The experiment was conducted on Haringhata Black Chicken breed to investigate the polymorphism of

exon-2 of Toll-Like Receptor-4 gene. Genomic DNA extracted from 82 birds was used for Polymerase Chain Reaction

(PCR). Amplified polymerase chain reaction (PCR) product (248bp) of exon-2 of TLR4 gene revealed two types of SSCP

banding pattern arbitrarily assigned as EF and FF genotypes on 10 percent polyacrylamide gel. The genotype frequencies

were found to be 0.634 for EF and 0.366 for FF genotype with respect to exon-2 of TLR4 gene. The frequencies of E and

F alleles were estimated as 0.317 and 0.683, respectively. The calculated Chi square value revealed that the population

was not in Hardy Weinberg Equilibrium with respect to TLR4 gene in Haringhata Black Chicken

Baidya S

Bhattacharya D.

Ghosh P. R.

Pan S

Sahoo A.K.

Saikhom R

Sarkar U.

Taraphder S

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154Exploratory Animal and Medical Research, Vol.8, Issue 2, December, 2018Research ArticleTOLL-LIKE RECEPTOR-4 (TLR4) GENE POLYMORPHISM IN HARINGHATABLACK CHICKEN BREEDSaikhom R, Sahoo A.K., Taraphder S*, Pan S1, Sarkar U., Ghosh P. R.2, Bhattacharya D.3, Baidya S4.Received 17 September 2018, revised 28 November, 2018ABSTRACT: The experiment was conducted on Haringhata Black Chicken breed to investigate the polymorphism ofexon-2 of Toll-Like Receptor-4 gene. Genomic DNA extracted from 82 birds was used for Polymerase Chain Reaction(PCR). Amplified polymerase chain reaction (PCR) product (248bp) of exon-2 of TLR4 gene revealed two types of SSCPbanding pattern arbitrarily assigned as EF and FF genotypes on 10 percent polyacrylamide gel. The genotype frequencieswere found to be 0.634 for EF and 0.366 for FF genotype with respect to exon-2 of TLR4 gene. The frequencies of E andF alleles were estimated as 0.317 and 0.683, respectively. The calculated Chi square value revealed that the populationwas not in Hardy Weinberg Equilibrium with respect to TLR4 gene in Haringhata Black Chicken.Key words: Haringhata Black Chicken, TLR4 Gene, Polymorphism, PCR-SSCP.Department of  Animal Genetics and Breeding, 1Department of Livestock Production and Management, 2 Department ofVeterinary Physiology,  3 Department of Livestock Products Technology, 4 Department of Veterinary Parasitology, Faculty ofVeterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37 Belgachia Road, Kolkata -700037, West Bengal, India.*Corresponding author. e-mail: subhash.taraphder@gmail.comINTRODUCTIONToll-Like Receptors popularly known as TLRs aremembers of cellular receptors belonging to the innateimmune system. These receptors recognize molecularpatterns known as pathogen associated molecular patterns(PAMPs) which are unique to microbes namely,lipopolysaccharide (LPS), double stranded RNA,flagellin, lipoteichoic acid (LTA) etc. TLRs are presentin the organs that are involved in immune responses andin the tissues that are generally exposed to pathogens,including skin, respiratory tract, intestinal and genito-urinary tracts, bladder, kidney, spleen and thymus(Slawinska et al. 2013).The chicken genomic Toll-Like Receptor-4 (TLR4) is5164 bp long, consists 2 introns (813 and 1370 bp) and 3exons (230, 167 and 2584 bp) (Huang et al. 2015). Thereare 13 members of TLR found in mammals (Beutler 2005)(man and mice). Among various TLRs, TLR4 andTLR5are observed in high levels in Indian Aseel andKadaknath breeds when compared to commercial broilers(Dhinakar et al. 2011). The higher levels of immunityobserved in indigenous chickens may be correlated tosuch an increased TLR mRNAs expression. Leveque etal. (2003) proposed that the polymorphism of chickenTLR4 was related to susceptibility to salmonellainfection. In the present study, polymorphism of exon 2region of chicken TLR4 gene was carried out onregistered breed Haringhata Black Chicken using