66 Acta argiculturae Slovenica, Supplement 5, 66–70, Ljubljana 2016 24th Int. Symp. “Animal Science Days”, Ptuj, Slovenia, Sept. 21st−23rd, 2016. COBISS: 1.08 Agris category code: L10 MICROSATELLITE MULTIPLEX METHOD FOR POTENTIAL USE IN BLACK SLAVONIAN PIG BREEDING Polona MARGETA 1, Vladimir MARGETA 2, Kristina GVOZDANOVIĆ 3, Dalida GALOVIĆ 4, Ivona DJURKIN KUŠEC 5, Goran KUŠEC 6 Microsatellite multiplex method for potential use in Black Slavonian pig breeding 1 J.J.Strossmayer University of Osijek, Kralja Petra Svačića 1d, Osijek, Croatia, e-mail: polona.frajman@gmail.com 2 Same address as 1, e-mail: vmargeta@pfos.hr 3 Same address as 1, e-mail: kgvozdanovic@pfos.hr 4 Same address as 1, e-mail: dgalovic@pfos.hr 5 Same address as 1, e-mail: idurkin@pfos.hr 6 Same address as 1, e-mail: gkusec@pfos.hr ABSTRACT Previous studies on Black Slavonian pig breed were focused on the main coat color MC1R locus and on develop- ment of the method for Black Slavonian cross-breeds identification, based on coat color genotype. Microsatellites have been used for population studies in pigs for last 25 years and are convenient markers for evaluating the genetic diversity of pig breeds, for parental analysis and identification of pigs as well as traceability of different pig breeds meat in meat products. We developed a PCR multiplex, containing 9 microsatellites in a single reaction. The method was tested on 50 Black Slavonian pig samples (30 homozygous and 20 heterozygous for MC1R locus). For the 9 microsatellite loci analyzed, 79 alleles were detected. The number of detected alleles was high for all loci, with the number of alleles per locus ranging from 5 for S0026, Sw24 and Swr1941 to 22 alleles for S0005. Our results demonstrate that described MS multiplex method may provide a reasonable amount of genetic information to be suitable for use in Black slavonian pig breeding programs in the future. Key words: pigs, breeds, Black Slavonian pigs, genetics, microsatellites, multiplex PCR 1 INTRODUCTION Black Slavonian pig is Croatian autochthonous pig breed, established at the end of the 19th century near Osi- jek in Slavonia by crossing a locally raised Mangalitsa pigs with Berkshire, Poland China and Large black pig breeds. It is black, resistant and convenient for keeping in extensive (pastures, woods) and half-extensive con- ditions (pens with some free space). It is also of great meat quality, convenient for producing traditional meat products like kulen (dry cured sausage), ham, bacon and other sausages (Karolyi et al., 2007). In the second half of the 20th century this pig breed was raised mostly in extensive conditions by local farm- ers, which provided opportunity for uncontrolled cross- ing of this pig breed with commercial pig breeds (Large White, Yorkshire, Pietrain, Duroc). Phenotypic distin- guishing between purebred and F1 crossbred pigs is not possible because of the dominant black color of Black Slavonian pig. Previous study of the Extension locus (melanocortin receptor 1) in Black Slavonian pigs revealed 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 (Margeta et al., 2013). The advantages of this meth- od are that it is fast, simple and low-cost. On the other hand, method is based only on one gene and could lead to excessive elimination of pigs from breeding program. Microsatellites have been intensely used for popula- tion studies in commercial as well as rare pig breeds in the last 25 years (Giuffra et al., 2000; SanCristobal et al., Acta agriculturae Slovenica, Supplement 5 – 2016 67 MICROSATELLITE MULTIPLEX METHOD FOR POTENTIAL USE IN BLACK SLAVONIAN PIG BREEDING 2006; Gama et al., 2013). Microsatellites have been pro- posed as convenient markers for evaluating the genetic diversity of domestic animals because of their abundant, even distribution in the genome, high level of polymor- phism and ease of genotyping. The