Study of Polymorphism in Candidate Milk Protein Genes and Their Association with Milk Traits in Gaolao Breed of Cattle
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Date
2020-11-25
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MAFSU, Nagpur
Abstract
The present research was undertaken to study the genetic
polymorphism of candidate milk protein genes in Gaolao cattle with the
objectives to investigate the polymorphism in - casein, -casein, -
lactalbumin and -lactoglobulin genes, to detect their relationship with milk
traits and to sequence their known polymorphism. Blood samples and milk yield data was collected from total 83 unrelated lactating Gaolao cattle
belonging to Bull Mother Farm, Pohara Dist. Amravati (n=60) and from
breeding tract i.e. villages of Wardha district (n=23) during 2015-2017. The
research work was carried out in the Molecular Genetic Laboratory,
Department of Animal Genetics and Breeding, Post Graduate Institute of
Veterinary and Animal Sciences, Akola. The milk yield (kg) was estimated by
the Test Interval Method following the guidelines of International Committee
for Animal Recording. The milk composition traits viz. Fat%, Protein %,
Lactose% and SNF % estimated using milk analyzer were subjected for daily
yields with adjustment of milking interval and stage of lactation.
The polymorphism in - casein gene was detected for loci 121 bp, 251
bp, 495 bp and 357 bp; in -casein for loci 379 bp (Exon III to V), 453 bp
(Exon IV), 350 bp (Exon IV) and synthetic primer of 350 bp; in -lactalbumin
for loci 309 bp, 166 bp, 268 bp (Exon III) and 133 bp and in -lactoglobulin
genes for loci 247 bp (Exon IV), 854 bp, 262 bp (Exon IV) and 119 bp,
respectively. All the loci were subjected for PCR-RFLP method, except locus
357 bp in -casein gene, 350 bp synthetic primer in –casein gene, for locus
133 bp in -lactalbumin gene and for locus 119 bp in -lactoglobulin gene,
which were screened by PCR SSCP method following silver staining
technique.
All the studied loci exhibited polymorphism except, the locus 495 bp
(Exon VII) of ß-casein gene showed monomorphic pattern (BB genotype).
Amongst 15 polymorphic loci, 121 bp of ß-casein and 854 bp of ßlactoglobulin
gene loci showed AA and AB genotypes only. However, rest of
other loci exhibited AA, AB and BB genotypes. The higher gene frequency for
A allele was observed for loci 357 bp (0.55) of ß-casein, 379 bp (0.93) and
453 bp (0.87) of -casein, 166 bp (0.88) of -lactalbumin gene, 854 bp (0.91),
262 bp (0.73) and 119 bp (0.60) of ß-lactoglobulin gene. However, higher
gene frequency for B allele was observed for loci 121bp (0.96), 495 bp (1.0)
and 251 bp (0.87) of ß-casein, 350 bp (0.64) and 350 bp synthetic (0.67) of -
casein, 309 bp (0.72), 268 bp (0.85) and 133 bp (0.72) of -lactalbumin gene,
247 bp (0.71) of ß-lactoglobulin gene Mean values for Total Milk yield (kg), Total Fat yield (%), Total
Protein yield (%), Total Lactose yield (%) and Total SNF yield (%) were
312.47 ± 85.52, 4.14± 1.15, 3.21±0 .86, 4.99±1.35 and 9.30± 2.47,
respectively in Gaolao cattle. All the loci exhibited effect of genotype as a non
significant source of variation in Gaolao cattle, except locus i.e. 251 bp of -
casein gene which exhibited significant influence of genotype (A2A2) on all
the studied milk traits except TFY. On the other hand, the effect of genotype
could not be estimated for locus 495 bp of -casein gene, due to its
monomorphic pattern. The effect of location and season of calving was also
found non-significant with respect to milk traits under study. However, the
effect of parity was found significant with TMY, TPY and TLY for all the loci
as well as with TFY for locus 453 bp of -casein gene and with TSNFY for
loci -casein 495 bp of -casein gene and 379 bp of -casein gene
respectively.
The representative polymorphic amplicons of 11 loci of studied milk
protein genes, belonging to different genotypes detected by PCR-RFLP
method only were outsourced for DNA sequencing for confirmation of
restriction sites and thereby the genotypes observed, with the help of Mega X
and Chromas softwares. Restriction sites were confirmed for loci 121 bp and
251 bp of ß-casein gene, 379 bp, 453 bp and 350 bp of -casein gene, 309 bp
and 166 bp of -lactalbumin gene and 247 bp, 854 bp and 262 bp of ßlactoglobulin
gene. However, the cutting site and genotype could not get
confirmed for locus 268 bp of -lactalbumin gene due to poor quality of
sequencing.
Thus, it can be concluded from present study that, polymorphic nature
of studied loci can be utilized as baseline information for future genomic
selection database for Gaolao cattle. The predominance of B allele for loci 121
bp and 357 bp and A2 allele for locus 251 bp in ß-casein gene was indicating
preponderance and thereby confirming the uniqueness of Gaolao breed as Bos
indicus germplasm for studied loci. The estimates of TMY, TFY, TPY, TLY
and TSNFY when compared with other recognized cattle breeds of
Maharashtra, were supporting the classification of Gaolao as draught purpose breed. Non specific trends or genotypes observed were suggestive of
differential expression of respective allele and or variation in sample size
under different genotypes of each locus. The automated DNA sequencing
confirmed the restriction sites for studied polymorphic loci of - casein, -
casein, -lactalbumin and -lactoglobulin genes identified by PCR-RFLP
method in Gaolao cattle.