Weinberg equilibrium and genotype and allele frequencies of the 16 SNPs were also studied in each of the investigated goat breeds and are shown in Table 2. All breeds were found to be in equilibrium (P>0.05) except one SNP in each breed (rs659842900 / IL1RN g.46358256A>G in Banat’s White, rs661165283 / TNF g.26141981T>A in Carpatina and rs648293427 / IL15RA g.10344025C>T in Hungarian Milking).
Levels of polymorphism were generally low in all studied breeds. The homozygous genotypes were more frequent than heterozygous in most cases. On average, from polymorphic SNPs identified, the Hungarian Milking breed showed the highest proportion of polymorphic SNPs (100%), whereas the Carpatina breed had the lowest proportion of polymorphic SNPs (87.5%) and presented fixed alleles in a number of 2 SNPs (rs669680484 and rs667413402). From the 16 polymorphic SNPs, four (25%) were found with an overall frequency of the rare allele lower than 5% (rs669680484/PTX3, rs667413402/IL8, rs647408958/ IL15RA and rs669561078/ TNFSF13). The frequencies of major alleles ranged from 0.53 for rs661943224/IL15RA to 0.99 (SNPs with rare allele lower than 5%). The highest value of minor allele frequency (MAF) was obtained for TLR3 g.14987931C>G (0.35), SOCS3 g.52626440T>G (0.46), IL15RA g.10343904C>T (0.47), IL15RA g.10344025C>T (0.45), and IL15RA g.10354813C>T (0.42).
Discussion
Kompetitive Allele Specific PCR technology was used to convert a set of 52 SNPs into assays for genetic characterization of indigenous goat breeds reared in Romania and Hungary with a special focus on genetic resistance to the mammary gland and gastrointestinal infections. Among the 52 SNPs, a number of 12 (23.08%) failing development and 40 (76.92%) were successfully genotyped of which 24 (46.15%) were monomorphic across Banat’s White, Carpatina and Hungarian Milking goat breeds. Current findings are in accordance with those previously reported in studies where the KASP assay success rates were ranging from 78.5% [25] to 80.9% [26]. With the failed and monomorphic assays removed, 16 SNPs (30.77%) were further validated and used for the current study. Of the 16 SNPs, a percent of 75% (12 SNPs) met the criteria for analysis (MAF ≥ 5% and call rate > 89%).
Genetic resistance to the mammary gland and gastrointestinal infection
The present study found 16 polymorphic SNPs across 10 genes that are relevant in pathways associated with parasite and mammary gland infection or are involved in production traits or have a potential contribution to an important metabolic condition. However, functional genes, such as CLEC4E, IL6, IL8, IL1RN, IL15RA, PTX3, SOCS3, TNF, TNFSF13 and TLR3 are related to host resistance to disease in different farm species.
The CLEC4E is expressed on the surface of macrophages [27] and play an important role in recognition of bacterial glycolipids by the immune system being an immune response gene associated with genetic resistance and susceptibility to a wide array of diseases [21]. Bhuiyan et al. [22] showed that CLEC4E was a down-regulated gene involved in immune response system in susceptible goats to nematodes infection. In others studies on goats, CLEC4E was up-regulated when was investigated in the in vivo transcriptional response of mammary epithelial cells at the early stages of infection with Staphylococcus aureus [21]. In this paper, were analyzed four SNPs in CLEC4E gene and found two to be polymorphic (CLEC4E g.93527308C>T and CLEC4E g.93538087T>C) in all three studied goat breed.
Pentraxin 3 (PTX3) is a glycoprotein expressed by diverse cell types upon primary inflammatory stimuli such as those mediated by IL-1β, TNFα and agonists of TLRs [28,29]. The PTX3 gene has as a primary function the regulation of innate resistance to pathogens and inflammatory reactions and acts as antimicrobial agents that could assist defense of the mammary gland against chronic and subclinical infections [11]. In our study, five SNPs were investigated in intron regions of PTX3 gene, of which four presented fixed alleles and one was found polymorphic (PTX3 g.108076746C>T). Moreover, the results showed that polymorphic SNP at the PTX3 gene was not presented in Carpatina or Banat’s White goat breeds. Different studies showed that the PTX3 represents the first line of immune defense in udder being significantly up-regulated in response to Staphylococcus aureus infection in goats [11,30]. Other studies conducted in different farm species revealed that the phenotype of PTX3 gene plays a protective role against several types of harmful microorganisms [29].
