36 Present address: 2 Ph.D. Scholar (virgoalka@gmail.com), 2,3 Principal Scientist (idgupta1959@gmail.com, archana.ndri @gmail.com), Dairy Cattle Breeding Division. 4 Senior Scientist (vohravikas@gmail.com), National Bureau of Animal Genetic Resources, Karnal . Dairy industry is suffering huge losses annually due to mastitis, which is one of the major production related diseases in cows. Mastitis is the single most important cause of financial loss to the dairy industry not only to India but worldwide and is clearly a global disease. According to an estimate loss due to mastitis approach $2 billion annually in the United States alone (Halloran 2009) and about `6,053.21 crore in India (Burman 2002). Lactoferrin protein is present in all external secretions including bovine milk and secondary granules of polymorpho nuclear cells and exerts several functions related to innate immunity and host defence. Level of lactoferrin protein in milk varies enormously between species and bovine milk is the most common source of commercially produced lactoferrin, concentrations ranging between 0.02 and 0.2 mg/ml in milk. Some reports indicated that lactoferrin (Lf) concentration in milk and serum would change during the infection of mastitis (Barkema 1998, Hirvoen 1999), suggesting that there was some association between Lf and mastitis. Therefore, observation on polymorphism of Lf gene by RFLP, and association between Lf and mastitis incidence could give some novel insight into theory and practice. In the present study, polymorphism of Lf gene promoter was investigated by created restriction site PCR-RFLP, and association between Lf genotypes and mastitis incidence was studied. Experimental animals, DNA isolation and polymorphism detection: Genomic DNA was isolated from Sahiwal (200) and Karan Fries (150) cattle maintained at cattle yard of the Institute, using phenol/chloroform method (Sambrook and Russel 2001). NCBI sequence accession number AY 319306 was used to design the primer. Forward and reverse primers (PF 5’- AACCTACACATGCTGCAATGGAAG- 3’ and PR 5’-TGCTTATCGTTCACTGATTGCAGG-3’) with T M of 61°C, were designed using Primer-3 software with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzyme for Indian Journal of Animal Sciences 84 (10): 1068–1070, October 2014/Short Communication Detection of allelic variants in lactoferrin gene promoter using created restriction site PCR-RFLP and its association with mastitis ALKA CHOPRA 1 , I D GUPTA 2 , ARCHANA VERMA 3 and V VOHRA 4 National Dairy Research Institute, Karnal, Haryana 132001 India Received: 30 May 2013; Accepted: 4 June 2014 Key words: Cattle, Lactoferrin gene, Polymerase chain reaction, Mastitis, Sahiwal, Karan Fries discriminating sequence variation. Restriction fragment length polymorphim genotyping was carried out using a tetra cutter restriction enzyme Hae III enzyme for a PCR amplified fragment size of 92 bp from promoter region of Lf gene. The optimization of PCR was done to get the best possible amplification of the product, at an annealing temperature of 59°C for 1 min. The detection results of allelic variation at SNP sites were based on the electrophoretic pattern of the restriction enzyme treated PCR products. Selected PCR products of all types of genotypes were sequenced using automated dye terminator cycle sequencing method with Ampli Taq DNA polymerase in a DNA sequencer and sequence comparison was made using CLUSTAL W (free available online software). Mastitis incidence, statistical and genotype analysis: Data on clinical mastitis was collected for all the screened cows, recorded in the treatment register maintained at organized herd of NDRI for a period of 12 years (2000 to 2012). Genotype and allele frequencies were calculated and compared by gene counting method (Falconer and Mackay 1996). Association of lactoferrin variants with affected and non-affected cows were calculated using Chi Square Test SAS EG 4.5. Variations and mutations in Lf promoter may play an important role in the transcription and regulation Lf gene function. Created restriction site PCR-RFLP of bovine lactoferrin gene was conducted in Sahiwal and Karan Fries cattle. Polymorphism was observed in a 92 bp PCR amplified fragment. Three different genotypes were detected by 2.5% agarose gel with EtBr staining,viz. EE, EF and FF in Sahiwal cattle whereas the EE genotype was absent in our herd of Karan Fries cattle. In Sahiwal cattle EE EF and FF genotypes were having a frequency 2.5, 37 and 60.5% respectively. The frequency of E and F allele in Sahiwal cattle were 0.21 and 0.79 respectively. In Karan Fries cattle EF and FF genotypes were found with genotype frequency 14 and 86%, respectively. EE genotype was absent in Karan Fries cattle. The frequency of E and F allele in Karan Fries cattle were 0.07 and 0.93 respectively. Our results showed marked variations with Huang et al. (2010) and does not agree with respect to these alleles in Chinese Holstein Friesian cattle where they reported E and F alleles with