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Molecular Characterization of Selected Local Cattle Using Random Amplified Polymorphic DNA (rapd) Markers

MM Khanam
FTI Project, GPC, BAU

MK Islam
HRC, BARI

KM Hossain
Biotechnology & Genetic Engineering Discipline, Life Science School, Khulna University

SMM Rahman
Biotechnology & Genetic Engineering Discipline, Life Science School, Khulna University

J Jasmin
Food Security Project, AED, BARI

M Ahmed
Vertebrate Pest Division, BARI. Gazipur

Abstract/ Executive Summary:

A genetic analysis using RAPD (Random Amplified Polymorphic DNA) markers was performed to determine the cattle-specific primers and generate RAPD fingerprints to find out genetic diversities of cattle breeds an important province for cattle. The DNA samples were isolated from a total of twenty animals from local cattle and three random primers were selected. Genetic relations among cattle were determined by RAPD polymorphisms. The study showed that the polymorphisms generated by RAPD-PCR enabled the determination of genetic relationships and fingerprints of local cattle. In this study, molecular characteristics were used to determine genetic relationship among these selected local cattle. The results obtained from amplified PCR products are more reliable than that of morphological characters. In this experiment the highest value of Nei’s genetic distance was found among Bag-03, Pir-85, Pir-10, Pir-01, Khu-09, and Khu-11and Khu-17 cattle. The lowest genetic distance was revealed between Bag-102 and Bag-69 cattle. A total of 14 bands were identified from PCR products which illustrated 86.66% of the polymorphism. These data provides a scientific basis for future studies on local cattle.

Keywords: Cattle breeds, Genetic diversities, Cattle-specific primers, RAPD fingerprints

Location: Animal cell culture and Molecular Laboratory in Biotechnology and Genetic Engineering Discipline, Khulna University, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

The present research work on molecular characteristics of selective local cattle using random amplified polymorphic DNA (RAPD) markers was undertaken to know the genetic diversity and relationships among local cattle.

Methodology:

The experiment was carried out at the Animal cell culture and Molecular Laboratory in Biotechnology and Genetic Engineering Discipline, Khulna University, Bangladesh. The details of materials and methods used are described as follows. Animal selection and blood sample collection - In order to minimize sampling errors two conditions were maintained while selecting local cattle: i. Related animals were not sampled, ii. Sampling of animals which represented different eco-regional populations. Bagerhat, Khulna and Pirojpur districts were considered suitable for these conditions because animals from different regions were frequently introduced and maintained in semi-intensive farming system. A total of twenty native cattle were selected for this study. Five ml blood was collected from each animal in a 10 ml EDTA vaccutainer tubeand kept in ice box during collection and then transported to the laboratory and stored at -20ºC until the isolation of genomic DNA. Extraction of genomic DNA from blood sample DNA was extracted from blood by following the modified method of Bruce (1996). The presence of DNA in the extracted samples was confirmed by running the sample in a 1 % agarose gel at 50V for 1.20 hours. The quantity of DNA was measured by spectrophotometer at 260 nm wavelength. PCR reaction, Primer selection - In this study 3 RAPD primers were selected after screening 10 primers (Bioneer). PCR amplification - The amplification conditions were based on standatrd method. PCR reactions were performed on each DNA sample in a 20 μl reaction mix containing 2 μl of template (4ng DNA), 2 μl of 10x reaction buffer with MgCl₂, 2 μl of dNTPs (250 mM each), 2 units of Taq DNA polymerase, 1 μl (100 picomol) of primer, 11 μl of sterile distilled water. DNA amplification was performed by thermal cycler (Eppendrof Master Cycler Gradient). The amplification program included an initial denaturation step of 94°C for 5 minutes (one cycle) followed by 40 cycles of 94°C (denaturation), 1 minute; 37°C (annealing), 1 minute and 72°C (extention) for 2 minutes. The final extension was performed at 72°C for 15 min (one cycle) and held at 8°C. Gel electrophoresis of PCR amplified Product - PCR products from each sample were confirmed by running 2 % agarose gel in 1x TAE buffer at 40V for 80 minutes. Loading dye (5 μl) was added to the PCR products and loaded in the wells. Two molecular weight markers DNA (ladder) were also loaded on one side of the gel. After electrophoresis, the gel was taken out carefully from the electrophoresis chamber, washed with 7 μl ethidium bromide, mixed with distilled water (500 mI) and placed on high performance ultraviolet light box (UV transilluminator) for checking the DNA bands and photographed using a digital camera. Data analysis - Although a large number of fragments were generated from each primer, only clearly distinguishable and reproducible bands were considered and data was entered in a computer file as a binary matrix “0” coded for absence and “1” for presence of a band. The scores obtained using all primers in the RAPD analysis were then combined to create a single data matrix. This was used for estimating polymorphic loci, gene diversity, gene frequency, genetic distance (COMPONENT ,Version 1.31). Dendrogram was constructed using Unweighted Pair Group Method using Arithmetic Averages (UPGMA).

Source of Information: Eco-friendly Agril. J. 8(03): 16-23, 2015 (March); ISSN 1999-7957

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The differences of genetic variations in the present study in twenty experimental cattle samples were high and therefore, there is a scope for future improvement of local cattle. More research needs to be done on the native cattle and the choice of cattle for conservation must take into account any available information on productivity traits of economic value, specific adaptive features (tolerance to heat, low quality feeds, disease etc.), presence of unique genes or phenotypes, local or regional importance of a breed in production systems, and the availability of resources and infrastructure for animal production.

Knowledge Management: Journal