Open Access
Research
(Published
online: 01-07-2016)
1.
Molecular characterization and combined
genotype association study of bovine cluster of differentiation
14 gene with clinical mastitis in crossbred dairy cattle -
A. Sakthivel Selvan, I. D. Gupta, A. Verma, M. V. Chaudhari and
A. Magotra
Veterinary World, 9(7): 680-684
doi:
10.14202/vetworld.2016.680-684
A. Sakthivel Selvan:
Molecular Genetics Laboratory, Dairy Cattle Breeding Division,
National Dairy Research Institute, Karnal, Haryana, India;
drasakthivel1987@gmail.com
I. D. Gupta:
Molecular Genetics Laboratory, Dairy Cattle Breeding Division,
National Dairy Research Institute, Karnal, Haryana, India;
idgupta1959@gmail.com
A. Verma:
Molecular Genetics Laboratory, Dairy Cattle Breeding Division,
National Dairy Research Institute, Karnal, Haryana, India;
archana.ndri@gmail.com
M. V. Chaudhari:
Molecular Genetics Laboratory, Dairy Cattle Breeding Division,
National Dairy Research Institute, Karnal, Haryana, India;
mvet99@gmail.com
A. Magotra:
Molecular Genetics Laboratory, Dairy Cattle Breeding Division,
National Dairy Research Institute, Karnal, Haryana, India;
ankitoms@gmail.com
Received: 18-02-2016, Accepted: 25-05-2016, Published online:
01-07-2016
Corresponding author:
I. D. Gupta, e-mail: idgupta1959@gmail.com
Citation:
Selvan AS, Gupta ID, Verma A, Chaudhari MV, Magotra A (2016)
Molecular characterization and combined genotype association
study of bovine cluster of differentiation 14 gene with clinical
mastitis in crossbred dairy cattle, Veterinary World, 9(7):
680-684.
Abstract
Aim:
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.
Materials and Methods:
Genomic DNA was extracted using blood of randomly selected 94 KF
lactating cattle by phenolchloroform 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 PCRRFLP analysis, and association study was done
using Chi-square (χ2)
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%.
Conclusion:
AACD typed cows were found to be least susceptible to mastitis
incidence as compared to other combined genotypes.
Keywords:
cluster of differentiation 14, combined genotypes,
Helicobacter pylori 188I,
Haemophilus influenzae I,
mastitis, single nucleotide polymorphisms.
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