International Society of Animal Genetics (ISAG) and the Food and Agricul- ture Organization (FAO) have recommended a set of 30 microsatellite loci (ISAG/FAO, 2011) for evaluating the genetic diversity of pigs. Microsatellites are also power- ful tool for parentage analysis in domestic pigs and wild boars (Costa et al., 2012; Nechtelberger et al., 2001). Mi- crosatellites have higher variability and consequently in- creased power for parentage assignment when compared to the same number of bi-allelic markers such as SNPs (Schlotterer, 2004). For the same reason they are used for traceability of different pig breeds meat in meat products (Jae-Don Oh et al., 2014). Because of its great quality, the Black Slavonian pig meat is indispensable in the production of traditional meat products. Last year, the process for protection of Black Slavonian pig meat at the base of origin (PDO- Protected Designation of Origin) was initiated. Present work follows the idea to develop a microsat- ellite based method, which could be potentially used for pedigree analysis, identification of Black Slavonian pigs and also for traceability of Black Slavonian pig meat in meat products. 2 MATERIALS AND METHODS 2.1 MICROSATELLITE MARKERS Nine microsatellite markers from the ISAG/FAO recommendation list were selected based on their size and annealing temperature. The markers used, chromo- some location, and expected sizes of fragments are sum- marized in Table 1. Primers were labeled with fluorescent markers (6-FAM, HEX and ATTO550) to distinguish between fragments of similar size, and microsatellite markers were grouped in a single multiplex reaction, ac- cording to PCR conditions and expected fragment sizes (Table 1). 2.2 SINGLE LOCUS PCR AMPLIFICATION A PCR program to amplify all loci individually was designed. Reaction mixture included 50 ng of genomic DNA, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1 µM of each primer and 1 U of Taq DNA polymerase (ThermoFisher scientific) per 20 µL reaction volume. Amplification was performed in the thermal cycler (Eppendorf) as follows: initial activation step 7 min at 95 °C, followed by 35 cy- cles of denaturation (45  s at 95 °C), annealing (50  s at 55 ± 3 °C) and extension (20 s at 72 °C). Cycling program ended with final extension 7 min at 72 °C. Results of PCR optimization were compared on 3 % agarose gel. Nr. Name Chr. Primer sequence (5’ -> 3’) F,A,H- indicate Dye labelled primer Annealing temperature Allele range 1.      S0026 16 F-AACCTTCCCTTCCCAATCAC CACAGACTGCTTTTTACTCC 55 °C 87–105 2.      S0155 1 F-TGTTCTCTGTTTCTCCTCTGTTTG AAAGTGGAAAGAGTCAATGGCTAT 55 °C 142–162 3.      S0005 5 F-TCCTTCCCTCCTGGTAACTA GCACTTCCTGATTCTGGGTA 55 °C 203–267 4.      Sw2410 8 A-ATTTGCCCCCAAGGTATTTC CAGGGTGTGGAGGGTAGAAG 50 °C 90–131 5.      Sw830 10 A-AAGTACCATGGAGAGGGAAATG ACATGGTTCCAAAGACCTGTG 50 °C 168–203 6.      S0355 15 A-TCTGGCTCCTACACTCCTTCTTGATG TTGGGTGGGTGCTGAAAAATAGGA 50 °C 244–271 7.      Sw24 17 H-CTTTGGGTGGAGTGTGTGC ATCCAAATGCTGCAAGCG 55 °C 95–124 8.      Sw632 7 H-TGGGTTGAAAGATTTCCCAA GGAGTCAGTACTTTGGCTTGA 55 °C 148–178 9.      Swr1941 13 H-AGAAAGCAATTTGATTTGCATAATC ACAAGGACCTACTGTATAGCACAGG 55 °C 202–224 Table 1: Microsatellite markers with corresponding chromosome location, indication of dye used for labeling (F- 6FAM, H- HEX, A- ATTO550), annealing temperature and allele range Acta agriculturae Slovenica, Supplement 5 – 201668 P. MARGETA et al. 2.3 MULTIPLEX PCR AMPLIFICATION A multiplex PCR reaction was performed with 2x QIAGEN Multiplex PCR Master Mix following the manufacturer instructions. Primers were dissolved in TE buffer (10 mMTris·Cl, 1 mM EDTA, pH 8.0) to ob- tain 100  μM stock solution. For easy and reproducible handling of nine primer-pairs used in multiplex PCR a primer mix containing all primers at equimolar con- centrations was prepared by mixing 5 μL of each primer stock