SOCS3 is a member of the suppressors of cytokine signaling (SOCS) family of proteins that have a negative effect on cytokine signaling [31]. Brenaut et al. [21] studied the contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus in goats and revealed that SOCS3 is a highly regulated gene with a high degree of outward connectivity to other genes that are for the most up-regulated. Moreover, polymorphism of SOCS3 gene was associated with mammary development pathway [32] and somatic cell score trait in cattle [33,34]. In the current study, the SOCS3 g.52626440T>G was polymorphic in all three studied breeds, with the highest frequency of T allele (0.51) being found in the Hungarian Milking goats.
The level of some cytokines such as TNF-α, IL6 and IL8 are reported to increase during infections [35]. The interleukin-8 (IL8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by several cell types such as macrophages epithelial and endothelial cells [36]. This chemokine is one of the major mediators of the inflammatory response and acts on CXCR1 and CXCR2 receptors [37]. In humans, the gene polymorphism studies indicate that regions within the gene, others than promoter region, may contribute to CXCL8 production and, potentially, susceptibility to certain infectious diseases [38]. In cattle, analyzing of key molecules of the innate immune system in mammary epithelial cells has been revealed that the chemokine IL8 showed a significant increase in expression level in Escherichia coli as well as in Staphylococcus aureus -affected cells [39]. Brenaut et al. [21] revealed that mammary epithelial cells play an important role in the recruitment and activation of inflammatory cells through the IL8 signaling pathway. Furthermore, several researches suggest that different polymorphisms of the IL8 gene are associated with increased risk of infection from Escherichia coli and Helicobacter pylori and due to infection, have elevated inflammatory responses and/or more clinically significant disease [40,41]. In the present research, both investigated SNPs in IL8 were polymorphic (IL8 g.86041868A>G and IL8 g.86040123G>A).
The interleukin-6 (IL6) is a cytokine with a wide range of biological activities that play an important role in immune regulation and inflammation. Data from several studies on humans suggest that IL6 plays a critical role in the B cell hyperactivity and immunopathology of several diseases [42,43]. In our study, IL6 g.29257937T>C was polymorphic in all three studied breeds, with the highest value of MAF found in Banat’s White goats (0.11).
The tumor necrosis factor (TNF), known as TNFα or TNF alpha, is involved in systemic inflammation. The primary role of TNF is in the regulation of immune cells. A recent study examined the diversity in the TNF-α exon 4 and 3'UTR in native Chinese domestic goats and three SNPs in the 3'UTR region [44] were identified. However, an investigation on North American goat breeds revealed that the same analyzed SNPs of TNF-α were monomorphic [45]. In cattle, the role of TNF-α has been reported also in an acute mastitis [46]. Here, the locus TNF g.26141981T>A was polymorphic in all breeds, having the same value of MAF (0.13).
The interleukin-1 receptor antagonist (IL-1RA), encoded by the IL1RN, belongs to the interleukin 1 cytokine family [47]. The protein encoded by this gene modulates a variety of interleukin 1 related to the immune and inflammatory responses [48]. In humans, the polymorphisms of this gene were reported to be associated with different diseases [49]. Brenaut et al. [21] investigated in goats the in vivo transcriptional response of mammary epithelial cells at the early stages of infection with Staphylococcus aureus and found a highly increased level of IL1RN expression. In our research, were analyze four non-synonymous SNPs in IL1RN gene and found IL1RN g.46358256A>G and IL1RN g.46353777G>T to be polymorphic in all studied animals.
The IL15RA gene encodes a cytokine receptor that specifically binds interleukin 15 (IL15) with high affinity. Among related pathways are the IL-15 signaling pathways and its primary biological effects on different immune cell types and innate immune systems. In humans, the SNPs in IL15RA were correlated with macro-pathogen richness and might indicate selection for improved intestinal clearance of nematodes [50]. However, no studies on goats were published up-to-date related to polymorphism in IL15RA. Current findings revealed four polymorphic SNPs in IL15RA gene (IL15RA g.10343904C>T, IL15RA g.10344025C>T, IL15RA g.10354726G>A and IL15RA g.10354813C>T) out of ten analyzed non-synonymous SNPs in exon regions. The values of MAF in three of IL15RA loci was >42%, those markers are therefore high informative.
