INVESTIGATION OF G+265C AND G-1539A SINGLE NUCLEOTIDE POLYMORPHISMS OF TOLL-LIKE RECEPTOR 4 GENE (TLR4) IN SOME CATTLE BREEDS RAISED IN TURKEY

Abstract

Toll-like receptors play an essential role in how the innate immune system reacts to pathogens. These receptors help the innate immune system recognise the antigenic structure of pathogens and initiate the inflammatory response. TLR4 is one of these receptors, and it has been identified as a candidate molecular marker for resistance to mastitis in cattle. This study aimed to describe G-1539A (BgII) in promoter and G+265C (MspI) in 5'-UTR polymorphisms in the TLR4 gene using Zavot (n=60), East Anatolian Red (EAR, n=49), Anatolian Black (AB, n=59), South Anatolian Red (SAR, n=42), Turkish Gray (TG, n=60) and Holstein (n=218) cattle breeds. For this purpose, we used polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) to genotype a total of 488 cattle. Two alleles (A and G) and three genotypes (GG, GA and AA) were examined by digestion of the PCR product with BgII restriction enzyme. The frequency of the G allele was higher in all investigated breeds except Zavot. The GA genotype was found to be the most common genotype in the EAR, SAR, TG, AB, Holstein, and Zavot breeds. Two alleles (C and G) and three genotypes (GG, GC and CC) were examined by digestion of the PCR product for the G+265C SNP with the MspI restriction enzyme. The most prevalent in terms of frequency was the C allele in all breeds with regard to G+265C SNP. All examined breeds were within the Hardy-Weinberg equilibrium (p>0.05). Consequently, the existence of two SNPs of the TLR4 gene has been reported for the first time in six cattle breeds raised in Turkey. The study demonstrated that the investigated breeds continue to exhibit variation in terms of these two SNPs. It might be possible to utilise these polymorphisms in efforts to breed herds resistant to significant breeding diseases, such as mastitis. Key words: cattle; PCR-RFLP; promoter; SNP; TLR4 RAZISKOVANJE ENOJNIH NUKLEOTIDNIH POLIMORFIZMOV G+265C IN G-1539A GENA RECEPTORJA TLR4 PRI NEKATERIH PASMAH KRAV, GOJENIH V TURCIJI Povzetek: Tolicni receptorji oz. receptorji TLR (iz angl. Toll-like receptors) igrajo bistveno vlogo pri reakciji prirojenega imunskega sistema na patogene. Ti receptorji pomagajo prirojenemu imunskemu sistemu prepoznati antigensko strukturo patogenov in sprožiti vnetni odziv. TLR4 je bil izmed skupine tolicnih receptorjev prepoznan kot kandidatni molekularni oznacevalec za odpornost na mastitis pri govedu. V studiji smo želeli opisati G-1539A (BgII) v promotorju in G+265C (MspI) v 5'-UTR polimorfizmih gena TLR4 pri razlicnih pasmah goveda: pasmi Zavot (n = 60), vzhodno anatolskem rdecem govedu (EAR, n = 49), anatolijskemu crnemu govedu (AB, n = 59), južno anatolskemu rdecemua govedu (SAR, n = 42), turskem sivem govedu (TG, n = 60) in govedu pasme Holstein (n = 218). V ta namen smo uporabili verižno reakcijo s polimerazo in polimorfizem dolžin restrikcijskih odsekov (PCR-RFLP) za genotipizacijo 488 govedi. Z restrikcijsko cepitvijo produktov PCR z restrikcijskim encimom BgII smo pregledali dva alela (A in G) in tri genotipe (GG, GA in AA). Pogostost alela G je bila vecja pri vseh raziskanih pasmah, razen pasmi Zavot. Ugotovljeno je bilo, da je genotip GA najpogostejsi genotip pri pasmah goveda EAR, SAR, TG, AB, Holstein in Zavot. Dva alela (C in G) in trije genotipi (GG, GC in CC) so bili pregledani z restrikcijsko cepitvijo produkta PCR za SNP G+65C z restrikcijskim encimom MspI. Alel C je bil najbolj razsirjen pri vseh pasmah. Vse pregledane pasme so bile znotraj Hardy-Weinbergovega ravnotežja (p > 0,05). V raziskavo torej porocamo prvic o obstoju dveh SNP gena TLR4 pri sestih pasmah govedi, vzrejenih v Turciji. Studija je pokazala, da so pri preiskanih pasmah vidne razlike v teh dveh SNP. Te polimorfizme bi bilo mogoce uporabiti pri prizadevanju za vzrejo cred, odpornih na pomembne bolezni pri govedu, kot je mastitis. Kljucne besede: govedo; PCR-RFLP; promotor; SNP; TLR4