title: Molecular characterization and combined genotype association study of bovine cluster of differentiation 14 gene with clinical mastitis in crossbred dairy cattle.
abstract: OBJECTIVE
  The present study was undertaken with the objectives to characterize and to analyze combined genotypes of cluster of differentiation 14 (CD14) gene to explore its association with clinical mastitis in Karan Fries (KF) cows maintained in the National Dairy Research Institute herd, Karnal.
  
  
  METHODS
  Genomic DNA was extracted using blood of randomly selected 94 KF lactating cattle by phenol-chloroform method. After checking its quality and quantity, polymerase chain reaction (PCR) was carried out using six sets of reported gene-specific primers to amplify complete KF CD14 gene. The forward and reverse sequences for each PCR fragments were assembled to form complete sequence for the respective region of KF CD14 gene. The multiple sequence alignments of the edited sequence with the corresponding reference with reported Bos taurus sequence (EU148610.1) were performed with ClustalW software to identify single nucleotide polymorphisms (SNPs). Basic Local Alignment Search Tool analysis was performed to compare the sequence identity of KF CD14 gene with other species. The restriction fragment length polymorphism (RFLP) analysis was carried out in all KF cows using Helicobacter pylori 188I (Hpy188I) (contig 2) and Haemophilus influenzae I (HinfI) (contig 4) restriction enzyme (RE). Cows were assigned genotypes obtained by PCR-RFLP analysis, and association study was done using Chi-square (χ (2)) test. The genotypes of both contigs (loci) number 2 and 4 were combined with respect to each animal to construct combined genotype patterns.
  
  
  RESULTS
  Two types of sequences of KF were obtained: One with 2630 bp having one insertion at 616 nucleotide (nt) position and one deletion at 1117 nt position, and the another sequence was of 2629 bp having only one deletion at 615 nt position. ClustalW, multiple alignments of KF CD14 gene sequence with B. taurus cattle sequence (EU148610.1), revealed 24 nt changes (SNPs). Cows were also screened using PCR-RFLP with Hpy188I (contig 2) and HinfI (contig 4) RE, which revealed three genotypes each that differed significantly regarding mastitis incidence. The maximum possible combination of these two loci shown nine combined genotype patterns and it was observed only eight combined genotypes out of nine: AACC, AACD, AADD, ABCD, ABDD, BBCC, BBCD, and BBDD. The combined genotype ABCC was not observed in the studied population of KF cows. Out of 94 animals, AACD combined genotype animals (10.63%) were found to be not affected with mastitis, and ABDD combined genotyped animals was observed having the highest mastitis incidence of 15.96%.
  
  
  CONCLUSIONS
  AACD typed cows were found to be least susceptible to mastitis incidence as compared to other combined genotypes.
authors.affiliation: Molecular Genetics Laboratory, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: A Sakthivel
authors.initials: AS
authors.lastname: Selvan
authors.affiliation: Molecular Genetics Laboratory, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: I D
authors.initials: ID
authors.lastname: Gupta
authors.affiliation: Molecular Genetics Laboratory, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: A
authors.initials: A
authors.lastname: Verma
authors.affiliation: Molecular Genetics Laboratory, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: M V
authors.initials: MV
authors.lastname: Chaudhari
authors.affiliation: Molecular Genetics Laboratory, Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, Haryana, India.
authors.forename: A
authors.initials: A
authors.lastname: Magotra
keywords: Haemophilus influenzae I; Helicobacter pylori 188I; cluster of differentiation 14; combined genotypes; mastitis; single nucleotide polymorphisms