The SNPs investigated in the present study, such as CLEC4E, IL6, IL8, IL1RN, IL15RA, PTX3, SOCS3, TNF, TNFSF13 and TLR3, may be potential markers for genetic resistance to the mammary gland and gastrointestinal parasite infection in goats. Several studies revealed a number of polymorphisms of these genes that proved to have crucial roles in pathogen recognition and influencing additional immunological processes and therefore playing an important role in infections. However, to date, the full importance of these SNPs variations is unclear and therefore, an increased understanding of those genes variation in each goat breed is important for determining the genes associated with parasite and mammary gland infection.
This study provides the first view of the polymorphisms in genes related to mastitis and parasite infection in Romanian and Hungarian goat breeds. By KASP assay was possible to identify 16 polymorphic SNPs in ten genes related to mastitis and parasite infection in Banat’s White, Carpatina and Hungarian Milking goat breeds. Although the use of this SNPs needs further studies related to marker associations and their marker-quantitative trait locus phase relationships in each population, in order to specify each SNP effect, the results obtained could prove valuable and contribute to future molecular markers studies related to parasites and mammary gland infections in goats.
Genetic diversity among Romanian and Hungarian indigenous goat breeds
The results of the current study showed a low genetic differentiation (FST) among studied breeds. The mean FST ranging from 0.009 for Banat’s White/Carpatina to 0.014 for Romanian breeds/Hungarian Milking. Those values of genetic differentiation among goat breeds are lower than values reported in previous studies on eight goat breeds from different European regions genotyped for 27 SNPs, where the FST values were variable within the range of 0.004–0.224 [19] or for 16 breeds from Italy, Albania and Greece assessed by 27 SNPs that revealed an overall FST of 0.063 [51]. Results obtained in the present research are comparable with other studies where the lower FST values were found among Argentinean goat populations [52]. However, the low level of genetic differentiation in the studied goat breeds could be the result of common origin, given the common border that Hungary and Romania have, and the shared common history.
For the SNPs markers, the PIC values in Romanian and Hungarian indigenous goat breeds ranged from 0.000 to 0.693 with an average value of 0.381. When individual PIC values were examined it was observed that a substantial portion (62,5%) of SNPs provided a moderate level of information (PIC≤0.50). Four SNPs (PTX3 g.108076746C>T, IL8 g.86041868A>G, IL15RA g.10354726G>A and TNFSF13 g.26523480A>G) possess low genetic diversity in all studied goat breeds, indicating that these loci are not suggested to be effective in evaluating genetic resources of the studied goat breeds. Low PIC values were earlier reported in Korean goats [53] using microsatellite analysis. However, due to bi-allelic nature of SNPs, their PIC values can result lower. Approximately 37% of the SNPs used in the present study were informative (PIC≥0.50). Our results revealed that SOCS3 g.52626440T>G, IL15RA g.10343904C>T, IL15RA g.10344025C>T and TLR3 g.14987931C>G are the most polymorphic markers and therefore can be utilized for molecular characterization of the studied goat breeds.
To conclude, KASP technologies were used in current research in order to investigate a total of 52 SNPs belonging to 19 genes involved in genetic resistance to the mammary gland and gastrointestinal infection. Almost the polymorphic SNPs investigated were non-synonymous SNPs that suggested their functional role in the immune response and inflammation. The results obtained in the present study may be a further step with respect to the development of SNP genotyping assay for genetic resistance to the mammary gland and gastrointestinal infection.
Supporting information
S1 Table. The collection sites and geographic coordinates of the Romanian and Hungarian goat breeds included in the study.
https://doi.org/10.1371/journal.pone.0197051.s001
(DOC)
S2 Table. Details of genes, chromosome location and genomic location at 52 SNP loci under study.
https://doi.org/10.1371/journal.pone.0197051.s002
(DOCX)
S3 Table. Success ratio of 52 SNPs investigated through KASP assay for 150 samples from Banat’s White, Carpatina and Hungarian Milking goat breeds.
https://doi.org/10.1371/journal.pone.0197051.s003
(DOCX)
Acknowledgments
The author would like to thank Dr. Sándor Kukovics and Dr. Gyula Veress for providing most part of the Hungarian samples.
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