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Genetic Diversity Analysis of Some Local Biodiesel Plant (jatropha Curcas L.) in Bangladesh

Fahmida Khatun
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Mahfuzur Rahman
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Ahasun Habib
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Shahidul Haque
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh-2202, Bangladesh

Harun-or Rashid
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh-2202, Bangladesh

Sumitra Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Arrafy Rahman
Department of Microbiology, International Institute of Applied Science and Technology, Rangpur-5400, Bangladesh

Abstract/ Executive Summary:

The present study was conducted to analyze genetic diversity among biodiesel producing plant Jatropha curcas genotypes using six RAPD primers. The J. curcas samples were collected from ten agro climatic regions in Bangladesh. The six primers produced 31 DNA bands. All the DNA bands (31) showed 100% polymorphism. Overall gene frequency of 10 genotypes of J. curcas was ranged from 0.10 to 0.90. The average Nei’s gene diversity and Shannon’s Information Index for all loci were estimated 0.2994 and 0.4650, respectively. The gene diversity value was ranged from 0.18 to 0.50 and Shannon’s Information Index was ranged from 0.3251 to 0.6931. Inter-germplasm similarity indices (Si) ranged from 5.56 to 66.67% with an average of 33.906%. The similarity coefficient range varied from 0.00 to 0.971. The UPGMA dendogram constructed from Nei’s (1972) genetic distance group indicated segregation of the ten J. curcas germplasm into three main clusters. Cluster I, II and III possessed 8, 1 and 1 germplasm, respectively. This study revealed that at least three different J. curcas genotypes are available in Bangladesh. The RAPD technique is, however, found to be useful in studying genetic variation among J. curcas genotypes of different regions in Bangladesh.

Keywords: Jatropha curcas, Genetic diversity, Biodiesel plant, RAPD

Location: Ten agro climatic regions in Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Plant(s)

Objectives:

The present study was designed to estimate the genetic variability among J. curcas found in Bangladesh.

Methodology:

Sample collection- Leaves of J. curcas samples were collected from 10 different regions of Bangladesh. Extraction of genomic DNA- Genomic DNA from each individual sample was extracted from young leaf tissue by using ATP^TM Genomic DNA Mini Kit (Plant) following the described protocol. Young and fresh leaves from each genotype were sliced with scissors and then distilled water and 100% ethanol were added to wash them before drying on fresh tissue paper for removal of microbial spore and any other foreign DNA. Protocol mentioned in ATP^TM Genomic DNA Mini Kit (Plant) (ATP biotech Inc., Taiwan) was followed accordingly. After DNA extraction, presence of genomic DNA was confirmed by agarose gel electrophoresis and DNA quantification was done by spectrophotometer. Primer selection- Eight primers were primarily used to produce polymorphic DNA bands, of which six primers i.e. OPP13, S1155, OPB02, S1239, S1027, and OPA02 gave reproducible, clear, bright and distinct polymorphic DNA bands. These primers were finally selected for determination of genetic diversity across the 10 J. curcas genotypes collected from different regions of Bangladesh. Preparation of PCR reaction mixture for final amplification- During the experiment the Thermo scientific PCR master mix (2x) were gently vortexed and then thawed and centrifuged. Then PCR tube was placed on ice. Working solution of DNA samples were mixed gently after thawing out. Dilute primers were ready for use and prepared working solution with nuclease free water and mixed gently. PCR reactions were carried out on each DNA sample in a 25 µl reaction mix containing 12.5 µl of 2x PCR master mixes, 2 µl of 10 pmol/µl primer, 2 µl of J. curcas genomic DNA and an appropriate amount of nuclease free water. All the pre-mixes were vortexed shortly, and then they were aliquot into the tubes. The PCR tubes were set on the wells of (BioRad-PTC200) thermocycler plate. Then the PCR reactions were programmed for 35 cycles with denaturation at 95°C for 1 min, annealing at 35°C for 1 min, extension at 72°C for 2 min and final elongation at 72°C for 10 min. The amplified PCR products were run using 1.5% agarose gel electrophoresis and photographed for documentation. Genetic data analysis- According to bands position on gel, all bands of RAPD markers were scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer. The size of each band was calculated using molecular weight ladder. The scores obtained using the primer in the RAPD analysis was then pooled to create a single data matrix. This was used to estimate population differentiation, genetic distance (D²), polymorphic loci, Nei’s (Nei, 1978) gene diversity, gene frequency (Elo et al., 1997) and to construct a UPGMA dendrogram among populations using a computer program, POP GENE (Yeh et al., 1999). Similarity index values between the RAPD profiles of any two individuals on the same gel were calculated from RAPD markers by using following formula: Similarity index (SI) = 2Nxy/Nx + Ny. Where, Nxy is the number of RAPD bands shared by individuals x and y respectively, and Nx and Ny are the number of bands in individual x and y, respectively

Source of Information: J Bangladesh Agril Univ 17(4): 437–445, 2019

Article Link (Online Journal): DOI: https://doi.org/10.3329/jbau.v17i4.44603

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The genotypes of same sub- cluster might be of same genotypic or parental source were revealed comparatively less genetic diversity. J. curcas is an economically important and promising plant for biodiesel production in Bangladesh. This importance necessitates its molecular characterization. The genetic diversity that has been revealed in the present study could be informative in the improvement, management and conservation point of view. The genotype pairs which showed high level of genetic distance could be used in the future research programme. Moreover, diversified gene pool of the studied J. curcas genotypes should be conserved effectively. So this study can be used as a relevant information source for further genetic diversity analysis.

Knowledge Management: Journal