Genetic diversity analysis of some local biodiesel plant (Jatropha curcas L.) in Bangladesh

Fahmida Khatun, Md. Mahfuzur Rahman, Md. Ahasun Habib, Md. Shahidul Haque, Harun-or Rashid, Sumitra Saha, Arrafy Rahman Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh 2202, Bangladesh Department of Microbiology, International Institute of Applied Science and Technology, Rangpur 5400, Bangladesh


Introduction
Jatropha curcas L. (Biodiesel plant) is a perennial and monoecious plant belonging to Euphorbiaceae family; native to America but distributed widely in the tropical and subtropical areas (Koushik et al., 2007). In Bengali, J. curcas is known as Verenda, Arenda, Chanda, Jamalgota etc. This plant can grow well under adverse soil and climatic conditions. J. curcas has magnetized a great deal of attention due to its potential as an incipient energy plant. Its seeds contain 30-45% oil in which a high percentage of polyunsaturated linoleic and monounsaturated oleic acid were found (Gubitz et al., 1999).
It is a minuscule tree which attaining a height of 5m and unisexual flowers and infrequently hermaphrodite flowers. Mature seeds are black, 2cm long and 1cm thick. Seed kernel contains about 33 -60% oil. After extracting from mechanical expeller, oil is filtered; processed and 20% of this oil can be utilized as biodiesel for running diesel Engineer. It is a renewable and safe energy source and a viable substitute to fuel wood, coal, furnace oil, kerosene, LPG, and diesel (Martin and Mayeux, 1985).
J. curcas is a second generation, drought tolerant biofuel crop. It can supersede fossil fuels in developed and in developing countries like Bangladesh while bringing superfluous income to poor farmers. Moreover, the country has a sizably voluminous area of uncultivable land which should be taken into consideration for propagating this consequential bioenergy crop to engender biofuel. It can be cultivated easily on wastelands and marginal land. As an oil bearing biomass feedstock, it can ascertain an alternative source of energy and reduce our dependency on fossil fuel. This plant can grow anywhere and can live for about 50 years (Henning, 2010). As a mature plant or tree, it can absorb around 18 lbs of carbon dioxide (CO2) per year, the plant plays an important role in climate change issues. So, Jatropha can sequester around 20 tons of CO2 annually in one hectare of land (Muok, 2008). J. curcas is as a sustainable source of second generation biodiesel feedstock species and different propagation technologies are used to increase their overall supply. In addition, the plant can grow in stressful environment including drought and its cultivated land pattern is suitable in Bangladesh (Nahar, 2011).
Since J. curcas plays an important role for the manufacture of biodiesel, researchers perceived a demand to develop noble cultivars. The breeding procedures of Jatropha are difficult, compared to breeding procedures for other oil producing species viz. peanut, cotton, castor, sunflower and soybean (Freitas et al., 2011). The success of a breeding procedure relies on the knowledge of estimates of genetic parameters within the main traits and of the genetic variability available. The existence of genetic variability is consequential in a breeding procedure so that breeders can select different genotypes to engender hybrids and analogous genotypes to engender lines. Now-a days, the DNA markers become the marker of choice for the study of genetic diversity has become routine, to revolutionized the plant biotechnology. RAPD, for Random Amplification of Polymorphic DNA, is a type of PCR reaction, where the segments of DNA that are amplified are random. The RAPD analysis is a commonly used molecular marker in genetic diversity studies. No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (Rafalski et al., 1994). Information about of genetic variability among J. curcas is not commonly available in our country. Therefore, the present study was designed to estimate the genetic variability among J. curcas found in Bangladesh.

Sample collection
Leaves of J. curcas samples were collected from 10 different regions of Bangladesh and listed in the Table 1.

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 (Table 2) i.e. OPP13, S1155, OPB02, S1239, S1027, and OPA02 gave reproducible, clear, bright and distinct polymorphic DNA bands (Tabassum et al., 2013;Kolade et al., 2016). 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 (D 2 ) (Nei, 1972), 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 (Chapco et al., 1992;Wilde et al., 1992;Lynch, 1990).

Results
The results that were obtained from the experiment using six RAPD markers on 10 J. curcas are mentioned below. Some of the data have been presented and expressed in tables and figures for ease of understanding.

Primer selection and RAPD pattern
Eight primers were initially screened. Within 8 primers, six primers (OPP13, S1155, OPAB02, S1239, S1027 and OPA02) produced distinct and clear bands. These six primers were selected for their better quality band and efficiency in detecting polymorphism. Then these six primers were used to screen all of the 10 J. curcas accessions. The RAPD profiles of J. curcas accessions using OPP13, S1155, OPAB02, S1239, S1027 and OPA02 primers are shown in Figure 1.

Score of polymorphic bands observed
All six primers produced a total of 31DNA loci with size ranging from 100bp to 1000bp; DNA fragments were found to be polymorphic. Parameters of six primers studied are presented in the Table 2. Primer OPP13 produced the highest number of polymorphic bands (9). Thus it showed a high level of polymorphism. S1155 primers produced 7 polymorphic bands. Similarly OPAB02, S1239, S1027 and OPA02 primers produced 5, 4, 3 and 3 bands, respectively. The banding patterns of J. curcas from different regions using primers OPP13, S1155, OPAB02, S1239, S1027 and OPA02 primers are shown in Figure 1(a), 1(b), 1(c), 1(d), 1(e) and 1(f), respectively. Among ten J. curcas germplasm, some of them showed inter monomorphic bands.

