india is of a fortune position of having the world 's best breeds of buffaloes for milk production .
special attention has to be focused on murrah breed of buffalo whose breed average milk production is about 2200  kg per lactation .
however , due to increased prevalence of infections , the realization of their true genetic merit has been hampered . among infectious diseases ,
mastitis , an inflammatory disease of the mammary gland generally caused by intramammary infections , is the most common , costly , and devastating disease in dairy animals .
therefore , attention needs to be focused to study the genes involved in disease resistance , especially for mastitis .
genes associated with immune responses of the mammary gland are potential markers because of their importance in mastitis .
the toll - like receptor-4 ( tlr-4 ) is an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections [ 1 , 2 ] .
its role in pathogen recognition and subsequent initiation of the inflammatory and immune responses , and highly polymorphic nature in the bovine species , make it a suitable candidate gene for use in marker - assisted selection for enhancing disease resistance in dairy animals .
the tlr-4 gene coding region is 2526  bp long consisting of 3 exons and is located on chromosome bta 8 .
bovine tlr4 has three exons , exon 1 includes coding base pairs 195 , exon 2 consists of base pairs 96260 , and exon 3 comprises base pairs 2612526 .
the whole genomic length is estimated to be approximately 11  kb , of which the first intron comprises about 5  kb and the second is 3  kb .
polymorphic studies and nucleotide sequencing of tlr-4 gene have been reported in cattle [ 4 , 5 ] . with the exception of the thesis by sonawane
, no such information is available in murrah buffalo . considering the importance of murrah buffalo in milk production
, the present study was undertaken to partially sequence the buffalo tlr-4 gene and to detect snp .
the animals included in the present study were from the herd of murrah buffaloes maintained at cattle yard of national dairy research institute , karnal , haryana , india .
ten  ml of blood was collected aseptically by jugular vein puncture in a sterile vacutainer tube containing 15% of 0.12  ml edta solution ( becton - dickinson vacutainer ) .
the samples were transported to the laboratory in an icebox and stored at 4c till further processing for dna isolation .
the blood samples were centrifuged and dna was isolated from the buffy coat alone using phenol - chloroform method , as described by sambrook et al .   with few modifications .
quality of dna was checked by electrophoresis by loading 2  l dna on 0.8% agarose in horizontal minielectrophoresis unit using 1xtbe as running buffer at 3040 volts for about one and a half hours .
after electrophoresis , the gel was stained with ethidium bromide solution ( 0.5  g / ml ) .
dna ( 2  l ) was dissolved in 98  l of double - distilled water and loaded into a 100  l cuvette . optical density ( od ) was determined at wavelengths 260  nm and 280  nm in a uv - vis spectrophotometer against distilled water as blank sample .
the sample possessing a ratio of less than 1.7 and more than 2.0 was subjected to proteinase k digestion and dna extracted with phenol chloroform isoamyl alcohol as described previously .
the primer pairs for exons 1 and 2 of tlr-4 gene were designed by using the primer 3 plus software , and primers 3 and 4 which are part of exon 3 were used as described by sonawane .
primers for tlr 4 gene are as follows : forward 5-catgctgatgatgatggcgcgtg-3 and reverse 5-cgtacgatcactgtacgcaagg-3 for exon 1 , forward 5-ttgttcctaacattagttacc-3 and reverse 5-ctggataaatccagcacttgcag-3 for exon 2 , forward 5-ggctggttttgggagaattt-3 and reverse 5-tgtgagaacagcaacccttg-3 for exon 3.1 , and forward 5-ccagagccgatggtgtatct-3 and reverse 5-cactgaatcaccgggcttt-3 for exon 3.2 . for amplification ,
25  l of pcr reaction was prepared by adding each primer , dntps , mgcl2 , 10 pcr assay buffer , dna template , and taq dna polymerase .
the amplification was carried out using a preprogrammed thermal cycler ( eppendrof mastercycler ) with the following conditions : initial denaturation of 2  min at 95c followed by 35 cycles of denaturation at 94c , annealing at 55c for primers 1 and 2 , 54c for primers 3 and 4 for 30 sec , extension at 72c each of 1  min 30 sec and lastly the final extension of 7  min at 72c .
after pcr amplification , 5  l the pcr product was checked on a 1.5% agarose gel to verify the amplification of target region .
