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Association of Different Gene Variants with Milk Production Traits and Their Uses as a Genetic Marker in Dairy Cattle.

M. M. R. Mufti
Animal Production Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. S. A. Bhyuian
Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh-2202.

K. S. Huque
Animal Production Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Abstract/ Executive Summary:

Breeding program relying on traditional techniques and selection criteria typically requires the investment of at least 4 years for cows and 8 years for bull or well pedigree recording systems. It would, therefore, be advantageous if additional methods or criteria or techniques that allow selection of superior bull, heifer or cow before showing their performances. Therefore, the research was taken with the following objectives: to identify the candidate genes related to milk production and their uses as marker for selection of dairy cattle. Sixty blood and milk samples were collected from RCC and BCB-1 at same management condition from BLRI farm and RCC habitat on the basis of body condition score. Milk sample were analyzed using Lactostar machine. Milk data (overall yields of milk, milk fat and milk protein, percent of milk fat, percent of milk protein and combined milk fat and milk protein percent) were recorded with their individual performances (quality and quantity). DNA was extracted from blood samples using DNA extraction kits (Invitrogen). Primer were selected from two sources, namely; previous researchers used in Bos indicus and Bos Taurus and primer designed from complete or partial cds (coding sequence) of selected genes collected from NCBI (National Center for Biotechnology Information) using Primer 3 Input (version 0.4.0) software package. Twelve primer pairs were selected from ABCG2, DGATI, OGAI, Prolactin and Somatotropin genes. Among the 12 primers, 10 primer were tested and 3 primers successfully amplified. PCR was carried out at 95? for 5 min., 35 cycles 30 s at 94?, 40 s at variable annealing temperature, 40 s at 72?; and a final 10 min extension at 72?. PCR products were electrophoresed at 2% agarose gel and the following picture represents the research findings of selected primer. The primers tested shown their band at ranges between 600-120 bp. Positive primers will be used for all samples and subsequently, RFLP technique will be followed for the endonuclease digestion at 37? for at variable incubation times. Digestion products will be separated electrophoretically in 4% w/v agarose gel. Frequencies of distribution of alleles within the herds would be compared using the Chi-square test. Database on milk production traits is completed and finally, statistical calculations would be performed using SAS producers. The effect of different gene genotypes on the milk production traits of cows would be analyzed using the general linear model (GLM) procedure in SAS (SAS Institute, V 6.4, 1986).

Keywords: Gene, Milk production traits, Genetic marker, Cattle, Milk.

Location: BLRI research farm and RCC habitat in Chittagong

Start Date: 01-07- 2009

End Date: 30-06-2010

Program Area: Variety and Species

Commodity: Cattle

Objectives:

1. To identify the candidate genes linked with milk production.

2. To sequence the specific gene involved in milk production.

3. Use of candidate genes as a marker for selection of dairy cattle.

Methodology:

Eighty blood and milk samples were collected from RCC and BCB-1 from BLRI research farm and RCC habitat in Chittagong. A. Collection of blood: Blood samples were collected by 10 ml stringe and kept in EDTA containing collection vial. The vials were gently tilting for mixing the blood with anticoagulant. The vial was marked with the date of collection, sex and genotype. During blood collection the vial was put into an ice box for preservation of blood until it is processed (not more than 5 hours). The vial was then stored at -20? temperature for long time preservation. B. Extraction of DNA: DNA was extracted using Easy-DNATM kit ( Invitrogen, USA) and the whole extraction protocol given as follows: (1) Pipette 350 µl of blood into each microcentrifuge tube. Blood samples mix to form a homogenous solution. (2) Add 500 µl of solution A to the tube. Mix by inversion several times. (3) Incubate at 65? for 6 minutes. (4) Remove sample from heat block or water bath and mix by inversion. (5) Add 900 µl of chloroform and vertex vigorously. The mixing is complete when the liquid portion of the sample flows freely and the hemoglobin looks like small chocolate-covered particles. (6) Add 500 µl of solution B and vertex briefly until the sample is uniformly viscous. (7) Centrifuge at 14,000 rpm in a microcentrifuge for 10 minutes at room temperature. (8) Pipette the clear, aqueous phase into a new 1.5 ml microcentrifuge tube. (9) Add 1 ml of room temperature 100% ethanol and mix by inversion until a precipitate forms. A precipitate is usually seen within 30-60 seconds. (10) Centrifuge at 14,000 rpm in a microcentrifuge for 5 minutes at room temperature. (11) Decant the supernatant and add 1 ml of room temperature 70% ethanol. (12) Centrifuge at 14,000 rpm in a microcentrifuge for 2 minutes at room temperature. (13) Decant the supernatant. Centrifuge at 14,000 rpm in a microcentrifuge for 1 minute at room temperature. (14) Remove residual ethanol with a pipette and invert tubes to dry. (15) Add 100-150 µl of autoclaved nuclease-free water to each tube. (16) Incubate at 65? for 5 minutes. C. Milk sample analysis: Milk sample were collected two times daily and five replications were followed for each cow. The samples were analyzed to quantify fat %, protein%, and lactose % by Lactostar machine and average value was considered for each individual. The lactation milk year, daily average milk yield of cows was collected from record book. D. Gene and primer selection: Milk production of cow controlled by several genes, but scientist identified few genes have significant effect on it. The genes that considered for this study was as below: (1) ABCG2: ATP binding cassette superfamily G member 2 transporter. (2) DGATI: Diacylglycerol O-acyltransferase 1. (3) OLRI: Oxidised low density lipoprotein receptor 1. (4) Prolactin (5) Somatotropin. E. DNA amplification and agarose gel electrophoretically: DNA amplification was performed by thermal cycler (Mastercycle Gradient, Eppendrof). E. PCR reactions: 10µl volume containing 200 µl dNTPs, IX buffer, 1.5mM Mgc12 4ng DNA template (40ng/ µl), IU Taq polymerase. F. PCR conditions: 94?-5 min, 94?-30s, 58-61?-40s, 72?-40s, 35 cycles, 72?-10 min. PCR products each sample were confirmed by running 2% agarose gel containing 5µl ethidium brodmide in IX TAE buffer at 80V, 300mA and 300W for 1.30 hrs. Loading dye (2.5 µl) was added to the PCR products and loaded in the wells. 100bp molecular weight marker was also loaded on either side of the gel. F. RELP: PCR products will be digested with the Restriction enzyme and digested products will be separated electrophoretically in 4% w/v agarose gel. G. Data analysis: (1) Frequencies of distribution of alleles within the herds would be compared. (2) Relationship between gene and milk traits will be determined using SAS procedures.

Source of Information: BLRI. Proceedings of the Annual Research Review Workshop 2009-2010, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The project was launched for two years and therefore, the results not come out this year. Next year, positive primers will be used for all samples and subsequently, RFLP technique will be followed for the endonuclease digestion at 37? for 3-5 hours. Digested products will be separated electrophoretically in 4% w/v agarose gel. Frequencies of distribution of alleles within the herds would be compared using the Chi-square test. Database on milk production traits is ongoing and a directory will form to compare with specific gene. Finally, statistical calculations would be performed using SAS producers. The effect of different gene genotypes on the milk production traits of cows would be analyzed using the general linear model (GLM) procedure in SAS.

Knowledge Management: Report/Proceedings