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ThesisItem Open Access MOLECULAR STUDIES ON TAPASIN GENE IN CHICKEN, DUCK AND GUINEA FOWL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) VARUNA P. PANICKER; Uma RThe present study was undertaken to investigate the genetic make up of exon 5 ā exon 6 region of avian tapasin gene in White Leghorn, White Plymouth Rock, Rhode Island Red, Kadaknath and Naked neck breeds of domestic fowl, Kuttanad and White Pekin breeds of duck and guinea fowl. The genomic DNA extracted from the blood of each breed was amplified using primers specific to exon 5 ā exon 6 of chicken tapasin. All the amplified products were of 400 -600 bp size. The products were extracted from gel and were cloned in PTZ57R/T vector. The cloned products were then sequenced by automated method. The tapasin exon 5 ā exon 6 sequence data of the various breeds studied varied both between species and between breeds in their length. The size of amplicon was 582 bp for all the chicken breeds except Naked neck which was found to be of 552 bp size. The PCR product size of the duck breeds Kuttanad and White Pekin were 440 bp and 445 bp respectively; and that of guinea fowl was 582 bp. All the obtained nucleotide sequences were then analyzed by comparing with tapasin exon 5 ā exon 6 sequence of chicken obtained from the database GenBank. The results showed that the GenBank sequence had maximum homology with the chicken breeds studied and, followed by guinea fowl and the least percent similarity was with the duck breeds. Among the exotic chicken breeds White Leghorn was 100 % identical to the database sequence where as White Plymouth Rock exhibited 99.1 % and Rhode Island Red showed 98.8 % homology to the published sequence. The native breeds Kadaknath and Naked neck were found to be 99 % and 98.2 % homologous respectively, to GenBank sequence. RE profile analysis of the sequence data showed that guinea fowl and all the chicken breeds were having almost similar restriction pattern for Sma I and Pvu II. Some chicken breeds had cut sites for Alu I. Duck breeds separated themselves out in that their DNA could be cut only by Alu I. Comparison of deduced amino acid sequences revealed variations between the different species studied. White Leghorn had the same amino acid sequences as those of AJ004999. Rhode Island Red varied from the GenBank sequence at three positions. All the other fowl breeds possessed four amino acid replacements and guinea fowl exhibited changes at nine positions when compared to the data base sequence. In the case of duck breeds, amino acid sequences obtained varied much from other species as well as in between the breeds due to variation in their nucleotide sequences. Phylogenetic analysis of the nucleotide sequences and the deduced amino acid sequences of different species and breeds revealed clustering of different chicken breeds along with guinea fowl as one main branch. Duck breeds clustered to a separate branch of the phylogenetic tree. The secondary structure of tapasin exon5 - exon6 region of various chicken breeds, duck and guinea fowl when predicted using the PAPIA system made it clear that the structures were similar in chicken breeds and guinea fowl reflecting their high degree of amino acid sequence identity while White Pekin and Kuttanad duck breeds showed variations in structure as expected owing to their amino acid sequence dissimilarities with respect to the other two species.