the amplified pcr fragments , namely , exon 1 , exon 2 , exon 3.2 , and exon 3.2 of tlr 4 gene were digested with dra i ( 5ttt / aaa3 ) , hae iii ( 5gg / cc3 ) , hind iii ( 5a / agctt3 ) , and hinf i ( 5g / antc3 ) restriction enzymes , respectively . the reaction mixture ( 20  l ) for each enzyme was kept for incubated at 37c for 4 hours .
restriction fragments were resolved on 2 - 3% agarose gel horizontal electrophoresis and visualized by ethidium bromide staining .
the ethidium bromide was added to the agarose gel of 1  l/100  ml of gel .
the agarose gel electrophoresis was performed in 1x tbe buffer at 100 volts for 30 , 60 , and 90 minutes till complete separation and visualization of all fragments of re - digested gene fragments , dna ladder and pcr marker .
the restriction - digested gene fragments were visualized on uv transilluminator and photographed with gel documentation system .
amplified pcr products were subjected to custom dna sequencing from both ends ( 5 and 3 ends ) .
representative samples from each of the variants obtained by rflp analysis were also custom sequenced ( chromous biotech pvt . ltd . ,
sequence data were edited using the editseq program , and multiple sequence alignments were performed with megalign program of lasergene software , respectively ( dnastar , inc , madison wi , usa ) .
the forward and reverse sequences for each pcr fragment were assembled to form contigs of the respective region .
the tlr-4 gene sequence of murrah was compared with that of bubalus bubalis ( eu386358 ) sequence to annotate different exonic regions putatively to identify snps in respective region .
the partial coding dna sequence of bubaline tlr-4 gene ( exons 1 , 2 , and 3 ) was conceptually translated and compared with that of the bubalus bubalis to detect amino acid changes in buffalo tlr-4 regions included in present study .
the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search ( blast ) at ncbi database to determine the sequence homology with the corresponding regions of other species .
the amplified pcr product was checked on 1.5% agarose to verify the amplification of target region .
the amplified sizes were estimated as 165  bp for exon 1 , 300  bp for exon 2 , 478  bp for exon 3.1 , and 409  bp for exon 3.2 .
polymerase chain reaction - restriction length polymorphism ( pcr - rflp ) analysis of each pcr product was carried out using dra i , hae iii , hind iii , and hinf i restriction enzymes for all 102 animals included in this study system .
restriction digestion of amplicon of exon 1 revealed two fragments of 110 and 55  bp exhibiting monomorphic ( bb ) pattern in all the animals under study .
however , exon 2 of tlr4 gene did not have any cutting site with dra i. restriction digestion of exon 3.1 resulted in resolution of 5 fragments , identified as aa ( 478 , 350 , 272 , 169  bp ) , ab ( 478 , 350 , 272 , 169 , 74  bp ) , and bb ( 272 , 169 , 74  bp ) genotypes .
tlr4-exon 3.2 exhibited aa ( 409  bp ) ab ( 409 , 246,163  bp ) and bb ( 246,163  bp ) genotypes with this restriction enzyme .
pcr - rflp of exon 1 with hae iii re yielded two genotypes ab ( 165 , 122 , and 43  bp ) and bb ( 122 and 43  bp ) .
exons 2 and 3.1 of tlr4 gene did not have any cutting site with hae iii re .
exon 3.2 exhibited aa ( 409 and 309  bp ) , ab ( 309 , 200 , 142 , and 100  bp ) , and bb ( 200 , 142 , and 100  bp ) genotypes .
pcr - rflp analysis of tlr4 gene using hind iii restriction enzyme did not reveal any cutting site .
pcr - rflp analysis of exon 1 of tlr4 gene using hinf i restriction enzyme yielded two fragments of 110  bp and 55  bp size .
exon 2 of tlr4 gene did not have any cutting site with hinf i re .
the only genotype exhibited by exon 3.1 of tlr-4 was bb with 291 and 187  bp restriction fragment size . for exon 3.2 , genotypes with restriction fragment
were identified as aa ( 409 , 308 , and 292  bp ) , ab ( 308 , 292 , 200 , 136 , and 100  bp ) , and bb ( 200 , 136 , and 100  bp ) .
the present findings of murrah buffalo could not be compared with other studies , as no such report on buffalo is available in the literature . in a recent study by sonawane   in the same buffalo herd , three genotypes aa , ab , and bb with variable frequencies using alu i , bsp 1286 i , and bshkai restriction enzymes were reported .
