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Study on Candidate Genes for Milk Production Traits of Red Chittagong Cattle

M.Y.A. Khan
Bangladesh Livestock Research Institute

M.P. Mostari
Bangladesh Livestock Research Institute

G. K. Deb
Bangladesh Livestock Research Institute

M.A. Alim
National Institute of Biotechnology, Savar, Dhaka, Bangladesh.

Abstract/ Executive Summary:

Advance biotechnology and molecular genetics open perspectives for the improvement of milk production traits in dairy cattle through detection of chromosomal regions or loci affecting traits of economic interest. The candidate gene strategy has been proven to be extremely powerful for studying the genetic architecture of complex traits and can detect loci even with small effects, provided that the candidate gene represents a true causative gene, which is more effective and economical method for direct gene discovery. We herein investigating the polymorphism of Stearoyl-CoA desaturase (SCD) and diacylglycerol acyltransferase 1 (DGATl) genes using pooled DNA sequencing and MALDI-TOF technique in 60 Red Chittagong Cattle (RCC) cow. Focusing on the effects of SNPs on milk production traits, the present study is ongoing; it has achieved the following progresses. Phenotypic variation within RCC breed has been identified on the basis of milk production traits so far average lactation yield varies from 385 to 1452 liters (n=31 ). Average milk yield per day ranges from 2.9 to 4.8 liter (n=31) and more than 32% (n=31) of lactating cows showed average milk yield per day> 4.0 liters. Consequence of pool DNA sequencing and genotyping of SNP of SCD and DGATl genes 14 set of primer were designed where 8 set from DGATl and 6 set from SCD. Among the 14 sets of primers 6 sets of SCD and 5 sets of DGATl primers were standardized and amplified well and the rest 3 sets of DGATl primers are on the way of standardization. DNA sequencing, genotyping of SNP and association analysis with milk production traits will be done by following year.

Keywords: Milk production trait, Candidate genes, Single nucleotide polymorphism, Red Chittagong Cattle

Location: Bangladesh Livestock Research Institute

Start Date: 00-00-2013

End Date: 00-00-2014

Program Area: Variety and Species

Commodity: Cattle

Objectives:

1.) To select high yielding female calf at early stage without waiting up to its lactation stage.

2.) To identify the potential DNA marker/SNPs in DGATI and SCD genes responsible for higher milk production in RCC

3 ) To use it/them in a marker assisted selection (MAS) program.

Methodology:

RCC herd consists of about 60 cows of which 31 cows were at lactation in different lactation stage and rests of them are dry cows. Phenotypic data of milk production traits (milk yield, fat yield, protein yield, SNF yield and lactose yield) were recorded from 31 lactating cows. For this study a complete lactation record is needed for evaluation of each cow. Milk yield data was collected from daily milk yield record book without considering the calf suckling. Full lactation lengths of all cows were not covered by this year, cows with full and partial (> 100 days) lactation length were used in lactation yield calculation. This study need complete lactation record of each cow so, recording of milk production traits will be continued for following year. Fat yield, protein yield, SNF yield and lactose yield of milk samples of morning and afternoon were analyzed using LACTOST AR in every 15 days interval. Blood samples were collected from 12 lactating cows and 3 breeding bulls for DNA extraction and PCR amplification test. DNA was extracted from blood samples using a commercial kit (QIAGEN DNA Mini Kit) following manufacturer instruction. In order to identify potential SNPs, 7 sets of reference and 7 sets of designed primers of DGATI and SCD genes were used to amplify all exons (some cases pecific exons and introns) and their partial flanking intronic sequences. Primers were designed based on the reference sequence of the bovine DGATI and SCD genes (GenBank Accession No. AY241932; and ACOOOI71) taken from NCBI and using Primer3 (v.0.4.0.) (http://frodo.wi.mit.edu/) and Net Primer (www.PremierBiosoft.com) web based Program. The primers were synthesized by Invitrogen (Invitrogen Life technologies, Beijing, China). PCR amplifications were performed with a programmable thermal cycler. Total 14 sets of primers were tested for PCR amplification in 15µ1 reaction mixture which contained 0.2mM dNTPs, 2mM MgCI₂, and 0.05U Taq DNA polymerase in final concentration. The amplification condition was: 5 min at 94°C for initial denaturing followed by 30 cycles at 94°C for 30 s; annealing at Tm (ºC) for 30 s, noc for 30 s; a final extension at noc for 10 min for all primers. Amplification' was confirmed by gel electrophoresis of the PCR products in 2% agarose gel followed by visualization under UV. The aforementioned works were completed but following will be finished in subsequent year. The PCR product will be sent to overseas laboratory for sequencing by using DNA Sequencer. Sequences will be generated with both the forward and reverse primers. To verify the sequences, chromatographs will be generated from sequencing and will be processed using BioEdit Sequence Alignment Editor, version 7.0.9.0. Both forward and reverse sequences will be aligned using the ClustalW multiple sequence alignment programs to determine the presence of any genetic polymorphisms. Then the identified SNPs will be genotyped in RCC cows using Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI- TOF) assay or any other suitable method. Identified genotype of each RCC will be used in statistical analysis. The kinship matrix (A-matrix) will be calculated using MATLAB software (version 7.1l.0.584). To assess the allelic and genotypic frequencies Hardy-Weinberg equilibrium tests will be performed using POP GENE software (version 1.32) and haplotypic frequencies and linkage disequilibrium will be calculated using HAPLOVIEW software (version 4.2). Finally, the effects of DNA marker on the milk production traits will be estimated using the mixed procedure of SAS 9.l.0 software with the animal model: Y=µ+hys+L+G+α+e, (where, Y is the phenotypic value of each trait of cows; µ is the overall mean; hys is a herd-year-season effect; L is fixed effect of lactation; G is a fixed effect corresponding to the genotype of polymorphisms; a is a random polygenic component account for all known pedigree relationships; e is a random residual). The difference between the effects of the polymorphism genotypes will be compared using multiple t-test with Bonferroni correction.

Source of Information: Proceedings of the Annual Research Review Workshop 2013-2014, BLRI, Savar, Dhaka. Publication No.261, pp: 47-53, October-2015.

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It is an on-going research programme preparatory part of DNA sequencing and SNP genotyping (primer designing, PCR amplification of designed primers) have been completed. Identification of phenotypic variation of milk production traits in RCC has partially completed in 2013-14.The rest of the work has extended in the following year but due to yearly reporting this article doesn't convey a complete message of the study.

Knowledge Management: Report/Proceedings