Morphological and molecular characterization of Ocimum species
O. tenuiflorum, O. gratissimum, O. basilicum and O. americanum was collected from kulumani, kallikudi, palakarai and palkalaiperur respectively within Tiruchirappalli district. The taxonomic identification of the collected Ocimum species was confirmed by Dr. S. Soosairaj, Assistant Professor, Department of Botany, St. Joseph’s College (Autonomous) Tiruchirappalli.
The Voucher Specimen (Herbarium Accession number for O. tenuiflorum − 2794, O. gratissimum − 2793, O. basilicum − 2795, O. americanum − 2792) was deposited in the Herbarium, Department of Botany, St. Joseph’s College (Autonomous) Tiruchirappalli. Morphological characters and their states of all studied taxa were summarized in Table 1. All taxa studied O. tenuiflorum, O. gratissimum, O. basilicum and O. americanum are herbs. All studied taxa are erect, branched, aromatic and usually tetragonous stem.
Table 1 Morphological characteristics of selected Ocimum Taxa
POLLEN MORPHOLOGY STUDIES:
The SEM micrograph of the studied pollen grains is illustrated in the figures. The Pollen Grains of the shape of the studied plants are prolate, oblate-spherical and percolate. All the plants taken for the studies showed they were reticulate type of exine in nature.
Table 2: Pollen Morphological characteristics of selected Ocimum Taxa
Molecular identification of Ocimum species
Dna Isolation, Purification And Quantification
DNA isolation of four different Ocimum species of leaves sample was carried out successfully using CTAB method and purified using sodium acetate ethanol precipitant. Figure 3a shows that the isolated genomic DNA was quantified in 0.8% agarose Gel electrophoresis. The genomic DNA of O. tenuiflorum, O. gratissimum, O. basilicum, and O. americanum shows that the genome is likely to similar.
Dna Amplification
The diluted genomic DNA was amplified using PCR and the amplicons were obtained. Figure 3b shows that amplicons obtained were approximately at the length of 700–800 bp for the rbcL gene, 300–450 bp for trnH-psbA and 830 bp for the trnL-trnF intergenic spacer region, evaluated by using 100–1000 bp as a DNA ladder.
The amplicons were eluted and sequenced using both forward and reverse primers and the sequence chromatograms were successfully obtained for all Ocimum samples. The length of the amplicons was identified in 1.5% agarose gel electrophoresis.
The rbcL, trnH-psbA and trnL-F DNA sequences for the plant O. tenuiflorum, O. gratissimum, O. basilicum and O. americanum was obtained. Nearly 12 plant sequences are obtained from three different regions which show there is difference in their molecular level identification.
Phylogenetics analysis of the sequence based on intraspecific molecular markers by interspecific Ocimum species
The NJ method using MEGA11 showed that the Ocimum species are closely related and comes under the Lamiaceae family. Figure 4–6 represents the phylogeny of the plant species based on molecular markers.
Phylogenetic tree of rbcL gene of Ocimum species.
Figure 4 denotes that phylogenetic tree of rbcL gene of Ocimum species. The tree was generated based on genetic marker into two clades O. tenuiflorum and O. gratissimum formed a first clade; Second clade formed by O. basilicum and O. americanum.
Phylogenetic tree of trnH-psbA gene of Ocimum species
Figure 5 denotes that phylogenetic tree of trnH-psbA gene of Ocimum species. The tree was generated based on genetic marker into three clades O. basilicum and O. americanum present in one clade, whereas O. tenuiflorum present in another clade and O. gratissimum present in-between two clades.
Phylogenetic tree of trnL-trnF gene of Ocimum species
Figure 6 denotes that phylogenetic tree of trnL-trnF gene of Ocimum species. The tree was generated based on genetic marker into three clades O. basilicum and O. americanum present in one clade, whereas O. tenuiflorum present in another clade and O. gratissimum present in-between two clades.
Comparative analysis of phylogenetics tree based on markers by interspecific Ocimum species
The phylogenetic tree obtained from MEGA11 software based on molecular markers interpret that rbcL gene plays a major role for molecular identification in species level while trnL-trnF and trnH-psbA intergenic spacer region as a supporting to the rbcL gene in molecular identification.
Phylogenetics tree was constructed by interspecific molecular markers with intraspecific Ocimum species
Phylogenetic tree of three chloroplast gene - O. tenuiflorum
Figure 7 denotes that phylogenetic tree of O. tenuiflorum based on rbcL, trnL-F and trnH-psbA molecular markers. The tree was generated based on genetic marker into two clades O. tenuiflorum trnH-psbA and trnL-trnF intergenic spacer present in one clade while rbcL present in second clade.
Phylogenetic tree of three chloroplast gene - O. gratissimum
Figure 8 denotes that phylogenetic tree of O. gratissimum based on rbcL, trnL-F and trnH-psbA molecular markers. The tree was generated based on genetic marker into two clades O. gratissimum trnH-psbA and trnL-trnF intergenic spacer present in one clade. While rbcL present in second clade.
Phylogenetic tree of three chloroplast gene - O. basilicum
Figure 9 denotes that Phylogenetic tree of O. basilicum based on rbcL, trnL-F and trnH-psbA molecular markers. The tree was generated based on genetic marker into two clades O. basilicum trnH-psbA and trnL-trnF intergenic spacer present in one clade, while rbcL present in second clade.
4.2.2.5.4 Phylogenetic tree of three chloroplast gene - O. americanum
Figure 10 denote a phylogenetic tree of O. americanum based on rbcL, trnL-F and trnH-psbA molecular markers. The tree was generated based on genetic marker into two clades O. americanum rbcL and trnL-trnF intergenic spacer present in one clade, while trnH-psbA present in second clade.
Comparative analysis of phylogenetics tree based on interspecific molecular markers by intraspecific Ocimum species
The phylogenetic tree obtained using MEGA11 for O. tenuiflorum, O. gratissimum, O. basilicum and O. americanum based on Ocimum species. The intergenic spacer region of trnL-F and trnH-psbA formed in one clade and rbcL gene present in another clade for O. tenuiflorum, O. gratissimum and O. basilicum except O. americanum. In O. americanum trnL-F and rbcL gene present in one clade while trnH-psbA present in another clade.
Phylogenetic analysis of 12 obtained sequences from interspecific Ocimum species and interspecific chloroplast gene
Figure 11 denotes that phylogenetic tree of O. tenuiflorum, O. gratissium, O. basilicum and O. americanum based on rbcL, trnL-F and trnH-psbA molecular markers. The tree was generated based on genetic marker. Hence Fig. 9 proved that there is a significant relationship between two molecular markers trnL-F and trnH-psbA due to intergenic spacer region. The rbcL marker present in different clade due to it as universal gene.
The evolutionary history was inferred using the neighbour joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 1.89865711 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Kimura, 1980).
The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kumar et al., 2016) and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 264 positions in the final dataset. Evolutionary analyses were conducted in MEGA11.
Sequences Submission
Sequence similarity tool - Blast
The sequences obtained were then employed to identify the other sequences published in literature with high degree of similarity to that of newly determined sequence by running BLAST tool.
Sequences Submission In Ncbi
The sequences obtained from the plants O. tenuiflorum, O. gratissimum, O. basilicum and O. americanum from three different loci are deposited in genbank by using bankit software. The accession numbers are obtained from the following Table 3.
Table 3: GenBank accession numbers for plant sequences
Here amplified PCR products were confirmed by agarose gel electrophoresis and the 4 PCR products were sequenced. Results obtained were compared with other sequences obtained from NCBI-genbank. From blast analysis, it was found that of the tweleve sequences, of the selected plant.