solution and adding TE buffer to the final volume of 250 μL. The multiplex PCR reaction mix was prepared containing 10  μL of 2x QIAGEN Multiplex PCR Mas- ter Mix,10x primer mix, 2 μM each primer, 2 μL of 5x Q-Solution (optional), 50–100 ng of template DNA and RNase-free water to the final volume of 20 μL. Microsat- ellite cycling protocol began with initial activation step 15  min at 95 °C, followed by 35 cycles of denaturation (30 s at 94 °C), annealing (90 s at 57 °C) and extension (60 s at 72 °C). Cycling program ended with a final exten- sion for 30 min at 60 °C. 2.4 MICROSATELLITE ANALYSIS Microsatellite multiplex PCR products were sent to Macrogen (Macrogen Inc., Netherlands), where they were analyzed using GeneScan350 ROX internal standard size marker on ABI3730XL capillary gene analyzer. Data pro- cessing of .fsa files was performed with the Peak Scanner (Applyed Biosystems) and the Peak Studio (Fodor Lab UNCC 2012) software. Markers and method parameters have been empirically optimized for specific peak detec- tion and precise sizing of amplified products. Analyzes were performed on 50 unrelated Black Sla- vonian pigs, 30 of them were homozygous for the MC1R locus (black coat color genotype) while 20 were heterozy- gous. Basic information like total number of alleles per marker, allele frequencies, observed and expected het- erozygosity (Nei, 1973), FIS were obtained with Genepop version 4.2 online software (Raymond and Rousset, 1995; Rousset, 2008). Microsatellite format conversions were conducted using MS toolkit software. 3 RESULTS AND CONCLUSION A quality microsatellite assay requires specific PCR amplification of microsatellite regions, automated specific peak detection and precise sizing of amplified fragments. Hereby we present optimized conditions for the amplification of nine microsatellite loci in one mul- tiplexed PCR reaction. We also show the results of two different programs for detection of specific peaks of am- plified products and precise sizing of microsatellite frag- ments in order to achieve reproducible results. 3.1 SINGLE LOCUS PCR AMPLIFICATION PCRs for all microsatellite primer pairs were first optimized in single locus amplifications with annealing temperature ranging from 52  ° to 58 °C. Checking the products on the agarose gel revealed that microsatellites were all well amplified when annealing temperature was 57 °C. 3.2 MULTIPLEX PCR AMPLIFICATION Based on results of the single locus PCR, we de- cided to optimize multiplex PCR reaction containing all nine primer pairs using gradient annealing tempera- ture between 55 ° and 58 °C and with two different reac- tion mixes, one without and one containing Q-solution. Again, best results with clearly visible bands on agarose gel and with low background were obtained with anneal- ing temperature 57 °C and with primer mix containing Q-solution. Although predicted annealing temperatures of 18 primers ranged from 50 to 55 °C, with optimiza- tion of the annealing temperature and ingredients of the reaction mix we were able to combine them into a single multiplex PCR. During optimization process it was also noticed that quality and quantity (between 50 and 100 ng per 20 μL) of template DNA play important role in ob- taining optimal results. 3.3 MICROSATELLITE ANALYSIS After analyzis on ABI3730XL capillary gene ana- lyzer the .fsa data were processed using the Peak Scanner (Applyed Biosystems) and the Peak Studio (Fodor Lab UNCC 2012) software (Fig. 1). For the 9 microsatellite loci analyzed, 79 alleles were detected in 50 individuals of the Black Slavonian pig breed (30 homozygous and 20 heterozygous for MC1R locus; Table 2). The level of polymorphism was high for all loci, with the number of alleles per locus ranging from 5 for S0026, Sw24 and Swr1941 to 22 alleles for S0005. Some alleles at 6 loci can only be found in MC1R heterozygotes, probably