PCR-SSCP technique.MATERIALS  AND  METHODSExperimental  birdA total of randomly selected eighty-two (82)Haringhata Black chicken at 22nd  weeks of age reared atintensive management system of the Haringhata PoultryFarm located at Mohanpur in Nadia District of WestBengal, India were used to investigate the geneticpolymorphism of Toll-Like Receptor 4 (TLR4) in thispresent study.DNA extractionApproximately 2 ml of blood sample was collectedaseptically from each individual from wing vein invaccutainer containing 200-300 µl of 10 percent EDTAas anticoagulant. The tubes were shaken gently tofacilitate thorough mixing of blood with theanticoagulant. The samples were then immediatelybrought to the laboratory in an icebox containing ice packsExplor Anim Med Res,Vol.8, Issue - 2, 2018, p. 154-158ISSN  2277- 470X (Print), ISSN 2319-247X (Online)Website: www.animalmedicalresearch.org155and stored at 4°C till further use. Genomic DNA wasextracted from white blood cells using standard saltingout procedure as described by Miller et al. (1988) withminor modifications. DNA samples were dissolved in0.1X TE buffer (pH 8.0). Horizontal submarine agarosegel electrophoresis was performed to check the qualityof genomic DNA using 0.8% w/v agarose (low EEO) geland visualizing the band under gel documentation system.The purity and concentration of DNA samples wereestimated by using UV-visible range spectrophotometer.All the DNA samples had 260/280 OD ratios in the rangeof 1.8 to 2.0, indicating high purity. The extracted DNAsamples were stored at -20°C till further use. DNAconcentration was adjusted to 30ng/µl before polymerasechain reaction (PCR) amplification.DNA  amplifications (PCR)The region of exon- 2 of TLR4 gene of HaringhataBlack Chicken was amplified by Polymerase ChainReaction (PCR) technique using the following primersas described by Liu et al. (2011). Primers for TLR 4:Forward 5’- ATC TGC CAC AGG TCA TCC-3’ Reverse:5’-AGC CAC GAG ACT CCA AA-3’. PCR was carriedout in a final reaction volume of 25 µl. PCR conditionsincluding the cyclic condition and the concentration ofdifferent ingredients of PCR reaction mixture wasstandardized for target gene fragment after carrying outPCR using several combinations. Then standardized PCRreaction mixture and standardized PCR thermocyclicconditions used for amplification of target fragment.Genomic DNA (2 µl) was amplified with 1 µl Taq DNApolymerase, 3.2 µl MgCl2, 1.5 µl dNTPs, 2 µl PCR bufferand 1 µl of each primer and Nuclease Free Water (NFW)13.3 µl in a total volume of 25 µl PCR mixture. The PCRmixture was properly mixed until homogeneous theninserted into the PCR machine for amplification. Theamplification for TLR4 gene was carried out using apreprogrammed thermal cycler (Eppendrof Mastercycler)with the following PCR condition: initial denaturation950C for 5 min followed by 35 cycles with each cycledenaturation 940C for 50 sec, annealing 60°C for 30 sec,xtension 720C for 50 sec and final extension at 720C for5 min. The amplified amplicons were run at 2% horizontalagarose gel electrophoresis at 70 volts for 120 minutes.Agarose gel (2%) was stained using ethidium bromide tovisualize the PCR product.Single strand conformational polymorphismThe PCR-SSCP technique for genotyping exon-2region of TLR4 gene revealed a good deal ofpolymorphism in it. The procedure of SSCP used in thisinvestigation was as per Orita et al. (1989) with minormodifications to study the sequence variation. PCRproducts (4 µl) from each individual sample werethoroughly mixed with 12 µl volume of SSCP loadingbuffer dye into 200 µl PCR tubes. The tubes were heatedin water bath at 94°C for 10 minutes for denaturation.The tubes were immediately snap cooled on ice for 10minutes to make the single strand DNA for itsonfirmation. The samples were then loaded immediatelyonto 10% polyacrylamide gel. The electrodes wereconnected properly with the power pack and the gel wasFig. 