Molecular Identification and Next‑Generation Sequence Analysis of Interspecies Genetic Variations among Three Varieties of Datura

Background: Datura is a well‑known plant used in Ayurveda. It is a widely growing plant from Solanaceae family, attributed to poisonous and medicinal values. It is used in the treatment of various skin diseases, fever, etc., It is also used for external application and rarely for oral administration. Objectives: The present study aims to compare three different varieties of Datura which include the species of Datura innoxia Mill., Datura metel L. and Datura arborea are used to study molecular marker and phylogenetic analysis. Materials and Methods: Whole‑genomic DNA was isolated from the leaves of Datura and the polymerase chain reactions amplification of DNA barcoding markers are rbcl, mat k, and internal transcribed spacer‑4 and 5 were analyzed by 0.8% agarose gel electrophoresis. Results: The DNA barcoding markers and next‑generation sequencing are able to identify the interspecies genetic variations among these closely related plant varieties of Datura . Conclusion: The interspecies genetic variations among these closely related three species of D. innoxia , metel, and arborea was closely related with Datura stramonium isolate NN003 chloroplast genome similarity of 98%, 99%, and 99%, respectively.


INTRODUCTION
Datura is mentioned in ancient classical literature like Charaka by the name of Kanaka. [1]Sushruta described Datura for the treatment of dog bites.Bhavamishra described Datura leaf externally for the treatment of lice, for analgesic and anti-inflammatory actions. [2]Vangasena described Datura seeds are used after purification processes by boiling in cow milk and shade dried in the room temperature and used it for filariasis. [3]atura is known by the name thorn apple is a poisonous herb.It is used in many Ayurvedic medicines after the purification method. [4]Leaves of Datura are used in alopecia, pain in the breast, tumor, rheumatism, and in inflammation and its flowers are anti-asthmatic.The leaves, seeds, and roots are anti-catarrhal and used in bronchial asthma, skin diseases, wounds, cough, burning micturition, mental illness, febrifuge, diarrhea, and dermatosis.Its chemical composition scopolamine, daturadiol, mentioned based on the flowering color of the plant (white, blue, black, red, and yellow). [6]In this study, three varieties of Datura which include the species of D. innoxia, D. Metel and Datura arborea were studied.The aim of the present study is to evaluate the molecular identification of rbcl, mat k, and internal transcribed spacer (ITS) 4 and 5 universal genes and next-generation sequencing (NGS) are used to explore the interspecies genetic variation among these closely related three plant varieties.

MATERIALS AND METHODS Chemicals
Plant DNA isolation kit was purchased from Favorogen, polymerase chain reactions (PCR) master mix (2X) from Genetix Biotech Asia Pvt. Ltd., New Delhi, India.Agarose, 6X gel loading dye and 100 bp and 1 Kb DNA ladder were procured from Himedia Laboratories PVT, LTD, Mumbai, India, Primers mat k, rbcl, and ITS-4 and 5 was synthesized by Eurofins Genomics India Pvt.Ltd, Bangalore, Karnataka, India.All other chemicals and solvents were of analytical grade and obtained from Himedia, Mumbai, India.

Plant collection
Fresh leaves of Datura are D. Innoxia, D. metel and D. arborea were collected from the Medicinal plant garden at Regional Ayurveda Institute for Fundamental Research (RAIFR), Kothrud, Pune, Maharashtra, India [Figure 1].The plant specimen was authenticated by Dr. Gajendra Rao, Research officer an expert in the field of Botany and live plants are being maintained in the Arborium/Garden of in RAIFR, Pune, Maharashtra, India, with a registration number of DMA-01, DMI-02, and DMD-03.

Extraction of whole-genomic DNA
The leaves of Datura varieties D. Innoxia Mill., D. metel and D. arborea 100 mg of each leaf were crushed with liquid nitrogen into a fine powder.Total genomic DNA was extracted from all the samples using the Favorgen Plant genomic DNA extraction kit.A Nanodrop was used to determine the concentration of genomic DNA.The integrity of DNA was confirmed by visualization on 0.8% agarose gel using ethidium bromide staining.

Polymerase chain reactions amplification
The extracted DNA was used as a DNA template in the PCR reaction.PCR amplification was conducted in 25 µl of PCR mixture containing 12.5 µl of PCR master mix (2X), 10 µM forward and reverse primer (2 µl each), 3.5 µl nuclease-free water, and 10 ng of template DNA.The thermal cycling conditions were initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 30-s annealing at 50°C for 1 min, extension at 72°C for 2 min followed by a final extension at 72°C for 10 min.The amplified products were detected by 0.8% agarose gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer and visualized with ethidium bromide staining.Primers used for amplification of ITS-5 and 4, mat k, and rbcl genomic regions shown in Table 1.

Amplification and next-generation sequence analysis
PCR was performed using the rbcl gene region.The reaction mixture was done in 25 µL consisting of the 12.5 µl of PCR master mix (2X), 0.25 µM forward and reverse primer, and 3.5 µl nuclease-free water and 10 ng of template DNA.The reaction mixture was incubated at 94°C for 1 min and amplification was performed with the following 35 thermal cycles; denaturation for the 30s at 94°C, annealing for 40s at 53°C, an extension for 40s at 72°C, and the final extension for 5 min at 72°C.The amplified PCR products were detected by 0.8% agarose gel electrophoresis in TAE buffer and visualized with ethidium bromide staining.Sequenced products were precipitated using two volumes of 80% propanol and then washed twice with 80% ethanol.The PCR-products were air-dried and resuspended in a denaturing buffer containing 15 µL of formamide.The final sequencing was done using Applied Biosystems DNA sequencer following standard protocol.Sequence analyzing was outsourced to Eurofins Genomics India Pvt.Ltd, Bangalore, Karnataka, India and was analyzed by NGS.The nucleotide sequence for rbcl gene sequence was subjected to sequence alignment using the Basic Local Alignment Tool (BLAST).The number of hits with homologous sequences is inferred based on similarity and alignment.