as a result of crossing with other pig breeds. In order to clarify the origin and thus the usefulness of these alleles for genetic studies in the future, the study was ex- tended to other pig breeds, which are currently raised in Acta agriculturae Slovenica, Supplement 5 – 2016 69 MICROSATELLITE MULTIPLEX METHOD FOR POTENTIAL USE IN BLACK SLAVONIAN PIG BREEDING B S0026 Sw24 Sw2410 S0155 Sw830 Sw632 S0355 S0005 Swr1941 Figure 1: Electropherograms of group I markers. The x-axis represents DNA fragment size in base pairs, and the y-axis represents fluorescence units. (A) Complete spectrum of group I markers labeled with specific fluorescent tags. Sw2410, Sw830 and S0355 labeled with Atto550, Sw24, Sw632 and Swr1941 labeled with Hex, and S0026, S0155 and S0005 labeled with 6-Fam. Internal size standard is 350Rox. Results are comparable when analyzed with Peak Scanner (Applyed Biosystems; A) and Peak Studio (Fodor Lab UNCC 2012; B) software. Similarly labeled fragments are distally spaced in size by assay design and nonspecific signals are kept at a minimum. MS locus Black Slavonian pigMC1R homozygotes (n = 30) Black Slavonian pigMC1R heterozygotes (n = 20) A HExp HObs FIS(W&C) FIS(R&H) A HExp HObs FIS(W&C) FIS(R&H) S0026 5 0.6514 0.57 0.132 0.109 5 0.6962 0.65 0.068 0.057 S0155 7 0.7384 0.67 0.099 0.037 9 0.8615 0.80 0.073 0.017 S0005 21 0.9119 0.87 0.050 0.034 22 0.8974 0.85 0.054 0.027 Sw24 5 0.4588 0.47 −0.018 0.042 5 0.3885 0.40 −0.031 −0.051 Sw632 5 0.5961 0.7 −0.178 −0.122 6 0.5026 0.50 0.005 −0.002 Swr1941 4 0.3961 0.43 −0.096 −0.051 5 0.2731 0.30 −0.101 −0.064 Sw2410 9 0.7040 0.73 −0.042 −0.033 10 0.7885 0.85 −0.080 −0.051 Sw830 8 0.6429 0.57 0.120 0.040 8 0.7218 0.70 0.031 0.002 S0355 8 0.7441 0.73 0.015 −0.023 9 0.6269 0.70 −0.120 −0.096 Table 2: Number of allels (A), heterozygosity (HExp, HObs) and inbreeding coefficient (FIS) for 9 loci of MC1R homozygous and MC1R heterozygous Black Slavonian pigs Acta agriculturae Slovenica, Supplement 5 – 201670 P. MARGETA et al. Croatia (PIC, Topigs, Yorkshire, Landras, Pietrain, Du- roc); the data are still being processed and are not shown in this work. The selected microsatellites represent high level of informativeness and the expected heterozygosity val- ues (Table 2) obtained at these loci are comparable with those reported in other MS studies in different pig breeds. This confirms the reliability of this set of markers and its power of resolution for genetic analyses. Genetic makers for identification of a certain breed need to have suffi- cient genetic diversity and power. Previous studies have indicated that for successful individual identification, HEx values need to be higher than 0.6. Our results show that HEx values are little lower for two MS markers (Sw24 and Swr1941), while for the other seven MS markers they are high enough, ranging even to 0.91 for S0005 MS marker. These results demonstrated that 7 out of 9 MS mark- ers tested may provide a reasonable amount of genetic di- versity and information to be suitable for the use in breed identification, not only in live animals, but also for the identification of meat origin in meat products. Another possible implementation of described MS multiplex set is also for parentage testing and pedigree analyses of Black Slavonian pigs. Certanly, further analyses includ- ing other pig breeds and testing of MS multiplex on Black Slavonian pig families are needed to show a sutability of described MS multiplex method for use in Black slavo- nian pig breeding in the future. 4 ACKNOWLEDGMENT This work has been fully supported by Croatian Sci- ence Foundation under the project number 3396. 5 REFERENCES Costa,  V., Pérez-González, J., Santos, P., Fernández-Llario, P., Carranza, J., Zsolnai, A., Anton, I., Buzgó, J., Varga, G., Monteiro, N., Beja-Pereira, A. (2012). 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