Genetic distance
Pair-wise Nei's genetic distances ranged from 0.0667 to 0.9714 (Table 7). The value of highest genetic distance (0.9714) was found between Jatropha genotypes sample number 3 vs. sample number 6 and sample number 9; while the lowest (0.0667) was found between Sample numbers 8 vs. sample number 10. Comparatively higher levels of genetic distance (0.9361) were observed in sample 3 vs. sample 5 and sample 3 vs. sample 9. Genetic distance 0.7949 found in sample 3 vs. sample 1 and sample 3 vs. sample 8.

UPGMA dendrogram
A dendrogram based on Nei's genetic distance Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated segregation of the 10 J. curcas germplasms into three main clusters (Figure 2). Cluster I was subdivided into three sub-clusters IA, IB, and IC. Sub-cluster IA comprised of sample 1 and sample 9; Sub-cluster IB comprised of sample 2, sample 7 and sample 4; and Sub-cluster IC comprised of sample 5, sample 8 and sample 10 of J. curcas. The cluster II comprised of only J. curcas sample 6 which was collected from Sadar, Mymensingh and cluster III has one sample i.e.; sample 3 which was collected from Rupsha, Khulna. The similarity coefficient range varied from 0.00 to 0.971. The highest value of similarity coefficient range is 0.971 and the lowest value of similarity coefficient is 0.00.  Nei's (1972) genetic distance, summarizing data on differentiation among 10 J. curcas genomes according to RAPD analysis

Discussion
In this study, genetic variation among ten (10) J. curcas germplasms collected from ten agro climatic regions was revealed using six RAPD primers. The primers generated 31 stable and reproducible DNA bands among 10 Jatropha genotypes. The band size varied from 100bp to 1000 bp, with average number of bands per primer was 5.17. A total of 31 bands were scored of which 100% (31 bands) were polymorphic across the 10 Jatropha genotypes. The number of polymorphic bands observed on an average 5.17, so the level of polymorphism observed in present study was fairly high (100%) indicating that a wide and diverse genetic distance existed among the genotypes of J. curcas found in Bangladesh.
Among the six primers, two primers produced several bands such as OPP13 primer produced total 9 bands and S1155 primer produced 7 bands. While the other primers OPAB02 produced 5 bands, S1239, OPA02 and S1027 produced 4, 3 and 3 bands, respectively. These findings revealed that all the six primers produced bands against the 10 J. curcas genomes which were collected from 10 agro climatic regions showed 100% polymorphism. Similar conclusion was made by Gupta et al. (2008) while accessing genetic variation in 14 accessions of J. curcas from several agro climatic zones of India using RAPD primers. They observed polymorphism (80.2%) in Jatropha species with 26 RAPD primers. 10 primers were used in five accessions of J. curcas from four agro climatic zones of Maharashtra to detect 125 DNA bands of which 96 (74.62%) were polymorphic (Gopale and Zunjarrao, 2013).
The genetic distance values observed in this study ranged from 0.0667 to 0.9714. These findings are in line with the results of Pamidiamarri et al. (2008) where the highest genetic polymorphism was observed in J. curcas through RAPD analysis. However, in another study moderate level of genetic variability was found in J. curcas germplasms in India (Basha et al., 2007). The findings from the present study revealed that J. curcas germplasms from different regions of Bangladesh have a wide range of genetic variation.
Pair-wise inter-genotype similarity indices (Si) is a measure of relatedness among genome pool that also demonstrated 10 Jatropha genotypes using the six primers. The overall average inter-genotype similarity index was 33.906%. The average highest inter-genotype similarity indices were found in six pair of genotypes for OPA02 primer and one pair of genotypes for S1027 and the value was 66.67% which showed less heterozygosity in other words they are homogenous. The germplasm pair S1vs. S9 showed the lowest average Si value (26.13%) considering all six primers. Pair wise similarity induces coefficient ranges varied from 0.00 to 0.971. Dhillon et al. (2006) reported that such a wide range in similarity coefficient values indicated that J. curcas germplasms represent a genetically diverse population and this might be attributed to a high level of cross pollination in this species. The diversity revealed by RAPD is in agreement with the conclusion that our breeding plant species retain considerable variability. The maintenance of a high genetic variance within population was favored by genetic systems of the species like gene flow, out breeding, mutation, high genetic load, etc.
The UPGMA dendrogram (Figure 2) showed that 10 genotypes were clustered into three groups. Cluster I was subdivided into three sub-clusters IA, IB, and IC. The cluster II comprised of only Jatropha sample 6 and cluster III comprised of sample 3. The cluster II and cluster III were collected from Sadar, Mymensingh and Rupsha, Khulna respectively. The cluster II and III showed more genetic diversity. The cluster I comprised other eight genotype samples, which were subdivided into three sub-clusters. The genotypes of same subcluster 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.