1. PCR amplified product of exon-2 region of TLR 4gene on 2% Agarose gel.Lane 1-5 : PCR Product (248 bp)Lane 6 : Negative ControlM  Lane : DNA Ladder (100 bp)Lane 1,4, 5 : EF GenotypeLane 2,3 : FF GenotypeFig. 2.  Different SSCP patterns of 248 bp fragment of exon2 region of TLR4 gene.Toll-like receptor-4 (TLR-4) gene polymorphism in Haringhata black chicken breed156Exploratory Animal and Medical Research, Vol.8, Issue 2, December, 2018run at the rate of 70 volt for 12 hours at room temperature.Silver staining of polyacrylamide gel was carried outfollowing the method as described by Byun et al. (2009)with few modifications. The gel was stained with asolution containing 10% ethanol, 0.5% acetic acid and0.2% silver nitrate for 20 minutes to identify the DNAsequence variations. The stained gel was then placed onthe U-V transilluminator within the gel documentationsystem (Syngene) and photographs were taken foridentification of different SSCP banding patterns. Theband patterns were visualized by silver staining anddocumented by gel documentation system.Genotypes identificationThe procedure for identification of particulargenotypes used in this study was adapted from thedifferent banding pattern of single strand conformationalpolymorphism (SSCP). The genotypes were detectedaccordingly by observing at every possible combinationof the SSCP patterns that could provide identification ofalleles. Each pattern was designated as a particulargenotype. Genotype frequencies of different SSCPpatterns were estimated from the combination of variousalleles generated based on the movement of DNAmolecules.Statistical  analysisThe following parameters were obtained: the genotypecategories and the gene frequency of the alleles thatcorresponded to the exon-2 of TLR4 genotype. Thefrequency of genotype and allele were computedfollowing standard procedure (Falconer and Mackay1996) of direct counting method for codominant loci. TheChi-Square (χ2) test for goodness of fit was used to findout difference among various genotypes and tested forHardy-Weinberg Equilibrium. The significance ofcalculated value was adjudged from the Table values ofSnedecor and Cochran (1994).RESULTS  AND  DISCUSSIONTLR4 PolymorphismIn the present study, eighty-two (82) DNA samplesfrom Haringhata Black chicken were screened to detectpolymorphism in amplified exon-2 fragment of TLR4gene using PCR-SSCP technique. The yielded PCRproduct was 248 bp (Fig. 1). The PCR product obtainedfor exon-2 of TLR4 gene agreed with that reported byLiu et al. (2011). However, Ulupi et al. (2013) obtaineda 220 bp size PCR product for the exon-2 region of TLR4gene. Good quality PCR amplified products (sufficientlyintense and without any spurious bands) as assessed byhorizontal submarine agarose gel electrophoresis wereused for SSCP analysis. The electrophoretic picture ofdifferent banding pattern and their mobilities duringpolyacrylamide gel electrophoresis were compiled in aphotograph and have been depicted in Fig. 2. The resultsof polymorphism of this present investigation onHaringhata Black Chicken were carried out by observingat every possible combination of the SSCP patterns thatcould provide identification of alleles. The bandingpattern of present investigation revealed two SSCPvariants for exon-2 of TLR4 gene, which was arbitrarilyassigned as EF and FF genotype. The EF genotypeshowed triple band while FF genotype showed singleband (Table 1).  