COBISS: 1.08 Agris category code: L10
HOW BLACK IS REALLY BLACK SLAVONIAN PIG?
Polona MARGETA 1, Vladimir MARGETA 1, Kristina BUDIMIR 1
abstract
Black Slavonian pig is Croatian autochthonous pig breed, established at the end of the 19th century by crossing locally raised Mangalitsa pigs with Berkshire, Poland China and Large Black pig breeds. Previous study of the main coat colour Extension locus in Black Slavonian pigs revealed the presence of the ED1 allele, typical for black pig breeds of Asian origin. Because the Black Slavonian pig is the only breed with ED1 genotype raised in Croatia, the genotyping of the MC1R gene was used to determine the purity of the Black Slavonian pigs. PCR-RFLP analysis using NspI restriction endonuclease performed on 179 pigs from 9 farms in Osjek-Baranja County shows that only 70 pigs were pure black while other 109 pigs were crossbreeds. The method used is simple, fast, low-cost and reliable and for that reasons it will be further used in the selection programme of Black Slavonian pig.
Key words: Black Slavonian pigs / MC1R gene / PCR-RFLP genotyping
1 introduction
Black Slavonian pig is Croatian autochthonous pig breed, established at the end of the 19th century near Osi-jek in Slavonia. Its establishment was a result of a planned selection under the supervision of Count Pfeifer, who crossed a locally raised Mangalitsa pigs with Berkshire, Poland China and Large black pig breeds. He created a pig which had excellent characteristics for that time, as confirmed by a gold medal won at Vienna World Exposition in 1873.
The breed was black, resistant and convenient for keeping in extensive (pastures, woods) and half-extensive conditions (pens with some free space). It was also characterised by meat quality, suitable for producing typical traditional meat products like "kulen" (dry cured sausage spiced with red paprika), ham, bacon and other sausages (Karolyi et al., 2007).
After World War II this pig breed was almost forgotten, raised mostly in extensive condition by local farmers, which provided the opportunity for crossmating with lo-
cal wild boar population, and also for uncontrolled crossing with modern pig breeds (Large White, Yorkshire, Pi-etrain, Duroc). Phenotypic distinction between purebred and F1 crossbred pigs is not possible because of dominant black colour of Black Slavonian pig.
Out of several genes affecting coat colour in domestic animals, the melanocortin receptor 1 (MC1R) locus is the most consistently polymorphic. MC1R is primarily expressed in melanocytes and plays a key role in melano-genesis by determining the switch between production of red/yellow pheomelanin and dark eumelanin (Robbins et al., 1993; Barsh, 1996). Loss-of-function mutations are associated with recessive red coat color, whereas dominant black coloring is linked with mutations causing the activation of MC1R signalling.
The molecular basis at the Extension (MC1R) locus in pigs is well defined (Kijas et al., 1998; Kijas, 2001) and described in numerous pig breeds (Fang et al., 2009). The wild-type (E+) allele allows full expression of both phe-omelanin and eumelanin. The dominant black colour results from two different mutations, each of them evolved
1 J.J.Strossmayer University of Osijek, Kralja Petra Svacica 1d, Osijek, Croatia, e-mail: polona.frajman@gmail.com
independently in Asia and Europe. The ED1 allele is Asian in origin and is associated with an L102P missense mutation, and ED2 is European and associated with a D124N substitution. The recessive red allele (e) controls two missense mutations A164V and A243T and allele EP causes black spotting on a red or white background.
Previous study of the Extension locus in Black Slavonian pigs revealed the presence of the ED1 allele (Margeta et al., 2009). Because the Black Slavonian pig breed is the only one with ED1 genotype raised in Croatia, the genotyping of the MC1R gene was used to determine purity of the Black Slavonian pigs. The method is simple, fast, low-cost and reliable.
2 MATERIALS AND METHODS
2.1 SEARCH FOR POLYMORPHIC SITES
The previously obtained (Margeta et al., 2009) sequence of the 5' part of the MC1R exon from Black Slavonian pig (ED1 genotype) was aligned using ClustalX programme (Larkin et al., 2007) with sequences of other pig breeds with different MC1R genotype: Wild Boar
(E+), Large White (EP), Hampshire (ED2) and Duroc (e) (Fig. 1). Polymorphic sites specific only for Black Slavonian pig breed were further searched for the presence of the restriction endonucleases recognition sites. The search was directed into commercially available endonu-cleases which cut the sequence once or mostly twice to obtain enzymes useful for fast and reliable screening and determination of the ED1 genotype.
