A dinucleotide substitution in exon-8 of DGAT1 has been identified to be responsible for variation in milk parameters in many cattle breeds of the world. Therefore, the present study was conducted in Sahiwal cows to investigate the genetic variation in DGAT1 gene through RFLP marker and to observe their association with milk performance parameters. Genomic DNA was extracted whole blood of 70 unrelated milking Sahiwal cows through spin column method. The 411bp fragment of DGAT1 was amplified and digested overnight with Eae I restriction enzyme to explore the genetic variability in DGAT1 gene. The lactation records of selected Sahiwal cows were tested for significance of association with genetic marker information. All the analyzed samples showed only ‘KK’ genotype and the gene and genotype frequency was observed fixed. The study concluded that exon 8 of DGAT1 gene was monomorphic in Sahiwal cattle and no association could be established in Sahiwal cows between DGAT1 gene and milk parameters.
Increased milk production has emerged as one of the main dairy breeding goals (Meredith et al., 2012) throughout the world. The genetic improvement in milk performance traits of native breeds could increase their potential values and improve production levels. The effective genetic improvement requires genetic information about the genetic variability and their effects on milk production. The potential value of indigenous livestock breeds for milk production traits must be analyzed and conserved to maximize the milk production so that they can become a self-sustainable resource. Livestock selection for improved production of milk has also influenced the evolution of animal breeds (Beja-Pereira et al., 2003) on global basis. Sahiwal is one of the most renowned milch breeds (Tolenkhomba and Yadav, 2012) of India which is famous for higher milk production. Many synthetic breeds throughout the world have been developed from animals of Sahiwal breed (Rehman et al., 2008). The candidate gene, Diacylglycerol aminotransferase (DGAT1) located on the centromeric end of the bovine chromosome 14 in the QTL region (Farnir et al., 2002) is considered to be directly responsible for 50% variation in milk fat content and milk yield in dairy cattle (Banos et al., 2008) and has strong functional and positional role in milk traits (Furbass et al., 2006). The knock-out trial for DGAT gene has shown reduced or inhibited milk secretion in mouse lines (Smith et al., 2000). A di-nucleotide polymorphism (GC/AA) in exon eight of DGAT1 gene harbours a non-conservative lysine to alanine substitution (K232A) with profound effect on milk fat and milk yield (Winter et al., 2002) in different bovine breeds (Sorensen et al., 2006). The polymorphic status of DGAT1 gene and their association with milk yield and milk fat has been amply reported in exotic cattle, however, there have been scant studies in Indian cattle.
The identification of specific pattern of allele and genotypic frequencies in indigenous cattle breeds and their association with lactation performance may result in detection of causal factors responsible for variation in performance for milk production. Restriction fragment length polymorphisms (RFLP’s) is one of the most abundant type ideal polymorphic genetic markers for the detection of genetic variation in polygenic traits (Jalving et al., 2004) and detects single nucleotide polymorphisms (SNPs) located at restriction site, easy to conduct and less cumbersome. Therefore, the present investigation was undertaken to with an objective to estimate the gene and genotypic frequency of DGAT1 gene in Sahiwal cattle and their association with milk performance traits.
Materials and Methods
Milking Sahiwal cows (N=75) with minimum of 120 days lactation and completed at least one parity were selected randomly from Livestock Research Station, Kodamdesar, Bikaner (Rajasthan). The biological material such as blood for analysis was collected from the same animals included in the study in accordance with the standard ethical procedures. The phenotypic information on different lactation parameters such as lactation yield, (LL), 305-day milk yield (305-DMY), daily milk yield (ADMY) and lactation length were collected from farm records and were standardized to reduce systematic errors and to make the data set uniform. Information on outliers animals and aberrant lactation was excluded from the present study. Animals with history of mastitis or dystokia were also excluded for the collection of biological material.
About 2ml of blood was taken from jugular vein in a sterile vacutainer tube containing Ethylenediaminetetraacetic acid (EDTA) as an anticoagulant under strict aseptic conditions from all the selected animals of Sahiwal cattle. Genomic DNA from the whole blood sample was extracted through spin column method as per standard method (Sambrook and Russell, 2001) under manufacturer’s protocols with slight modification. The quality and quantity of extracted genomic DNA was assessed through Nano-drop spectrophotometer and 0.8% agarose electrophoresis. The well-established dinucleotide AA/GC substitution in exon 8 of DGAT1 gene was selected as genetic marker for Sahiwal population study. Species specific primers were used to amplify the selected genomic regions of exon-8 of DGAT1 gene on the basis of available sequences of DGATI gene in NCBI GenBank database. The sequences of primers, the accession number of reference sequence and expected fragment length of different selected region are represented in Table 1.
Table 1: Primer sequences and expected fragment size of PCR products of DGAT1 gene
|Selected Region||Primer Sequences||GenBank Accession No.||Expected Fragment Length||References|
|DGAT1 exon-8 region||Forward||AJ318490||411-bp||Winter et al. (2002)|
The selected 411bp region of the cattle DGAT1 gene was amplified by PCR in a reaction mixture containing 5X PCR buffer (5μl), 1.5mM MgCl2 (3μl), 10 Mm dNTP’s mix (1μl), forward primer 70pmol/μl (1μl), reverse primer 70 pmol/μl (1μl), genomic DNA 25 ng/μl (4μl), Taq DNA polymerase 5U/μl (0.2μl) and DNAase free water (10.8μl). 5% dimethyl sulphoxide (DMSO) was added to the amplification mixture for equal amplification of both alleles as suggested by Winter et al. (2002). The PCR programme used for the amplification of DGAT1 gene is presented in Table 2.