It was found that out of 82 studiedHaringhata Black chicken 52 birds had EF genotype, 30had FF genotype. The research finding in this presentinvestigation revealed that 63.4 percent of the studiedbirds had EF genotype, 36.6 percent had FF genotypeindicating higher frequency of EF genotype followed byFF genotype for exon-2 region of TLR4 gene inHaringhata Black chicken population under present study.This finding was in contrast with those of Liu et al. (2011)and Yan et al. (2011) who reported all the three genotypesEE, EF and FF in Chinese chicken breed. The absence ofEE genotype in the present study might be due to limitedsize of the sampled Haringhata Black chicken population.However, Yan et al. (2011) observed higher frequency ofEF genotype in Chinese chicken bird.Genotypic  frequencyIn the analysed flock of Haringhata Black chicken, thegenotype and gene frequencies with respect to exon-2region of TLR4 gene were calculated and have beendepicted in Table 2. In the present study, genotypefrequency for EF was found to be higher than that of FFgenotype in Haringhata Black chicken with respect toexon-2 region of TLR4 gene. The genotype frequenciesfor EF and FF genotypes were 0.634 and 0.366respectively for exon-2 region of TLR4 gene. Thegenotypes observed in present investigation differed fromthose of Liu et al. (2011) and Yan et al. (2011) whoreported all the three genotypes EE, EF and FF in Chinesechicken breeds. Liu et al. (2011) found the genotypefrequencies of EE, EF and FF to be 0.251, 0.373 and0.391 respectively. Yan et al. (2011) found the genotypefrequencies of EE, EF and FF to be 0.314, 0.400 and0.286, respectively. However, Yan et al. (2011) reportedhigher frequency of EF genotype (0.400) than that of EE(0.314) and FF (0.286) genotypes. The genotypefrequencies observed in the present investigationsuggested that Haringhata Black chicken have less diversegenotypes for exon-2 region of TLR4 gene in the sampledpopulation, as compared to the earlier reports.157Allelic frequencyIn case of exon-2 region of TLR4 gene, the frequenciesof E and F alleles were 0.317 and 0.683 respectively(Table 2). The present finding revealed a higher frequencyof F allele which was predominant in the studiedHaringhata Black chicken population of the farm. Thepresent finding agreed with those of Liu et al. (2011)who reported higher frequency of F allele (0.563) thanE allele (0.437) in Chinese Chicken.  However, Yan etal. (2011) reported higher frequency of E allele (0.400)than F allele (0.286) in Chinese Chicken which was incontrary with the present finding. PCR-SSCP analysis ofToll-Like Receptor 4 gene showed polymorphism withtwo distinct alleles for TLR4 gene and F allele for TLR4gene was prevalent in the studied Haringhata Blackchicken population maintained in the farm.Hardy-Weinberg equilibriumTwo genotypes EF and FF were observed in the presentinvestigation for exon-2 region of TLR4 gene. Theobserved number and expected number of birds with EFand FF genotypes have been depicted in Table 3. It isevident that the observed and expected number of EEgenotype was 0.00 and 8.24 respectively. The observedand expected number of EF genotype was 52 and 35.51respectively and that of FF genotype was 30 and 38.25respectively. Genotypic frequencies were tested forequilibrium using Chi-square test. The calculatedChi square value obtained was 17.676 which is higherthan the tabulated value of 3.84 at 5 percent level ofsignificance at 1 degree of freedom (Table 3). Therefore,it can be concluded that the population of HaringhataBlack chicken was not in Hardy Weinberg equilibriumwith respect to exon-2 region of TLR4 gene. This resultindicated that the population might have under theinfluence of evolutionary forces like migration, mutationand selection. The present finding was in contrary to thefindings of Liu et al. (2011).In conclusion, it is inferred that Toll-Like Receptor-4gene of Haringhata Black chicken breed was polymorphicthat invites further attempt to investigate the associationstudy with important economic traits of this importantbreed of West Bengal as they performed better underintensive management for generating higher return in welladapted local environment vis-a-vis conservation of thisprecious