DNA barcoding markers to identify intraspecies genetic variations in three ecotypes of Abrus Precatorius L.
Srinivasan Marimuthu1, GP Pratap2, Goli Penchala Prasad3, Shyam Baboo Prasad4, Anupam Kumar Mangal5, N Srikanth6
1 Research Officer (Biochemistry), Regional Ayurveda Institute for Fundamental Research, Pune, Maharashtra, India
2 Survey of Medicinal Plants Unit, Central Research Institute for Unani Medicine, Hyderabad, