Full Length Research Paper - International Research Journal of Plant Science ( 2021) Volume 12, Issue 1
, DOI: 10.14303/irjps.2021.001
Syzygium (family:Myrtaceae) is a genus of plants with many benefits in economic and health. Information and publications on the classification of Syzygium still few in number. Grouping several plant species in this group is still problematic, among other there is overlapping of Syzygium and Eugenia and the position in the classification of some plants are yet unknown. Our study demonstrates that phylogenetic analysis can provide an efficient method for species level identifications and contribute powerfully to taxonomic and biodiversity research.
Syzygium, MEGA, rbcl , Phylogenetic analysis
Syzygium is the most species rich genus of woody plants in Southeast Asia with around 1000 species but little is known of the genus in Wallacea, the biogeographically important transition zone between the Asian and Australian continental areas. The Syzygium is considered to be difficult with respect to higher level classification, reflecting a predominance of "local" taxonomic treatments, while there are few morphological characters which can consistently relate species into species groups.
Collection of samples
Two different species of syzygium plants were collected from Madurai in Tamil Nadu. They were identified as species of syzygium. The plant specimen were shade dried and powdered for further use.The voucher specimens were deposited at the herbarium of the Department of Botany,Queen mary’s College, Chennai-600 004.
Genomic DNA isolation
Isolation of genomic DNA:
The genomic DNA was isolated from plant sample by following the method (Figures 1 and 2) (Moller et al., 1992).
Figure 1. Genomic DNA from plant samples
Figure 2. PCR amplification of rbcl gene of plant samples
PCR analysis
PCR amplification was performed using a 50 μL reaction mixture containing 100 ng of template DNA, 20 μmol of rbcl primers, 200 μM of dNTPs, 1.5 mM of MgCl2, 1U of Taq DNA polymerase (MBI Fermentas) and 10 μL of 10x Taq polymerase buffer. The sequences of rbcL primers used were as follows.
rbcLa-F: (5'-ATGTCACCACAAACAGAGACTAAAGC-3') - Forward
rbcLa-R: (5'-GTAAAATCAAGTCCACCRCG-3') - Reverse
Amplification was carried out with an initial denaturation at 98°C for 45 sec followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec, extension at 72°C for 40 sec and final extension at 72°C for 10 min using a thermocycler (iCycler; Bio-Rad Laboratories, CA). PCR products were analyzed on 1% agarose gel for rbcL amplicons in 1x TBE buffer at 100 V (Kimura M1980).
Sequence analysis of PCR products
The rbcl amplified fragments were purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA) from the agarose gel and sequencing using automated DNA sequencer (Model 3730, Applied Biosystems, USA). The sequences were analyzed using the option Basic Local Alignment Search Tool (BLAST) software available in NCBI.
Phylogenetic analysis
The sequences of these rbcl genes were compared against the sequences available from GenBank using the BLASTN program and were aligned using CLUSTAL W software Thompson 1994. Phylogenetic trees were constructed using the maximum parsimony method (Eck RV and Dayhoff MO 1966). Bootstrap analysis was done based on 1000 replications (Felsenstein J 1985). The MEGA4 package was used for all analyses
1. In the present study two different plants of syzygium namely were collected from in and around Chennai, Tamil nadu.
2. The genomic DNA was isolated from all the two samples in good condition. The isolated DNA was amplified and PCR products of appropriate size were recovered for all the samples.
3. In order to confirm the genus of the collected samples PCR amplification was performed using rbcLa (Forward primer) and rbcLa (Reverse primer) primers.
4. Molecular evolutionary genetic analysis (MEGA) electropherogram based on these sequences illustrated the levels of divergence between the morphologically identified genera.
5. Phylogenetic analysis using Maximum parsimony method resulted in well resolved phylograms.
6. Thus, it can be concluded that our finding are having separate evolutionary entities.
Based on the BLAST analysis and phylogeny analysis clearly revealed that the given samples were belongs to the taxa as follows:
S1: Syzygium caryophyllatum
S2: Syzygium nervosum