Phylogenetic tree
Molecular Evolutionary Genetics Analysis version 10, was used to construct a maximum likely hood tree for the obtained sequences to identify its interspecies relationships.

Genomic DNA quantification
The extracted plant DNA was analyzed using Nanodrop Eppendorf.The obtained DNA concentration from the D. innoxia was 20 ng/µl, D. metel was 25 ng/µl and D. arborea was 22 ng/µl, respectively.Hence, due to the good quality of genomic DNA concentrations in all three varieties of Datura was used as a DNA template for rbcl, mat k, and ITS-4 and 5 for further molecular marker analyses.

Internal transcribed spacer 4 and 5 gene amplification
ITS is the spacer DNA situated between the small subunit ribosomal RNA (rRNA) and large subunit rRNA genes in the chromosomes.Figure 2 shows the changes in the ITS-4 and 5 universal gene amplification to generate the PCR product approximately 670 base pairs (bp) in size of agarose gel electrophoresis.The gene was amplified successfully in D. innoxia, D. metel and D. arborea.

Rbcl and Mat k gene amplification
Figures 3 and 4 show the changes in the rbcl and mat k gene amplification to generate the PCR product of agarose gel electrophoresis.The results of DNA barcode performances with mat k and rbcl regions in the three varieties of Datura.The amplification of the gene was successful only in the rbcl gene region, The fragment sizes are about 550 bp.Mat k gene expression was less amplified when compared with rbcl gene.All the varieties of Datura, rbcl gene amplification are similar when compared with mat k gene.

Next-generation sequence analysis
Table 2 shows the statistical simulation of BLAST sequence homology of D. innoxia, D. metel and D. arborea species with rbcl primers, respectively.Sequence homology of the amplified sequence was detected using BLAST.The sequence length of rbcl was 614, 611 and 610 nucleotides with D. innoxia, metel and arborea varieties, respectively.Similarily with rbcl sequence homology of all the three species was 98%, 99%, and 98%, respectively.Table 3 shows the identification of single gene mutation of three varieties of D. innoxia, metel and arborea varieties with rbcl primers, respectively.We observed that species-specific substitution as marker nucleotides because they may be crucial for identifying each species.In D. innoxia, there is a mismatch sequence which was observed at the position of 125, 129, 444, 459, 592, and 605 and insertion was observed at the position of 130-132 and 30134.In D. metel species the mismatch sequences were observed at the position of 437 and 607 nucleotide sequences and the insertion was observed at the position of 111, 25481, 58541-58542.In D. arborea species we found that mismatch sequences were observed at the position of 10, 12, 46, 437, 605, and 606, respectively, and the insertion was observed at the sequence of 141, 597-599.

Phylogenetic analysis
To evaluate the phylogenetic relationships among the three varieties of D. innoxia, metel and arborea, phylogenetic trees were constructed by applying the NJ method to the rbcl sequences.As shown in the result of the phylogenetic tree [Figure 5], when all the three DNA barcodes sequences were employed, Datura metel and D. innoxia were closer genetically similar when compared to D. arborea.Therefore, the rbcl barcode sequences provided higher resolution for the identification of the clusters that constitute clades within the same species.From these phylogenetic analyses, we confirm that the identification of the three varieties can be achieved using rbcl universal gene.

DISCUSSION
DNA barcoding is a popular method for species identification of fresh tender leaves.It is mainly used to verify plants that lack flowers or have incomplete morphological characteristics.It is also to be used in samples such as processed food, fossil remains and herbarium specimens, in which the DNA has been highly degraded. [7]It involves sequencing a standard region of DNA as a tool for species identification. [8]Rbcl, mat k, and ITS-4 and 5 possess attributes that are highly desirable in a plant DNA barcoding system, although none of the three loci fits all three criteria perfectly.Chase, in 2007, reported that using two barcoding region rbcl and mat k options as a standard protocol for barcoding of all land plants. [9]Consortium for the barcode of life's plant working group reported that DNA barcodes with mat k and rbcl gene loci are the use of two regions of plastid DNA as a standard protocol for the core barcoding of land plants. [10]In this study, we assessed the molecular markers of ITS-4 and 5, mat K, and rbcL and next-generation sequences are used to identify the interspecies genetic variations among these closely related three species of Datura.

Conflicts of interest
There are no conflicts of interest.

Figure 1 :
Figure 1: Photography images of three varieties of Datura species

Figure 2 :
Figure 2: M represents marker 1 kb DNA ladder and Lanes L1 -D.innoxia of internal transcribed spacer, L2 -D.metel of internal transcribed spacer and L3 -D.arborea of internal transcribed spacer

Figure 4 :
Figure 4: M represents marker 1 kb DNA ladder and Lanes L1 -D.innoxia of mat k gene, L2 -D.metel of mat k gene, L3 -D.arborea of mat k gene

Table 2 :
Statistical simulation of Basic Local Alignment Tool sequence homology of D. innoxia, D. metel and D. arborea varieties with rbcl primers, respectively