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Study on Genetic Diversity of BLRI Cattle Breed -1 Using Microsattelite Markers

G. K. Deb
Senior Scientific Officer
Biotechnology Division BLRI,Savar,Dhaka-1341

M. R. Mufti
BLRI,Savar,Dhaka-1341

M. R. Rahman
Biotechnology Division BLRI,Savar,Dhaka-1341

S. R. Dey

N. R. Sarker
PSO
Animal Production Research Division BLRI,Savar,Dhaka-1341

Abstract/ Executive Summary:

Identification of intra and inter-genetic variability is essential for planning conservation strategy and genetic improvement program of livestock species. Microsatellite markers are widely used for genetic characterization of populations as they are highly polymorphic single locus DNA sequences scaterred throughout the genome and are readily adaptable to Polymerase Chain Reaction (PCR). Present study aimed to characterize BLRI Cattle Breed 1 (BCB-1) at genetic level and to find out their genetic distances with other native cattle (Red Chittagong, RCC and Progenitor cattle of BCB-1 of Pabna and Sirajgonj districts, Progenitor) of Bangladesh using three pairs of bovine microsatellite markers (ETH152, INRA32 and INRA63). Blood samples were collected randomly from fifteen animals of each group (BCB-1, RCC and Progenitor). Genomic DNA was extracted using BLRI developed DNA extraction kit. All the primer pairs were successfully amplified microsatellite loci. The PCR products were checked on 3% agarose for verification of quantity and size of amplicon. Data were analyzed using PopGen32 (Version 1.31) computer program. Results showed the highest and lowest gene frequency values (0.850 and 0.150) were observed in allele A and allele B with ETH152 in RCC and BCB-1 cattle and INRA63 in BCB-1 cattle.. All the three cattle populations showed significant deviation from Hardy-Weinberg equilibrium for at least one locus (RCC for ETH152 and INRA32, BCB-1 for ETH152 and INRA63 and Progenitor for INRA63). The estimated mean observed (Ho) and expected heterozygosity (He) were 0.167±0.134, and 0.377±0.134 in RCC vs. 0.167±0.116 and 0.339±0.122 in BCB-1 vs. 0.800± 0.200 and 0.393±0.097, respectively. However, mean heterozygosity of all three loci was same (0.351±0.111) in the studied cattle populations. All the studied microsattelite showed two polymorphic loci each. Average effective number of alleles was 1.604±0.349, 1.507±0.349 and 1.621±0.261 in RCC, BCB-1 and Progenitor cattle population respectively. The mean SI was 0.539±0.139 in RCC vs. 0.498±0.130 in BCB-1 vs. 0.558±0.100 in Progenitor cattle. The mean heterozygote deficiency (FIS) values were 0.528 in RCC vs. 0.519 in BCB-1 vs. 0.514 in Progenitor cattle. Among the three cattle populations genetic distances were low between BCB-1 and its progenitor. Dendrogram indicated segregation of the 3 cattle populations in to two main clusters. Cluster 1 consisted of RCC and that of cluster 2 consisted of BCB-1 and its progenitor. Our preliminary data inferred that BCB-1 is genetically separated from RCC and it has close relation with progenitor. This study needs further investigation with numbers of news microsattelite markers for increasing accuracy of the present results.

Keywords: BCB-1, Dandogram, Genetic distances, Heterozygosity, Fixation index

Location: APRD, BLRI, Savar, Dhaka

Start Date: 01-06-2011

End Date: 30-05-2012

Program Area: Variety and Species

Commodity: Cattle

Objectives:

To identify the genetic diversity of BCB-1 with other indigenous stock of Bangladesh and to find out marker genes for selection of breeding animals.

Methodology:

Collection of blood

Blood samples were collected from BCB-1, Red Chittagong (RCC, BLRI research farm and Farmer’s community at Chittagong district) and BCB-1 progenitor (Sahzadpur upazilla of Sirajgonj and Bhangura, Faridpur and Sathia upazilla of Pabna district) cattle populations in EDTA containing 10 mL collection vials using 21G needle attached with a disposable 10 ml plastic syringe. The vials were gently tilted for mixing the blood with anticoagulant. The vial was marked with the date of collection, sex and genotype. The blood samples were carried into lab in ice box and stored at -20^oC until extraction of genomic DNA (gDNA).

gDNA extraction

gDNA was extracted using BLRI developed DNA extraction kit from frozen thawed blood samples. The purity of gDNA was tested by electrophoresis of DNA sample on agarose gel using 1x TBE buffer. Six primers were tested for specificity and PCR analysis with three set microsattelite markers (ETH, INRA32 and INRA63) were completed for this report.

DNA amplification and agarose gel electrophoresis

Amplification was performed in 20µl reaction volume (200µm dNTPs, 1X PCR buffer, 500 µm forward and 500 µm reverse primer and 2 µL DNA templates, 1U Taq polymeras). The PCR reactions were started with an initial heating at 94⁰C for 5 min, followed by 35 cycles of 30s at 94?C, 40s at 56-60?C and 40s at 72?C and ended with a final extension at 72 ?C for 10 min. PCR products from each sample were confirmed by running on 3% high resolution agarose gel at 60V, 300mA and 300W for 1.30 hrs and visualized by ethidium bromide staining in 1x TBE buffer.

Data analysis

Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. The data were used to estimate allelic frequency, genetic distances and dendogram using PopGen32 (Version 1.31) computer program.

Source of Information: BLRI. Annual Research Review Workshop, 2011-12. BLRI, Savar, Dhaka.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Our preliminary data inferred that BCB-1 is genetically separated from RCC and it has close relation with its progenitor cattle.

Knowledge Management: Report/Proceedings