however , exon 2 in that study was also observed as highly conserved part of the gene .
hence , no cutting site was observed using 7 enzymes ( 3 res by sonawane , and 4 in the present study ) .
sharma et al .   reported cc , cg and gg , genotypes in the promoter region ( p 226 ) of holstein cattle .
wang et al .   reported moderate occurrence of polymorphism with alui in chinese simmental , holstein , and sanhe cattle .
nucleotide sequencing of amplified fragments of tlr-4 gene of buffalo was performed ( figures 1 , 2 , 3 , 4 , and 5 ) .
the coding dna sequence of bubaline tlr4 gene compared with that of this sequence was compared to the reported sequence of bubalus bubalis with ncbi accession number eu386358 .
the sequence obtained for murrah was compared and aligned custom sequenced using the megalian program of dnastar software . amplified regions of the 4 contig regions
clustal w multiple alignments with bubalus bubalis sequence revealed a total of 12  bp changes , one in exon 1 and 11 in exon 3 .
multiple alignment revealed a total of 12 mutations : 1 in exon1 and 11 in exon 3 .
out of these 12 mutations , six were nonsynonymous resulting in change in threonine to methionine , valine to arginine , tyrosine to serine , glutamine to histidine , and aspartic acid to glycine ( at two positions ) ( figure 6 ) .
sequence analysis revealed 12 snps in the coding ( exonic ) region of tlr-4 gene given in table 1 .
the coding dna sequences of murrah tlr-4 gene ( exon 1 , 2 , and 3 ) were conceptually translated and compared with those of bubalus bubalis reported sequences ( ncbi accession number eu386358 ) . at position 75nt of exon 1 , only one snp ( t to c )
however , a total of 11 snps have been identified in exon 3 at nucleotide positions 311 , 315 , 316 , 318 , 386 , 401 , 411 , 551 , 555 , 636 , and 994 .
only five of these nucleotide changes result into changes in amino acids leading to nonsynonymous snps . in the only report available till date ,
sonawane   has reported a total of six snps , out of which 4 are nonsynonymous , two each in exons 1 and 3 .
however , in holstein cattle , sharma et al .   have reported 3 snps : 1 in promoter region ( p-226 ) and 2 in exon 3 ( 1656 and 2021 ) .
wang et al . , 2007 have reported 31 snps scattered through the 5 flanking region to exon 3 .
the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search tool ( blast ) at ncbi database to know the sequence homology with the corresponding regions of other species .
it revealed 97% , 97% , 99% , 98% , and 80% homology with bos indicus , bos taurus , ovis aries , capra hircus , and homo sapiens respectively .
sequence alignment and homology across species using basic local alignment search tool ( blast ) analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively .
in conclusion , nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo revealed twelve snps : 1 in exon1 and 11 in exon 3 .
murrah is an indigenous buffalo breed , and the presence of the nonsynonymous snp is indicative of its unique genomic architecture .
sequence alignment and homology across species using basic local alignment search tool ( blast ) analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively .
 toll - like receptor-4 ( tlr-4 ) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections . 
 the present investigation was carried out to study nucleotide sequencing and snp detection by pcr - rflp analysis of the tlr-4 gene in murrah buffalo . 
 genomic dna was isolated from 102 lactating murrah buffalo from ndri herd . 
 the amplified pcr fragments of tlr-4 comprised of exon 1 , exon 2 , exon 3.1 , and exon 3.2 were examined to rflp . 
 pcr products were obtained with sizes of 165 , 300 , 478 , and 409  bp . 
 tlr-4 gene of investigated murrah buffaloes was highly polymorphic with aa , ab , and bb genotypes as revealed by pcr - rflp analysis using dra i , hae iii , and hinf i res . 
 nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo was done . 
 twelve snps were identified . 
 six snps were nonsynonymous resulting in change in amino acids . 
 murrah is an indigenous buffalo breed and the presence of the nonsynonymous snp is indicative of its unique genomic architecture . 
 sequence alignment and homology across species using blast analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively . 
1. Introduction
2. Materials and Methods
3. Custom DNA Sequencing
4. Results and Discussions
5. Analysis of Sequencing Data
6. SNP Identification
7. Sequence Alignment and Homology Across Species
8. Conclusion