Genotyping of various buffalo breeds for prolactin locus using PCR-RFLP
Ladani, Dineshkumar (2000) Genotyping of various buffalo breeds for prolactin locus using PCR-RFLP. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Among several hormones that regulate lactation and reproduction in cattle and buffaloes, prolactin plays an important regulatory function in milk production and reproduction. The present study was under taken with preliminary objective to genotype three different Indian buffalo breeds viz. Mehsani, Surti and Jaffarabadi for prolactin locus using Polymerase Chain Reaction-Restricted Fragment Length Polymorphism technique. Two prolactin gene specific primers Primer-I forward: 5' -' CGA GTC CTT A TG AGC TTG A TT CTT - 3', reverse: 5' - GCC TTC CAG AAG TCG TTT GTT TTC - 3' and primer-II forward: 5' - ATT ATC TCT CTC ATT TCC TTT CA - 3', reverse: 5' - ACT CTG CTG TCA CTG TCT GTA TT - 3' were used to amplify 156 bp and 857 bp PCR product respectively. Genomic DNA was isolated from blood samples of 23 Jaffarabadi, 30 Surti and 44 Mehsani buffaloes and two semen samples of Jaffarabadi buffaloes by ABSTRACT "Genotyping of various buffalo breeds for prolactin locus using PCR-RFLP" Name of the student Dineshkumar D. Ladani Major Advisor Dr.P. H. Vataliya, Ph.D Senior Scientist Department of Animal Genetics & Breeding College of Veterinary Science and Animal Husbandry Gujarat Agricultural University Anand Campus, Anand phenol:chlorophorm method. The average yield of DNA from blood and semen samples was 68.57 ± 9.80 μg and 14.46 ±6.46 μg per ml of blood and semen respectively. The PCR was carried out in a final reaction volume of 25 μl and subjected to 36 cycles comprising of 94°C for 1 minute, 56°C for 1 minute and 72°C for 1 minute. Primer-I A 156- bp PCR product was digested with Rsa I enzyme to detect A (156 bp) and B (74+82 bp) alleles on 3% agarose gel electrophoresis. In Mehsani buffaloes only AB genotype were observed, while in Surti and Jaffarabadi buffaloes AB and BB genotypes were identified. Genotype AA was not identified in any of the breed studied. The frequencies of AB and BB genotypes in Surti and Jaffarabadi were 0.965, 0.035 and 0.87, 0.13 respectively. The frequencies of A allele in Mehsani, Surti and Jaffarabadi were 0.50, 0.48 and 0.43 respectively. The frequencies of A allele were observed to be lower than those reported earlier in buffaloes. Primer-II Another set of bovine prolactin primer (primer-II) amplify 857 bp segment of buffalo prolactin gene. This primer-II also produced the same band pattern in two different known bovine DNA samples of AA and AB genotype typed by Primer-I. On Rsa I digestion of PCR product of all three breeds there were similar band pattern was observed in individual as well as pooled samples of Surti and Mehsani buffaloes, while Jaffarabadi pooled samples revealed a different band pattern than other two breeds. On digestion with Hae III only Jaffarabadi pooled sample revealed restriction site in the PCR product, while in other two breeds PCR product remained uncut which indicated absence for Hae III site unlike cattle. This indicated that there might be two more additional restriction sites for Rsa I in prolactin sequence of buffalo. Also there is sequence variation on the prolactin gene between Jaffarabadi and other two buffalo breeds, which can be ascertained by sequencing the PCR products. The results of the present study has demonstrated application of PCR-RFLP in genotyping of buffalo breeds for prolactin gene by two different primer sets. This technique can be used further for prolactin genotyping of large population of buffaloes.
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