germplasm is also essential for posterity use.ACKNOWLEDGEMENTThe financial support of this research work was fundedby the Department of Science and Technology under theMinistry of Science and Technology, Government ofIndia, by awarding the inspire fellowship to the firstauthor during Ph.D. Degree Program without which thiswork could have not been completed.REFERENCESBeutler B (2005) The Toll-like receptors: analysis by forwardgenetic methods. Immunogenetics, (PMID: 16001129) 57: 385-392.Byun SO, Fang Q, Zhou H, Hickford JGH (2009) Aneffective method for silver–staining DNA in large numbers ofpolyacrylamide gels. Analytical Biochem 385: 174-175.Dhinakar RTM, Rajanathan C, Kumanan K, Elankumaran S(2011) Expression profile of Toll- Like Receptor mRNA in anindigenous Aseel breed of chicken in India. Int J Poult Sci8: 651-655.Falconer S, Mackay TFC (1996) Introduction to quantitativegenetics, 4th edn. Longman group Ltd. Essex. England.Pattern Genotype No. of BirdThree Band EF 52Single Band FF 30Table1. Frequency of SSCP banding patterns of exon-2region of TLR4 gene in Haringhata Black chicken.EE 0 0.000 E 0.317EF 52 0.634 F 0.683FF 30 0.366Genotype No. of Bird Genotype Allele AllelicFrequency FrequencyTable 2. Genotype and allelic frequencies of exon-2 regionof TLR4 gene in Haringhata Black chicken.EE 0 8.24 8.240EF 52 35.51 7.657FF 30 38.25 1.779d.f. =1 χ2 value=17.676Genotype GenotypeObserved ExpectedNumber Number(O-E)2ETable 3. Observed and expected number of genotypes withrespect to exon-2 region of TLR4 gene in Haringhata Blackchicken.Toll-like receptor-4 (TLR-4) gene polymorphism in Haringhata black chicken breed158Exploratory Animal and Medical Research, Vol.8, Issue 2, December, 2018Huang HB, Xiao K, Lu S, Yang KL, Ansari AR, Khaliq Het al. (2015) Increased Thymic Cell  Turnover under BoronStress may bypass TLR3/4 pathway in African ostrich. PLoSONE 10(6): e0129596.Leveque G, Forgetta V, Morroll S, Smith AL, Bumstead N,Barrow P, Loredo-Osti J, Morgan K, Malo D (2003) Allelicvariation in TLR4 is linked to susceptibility to Salmonellaenterica serovar Typhimurium infection in chickens. InfectImmun 71: 1116-1124.Liu Y, Chang GB, Hu GS, Li Q, Xu Q, Chen GH 2011)Genetic variation at Exon2 of TLR4 gene and its associationwith resistant traits in chicken.  African J Biotech 10(42): 8260-8266.Miller SA, Dykes DD, Polesky HF (1988) A simple saltingout procedure for extracting DNA from human nucleated cells.Nucleic Acids Res 16 (3): 1215.Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T(1989) Detection of polymorphisms of human DNA by gelCite this article as: Saikhom R, Sahoo AK, Taraphder S, Pan S, Sarkar U, Ghosh PR, Bhattacharya D, Baidya S (2018) Toll-LikeReceptor-4 (TLR4) gene polymorphism in Haringhata Black chicken breed. Explor Anim Med Res 8(2): 154-158.electrophoresis as single-strand conformation polymorphisms.Proc Natl Acad Sci USA 86(8): 2766 -2770.Slawinska  A,  D’Andrea M,  Pilla F,  Bednarczyk M, SiwekM (2013) Expression profiles of Toll-like receptors 1, 2 and 5in selected organs of commercial and indigenous chickens.J Appl Genet  54(4): 489-492.Snedecor GW, Cochran WG (1994) Statistical methods8th edn. Affiliated East-West Press and Iowa State UniversityPress.Ulupi N, Muladno SC, Wibawan, IWT (2013) Associationof TLR4 gene genotype and resistance against Salmonellaenteritidis natural infection in Kampung chicken. Int J PoultSci 12(8): 445-450.Yan L, Guobin C, Wenbin B, Kehua W, Xueyu Z, GuohongC (2011) Polymorphism of Exon 2 of TLR4 gene and itscorrelation with some commercial traits in Anka chicken.Chineese J Anim Sci 2011-05.

TOLL-LIKE RECEPTOR-4 (TLR4) GENE POLYMORPHISM IN HARINGHATA BLACK CHICKEN BREED

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TOLL-LIKE RECEPTOR-4 (TLR4) GENE POLYMORPHISM IN HARINGHATA BLACK CHICKEN BREED

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