Breed identification and authentication of Black Slavonian pigs by DNA analysis

Istraživanje je provedeno na 107 životinja od kojih je bilo 50 svinja crne slavonske pasmine (CS), 15 PIC hibrida, 12 pietrena (P), 10 topigsa (TP), 10 velikih jorkšira (J), 5 landrasa (L) i 5 duroka (D). Od ukupno analiziranih 100 svinja crne slavonske pasmine kod kojih smo odredili genotip MC1R gena odabrano je 30 životinja homozigota za crnu boju (CS), te 20 životinja heterozigota (CSX). Cilj istraživanja bio je utvrditi genetski status crne slavonske svinje pomoću seta od 26 mikrosatelitska markera te optimizirati najmanji broj mikrosatelita dovoljnih za identifikaciju pasmine te autentifikaciju proizvoda načinjenih od iste. Prema preporuci ISAG-FAO odabran je set od 26 mikrosatelitna lokusa (S0026, S0155, S0005, Sw2410, Sw830, S0355, Sw24, Sw632, Swr1941, Sw936, S0218, S0228, Sw122, Sw857, S0097, sw240, IGF1, Sw2406, Sw72, S0226, S0090, Sw2008, Sw1067, S0101, S0178, Sw911 i S0002) grupirana u tri združene PCR reakcije. Dobiveni PCR produkti su analizirani pomoću ABI 3730XLs (Applied Biosystems). Analizom genetske strukture populacija utvrđena je niska genetska udaljenost između CS i CSX skupina, no visoka genetska udaljenost s ostalim pasminama. Analizom frekvencije gena identificirano je 15 privatnih alela specifičnih za skupinu crnih svinja homozigota na MC1R genu, te 10 privatnih alela specifičnih samo za heterozigote na MC1R genu. Prosječan broj alela po lokusu se kretao od 2,61 do 6,54 što ukazuje na veliku genetsku raznolikost. Optimizacijom broja mikrosatelitskih lokusa temeljenom na frekvenciji alela, broju privatnih alela, vrijednosti informacijskog sadržaja polimorfizma te izračunu vjerojatnosti identiteta utvrđen je set od pet markera koji su dovoljno informativni za identifikaciju pasmine i autentifikaciju proizvoda. Koristeći navedeni set moguće je identificirati crnu slavonsku pasminu od koje su proizvedeni proizvodi napravljeni od pojedinačnih životinja (šunka ili pršut), međutim identifikacija u kobasičarskim proizvodima nije izvediva zbog većeg broja životinja korištenih pri proizvodnji ovakvih proizvoda, što za ovaj tip analize i veliku polimorfnosti mikrosatelitskih markera nije pogodno.The study was conducted on 107 animals, of which there were 50 pigs Black Slavonian breed (CS) 15 PIC hybrids, 12 Pietrain (P), 10 Topigs (TP), 10 Large White (J), 5 Landrace (L) and 5 Duroc (D). The aim of this study was to determine the genetic status of the Black Slavonian pig with a set of 26 microsatellite markers that were grouped into 3 multiplex PCR reactions and optimize the smallest number of microsatellite markers that are sufficient for breed identification and product authentication. Isolation of DNA from the collected samples were performed using Thermo Scientific Gene Jet Genomic DNA Purification Kit. Insulation performance was checked by electrophoresis on 1.0% agarose gel. After checking, the isolated DNA was stored at - 20°C and was subsequently used for the genotyping of microsatellite loci. According to the recommendation ISAG-FAO selected set of 26 microsatellite loci were: S0026, S0155, S0005, Sw2410, Sw830, S0355, SW24, Sw632, Swr1941, Sw936, s0218, s0228, Sw122, Sw857, S0097, sw240, IGF1, Sw2406, Sw72 , s0226, S0090, SW2008, Sw1067, S0101, S0178, S0002 and Sw911. Microsatellites are grouped into 4 combined with the reaction due to their length. In a joint response to each of three microsatellites were marked with the same colour (F-6-FAM - blue, ATTO550- yellow, H HEX green). By analysing the genetic structure of populations showed low distance between the two groups of Black Slavonian breed, but a high genetic distance to other breeds. Analysis of allele frequency was found 15 private allele specific for black pigs homozygous the MC1R gene, and 10 private alleles specific for hybrids. The average number of alleles per locus ranged from 2.61 to 6.538 indicating a great genetic diversity. The optimization of the number of microsatellite loci was based on the frequency of alleles, the number of private alleles, the value of polymorphism information content and calculation of probability of identity. The number of microsatellite loci was optimized and analysis was provided a set of five microsatellite markers that were sufficiently informative for breed identification and product authentication. With a set made of 5 microsatellite markers, it is possible to identify the breed from which are certain food products are produced, such as ham. Identification in other kind of products is not possible because such products are produced by a large number of animals witch is not suitable for this type of analysis because of high microsatellite polymorphism