2.2 PCR AMPLIFICATION AND RESTRICTION
Samples of 179 Black Slavonian pigs were collected on 9 farms in Osijek-Baranja County. Genomic DNA was isolated from samples with Genomic DNA Miniprep Kit (Bio Basic Inc.). A primer pair MERL1 (5'-RGTGCCT-GGAGGTGTCCAT-3') and EPIG2 (5'-CGCCCAGAT-GGCCGCGATGGACCG-3') was used to amplify a 428-bp product from the 5' half of the single exon of MC1R gene (Kijas et al., 1998). PCR was performed and 10 ^l of the PCR-products from all 179 animals were digested by restriction endonuclease NspI (Thermo Scientific) to determine genotype at nucleotide position 282. Only pigs
Hampshire(ED2)	GGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCT 248
Large White(Ep)	GGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCT 248
Wild Boar (E+)	GGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCT 248
Duroc (e)	GGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCT 2 48
Black Slavonian(ED1)	GGCCGCCATCGCCAAGAACCGCAACCTGCACTCGCCCATGTACTACTTCGTCTGCTGCCT 248
Hampshire(ED2)	GGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACGTGCTGGAGACGGCCGTGCTGCTGCT 308
Large White(Ep)	GGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACGTGCTGGAGACGGCCGTGCTGCTGCT 308
Wild Boar (E+ )	GGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACGTGCTGGAGACGGCCGTGCTGCTGCT 308
Duroc (e)	GGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACGTGCTGGAGACGGCCGTGCTGCTGCT 308
Black Slavonian(ED1) GGCCGTGTCGGACCTGCTGGTGAGCGTGAGCAACATGCTGGAGACGGCCGTGCTGCCGCT 308
********************************** ********************* ***
Hampshire(ED2)	GCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAATGTCAT 3 6
Large White(Ep)	GCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAATGTCAT 3 6
Wild Boar (E+)	GCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAATGTCAT 36
Duroc (e)	GCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAATGTCAT 3 6
Black Slavonian(ED1) GCTGGAGGCGGGCGCCCTGGCCGCCCAGGCCGCCGTGGTGCAGCAGCTGGACAACGTCAT 36
****************************************************** *****
Hampshire(ED2)	GAACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGT 428
Large White(Ep)	GAACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGT 428
Wild Boar (E+)	GGACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGT 428
Duroc (e)	GGACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGT 428
Black Slavonian(ED1) GGACGTGCTCATCTGCGGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGCGCCATCGCCGT 428
* **********************************************************
Figure 1: A part of a CLUSTAL 2.1 multiple sequence alignment of the 5' part of the MC1R exon1of pig breeds with different Extension locus genotype. Polymorphic sites specific for the Black Slavonian pig breed (genotype ED1) are shaded and NspI recognition site is underlined.
with ED1 allele holds Nspl restriction site (RCATGY). The digestion-reactions were checked on 2% agarose gel.
3 RESULTS AND DISCUSSION
3.1 SEARCH FOR POLYMORPHIC SITES
Multiple sequence alignment (ClustalX) of the 5' part of the MC1R exon (from nucleotide position 128 to nucleotide position 428 from transcriptional start site) from different pig breeds revealed tree polymorphic sites specific for the Black Slavonian pig breed: A-G substitution at nucleotide position 282, T-C substitution at position 305, and T-C substitution at nucleotide position 363, respectively (Fig. 1).
All tree polymorphic sites were further searched for the presence of the restriction endonucleases recognition sites. The search directed into commercially available endonucleases which cut the sequence once or mostly twice revealed only one restriction endonuclease with mentioned characteristics. Restriction endonucle-ase Nspl with recognition sequence RCATGY recognizes the polymorphic site at nucleotide position 282 and cuts the sequence in Black Slavonian pigs (ACATGC), while simple substitution abolishes recognition sequence in pig breeds of other MC1R genotypes (ACGTGC).
3.2 PCR AMPLIFICATION AND RESTRICTION
PCR amplification of the 428 bp from the 5' site of the MC1R exon was performed on all 179 collected samples of Black Slavonian pigs. Results of the restriction digestion of the PCR product with NspI restriction en-donuclease showed, that out of 179 pigs included in the study, 70 (39.1%) were pure Black Slavonian pigs (genotype ED1), while other 109 (60.9%) pigs were crossbreeds (Fig. 2). The higher number of crossbred pigs was expected, because of unplanned crossings with commercial pig breeds like Large White, Yorkshire, Pietrain and Duroc in the near past. Because of extensive and half-extensive keeping, there were also chances for contact with local wild boar population.
By comparing results from each farm, it could be observed that farms with higher number of pigs had also higher number of crossbred pigs. There was only one farm on which all eight analysed pigs were pure black. Interestingly, that was the farm were we performed MC1R genotyping few years ago (Margeta et al., 2009). The owner decided at that time to keep the pigs shown with PCR-RFLP analysis to be pure black and those included in the present study are their descendants.
Figure 2: Columns on the chart represent numbers of pure and crossbred pigs on each farm, and the chart-pie represents the percentage of pure Black Slavonian pigs and crossbreeds among all 179 analysed samples
4	CONCLUSION
In the present study, we were able to identify a simple PCR-RFLP method, based on different coat colour MC1R gene genotypes, enabling us to detect potential crossings of autochthonous Black Slavonian pig with modern pig breeds or wild boars. Analysis of 179 pigs showed that the majority of Black Slavonian pigs today are crossbred and for that reason the MC1R genotyping will be further used in selection of pure Black Slavonian pigs.
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