Table 2: PCR programming for amplification of exon-8 of DGAT1 gene
|Steps||Temperature||Time||No. of Cycle|
|I. Initial Denaturation||95oC||5 min.||1 cycle|
|(i) Denaturation||95oC||1 min.|
|(ii) Annealing||52oC||30 sec.||40 cycles|
|(iii) Synthesis||72oC||1 min.|
|III. Final extension||72oC||10 min.||1 cycle|
|IV. Hold||4oC||5 min||1 cycle|
The quality and size of the PCR amplicons for studied locus was assessed on 1.5% agarose gel containing ethidium bromide (1% solution) (Fig. 1). The 411-bp amplicons for the respective DGAT1 gene were digested with 5U of Eae I (1μl) restriction enzyme in a 40μl reaction mixture containing 10X buffer (5μl) and nuclease free water (35μl). The reaction mixture was mixed properly through spinning and kept under water bath at 37ºC for overnight digestion. The different DGAT1 genotypes were analyzed as per method of Winter et al. (2002) on 8% polyacrylamide gel. The results of electrophoretic separation were visualized and documented through GelDOC Unit after staining with ethidium bromide dye. The lactation records of selected Sahiwal cows were tested for significance of association with genetic marker information using least square method of SPSS ver. 20.0 for Windows (SPSS Inc., Chicago, IL, USA).
Fig.1: PCR amplicons of exon-8 of DGAT1 gene (Lane 1- 7: PCR Amplicons of 411 bp, MW: Molecular weight marker)
Statistical analysis was carried out under single gene model to estimate the effect of genotype on the traits. The following univariate analysis of variance was carried out through general linear model (GLM) procedure to analyse the differences among genotypes:
Xij = μ + gi + eij
Xij = mean observed value of milk performance parameter; μ= general mean
gi= fixed effect of the ith genotype ; eij= random error, eij ~NID (0, σe2)
Least squares means for each lactation traits and their corresponding standard errors were also computed.
Results and Discussion
The genetic variation in exon-8 region of DGAT1 gene in Sahiwal cattle (Bos indicus) was reported in the present study. The RFLP analysis enables interpretation of genetic information on wider basis for the above mentioned QTL on BTA14 for GC/AA dinucleotide base pair substitution in DGAT1 gene leading to non-conservative lysine to alanine amino acid substitution (K232A). The in vitro amplification of genomic DNA from all the samples revealed amplification band of 411bp of DGAT1 exon-8 coding region using species specific oligonucleotide primers. The restriction digestion of PCR amplified products of DGAT1 gene through Eae I restriction enzyme revealed the revealed the presence of only one undigested intact band of 411bp, representative of ‘KK’ genotype in all the seventy five animals studied. None of the animal included in the study revealed polymorphism in the DGAT1 gene of Sahiwal cattle. Eae I restriction enzyme digests the ‘A’ allele but not ‘K’ allele. The digestion of the ‘A’ allele produces two fragments of 208 and 203 bp. The present study provides evidence that mutation is not present at Eae I restricted site in DGAT1 exon 8 loci in Sahiwal cattle. The present study is in agreement with the findings of Kaupe et al. (2004) in Nellore cattle, Tantia et al. (2006) in Rathi, Sahiwal, Tharparkar, Deoni, Red Kandhari and Punganur cattle and Ganguly et al. (2013) in Sahiwal cattle for DGAT1 locus. Most of the earlier studies which established the conserve nature of DGAT1 gene in indigenous cattle were conducted on limited number of animals. The DGAT1 allele ‘A’ and genotype ‘KA’ was reported to be most frequent in crossbred population (Molee et al., 2015).
The mean lactation performance of the selected milking animals for average for all parity for different lactation traits was analyzed and the breed wise results are displayed in Table 3. The average lactation yield, daily milk yield, 305 day milk yield and lactation length were observed to be 1441.80 kg, 1824.89 kg, 5.98 kg and 240 days, respectively.
Table 3: Lactation performance (LSM±SE) of studied Sahiwal animals
|Total milk yield (kg)||305 day Milk Yield (kg)||Daily milk yield (kg)||Lactation length (days)|
The nationwide higher number of graded animals in Sahiwal cattle population (77.62%) could be a contributing factor for low performance of Sahiwal animals (Anonymous, 2013). An association between genotype of DGAT1 gene and milk performance could not be established for K232A polymorphism through RFLP analysis due to absence of alanine coding allele ‘A’ in each of the studied breeds examined. The reports of Bennewitz et al. (2004) also indicated that K232A polymorphism of DGAT1 is not sole responsible for all the genetic variation in QTL located at the centromeric end of chromosome 14 for milk related traits. The fix nature of ‘K’ allele in indigenous cattle breeds of Rathi, Sahiwal and Kankrej reflects their exceptional adaptive value. The greater overall immunity of indigenous cattle against many diseases and their survival adaptation to coarse roughage with low requirement of green could be due to enhanced acyltransferase activity of DGAT1 gene which catalyzes the synthesis of retinol esters and thus regulates the synthesis of Vitamin A or retinol. The significant and favourable association of DGAT1 ‘K’ allele with low somatic cell count in lactating cattle was reported by Manga and Riha (2011) that partially explains the exceptional genetic resistance of indigenous cattle against mastitis as the triglycerides are also one of the main constituent of cell membrane in immunocompetent cells (Liu et al., 2007).
The present study concluded that native Sahiwal cattle is lacks variation in exon 8 of DGAT1 gene through RFLP assay and are selected for fat enhancing lysine allele of DGAT1 gene and variation in other region of DGAT1 gene should be explored through alternate technique to observe their impact on milk performance parameters.
Authors are thankful to Rajasthan University of Veterinary and Animal Sciences, Bikaner (Rajasthan) for providing financial support and necessary infrastructure.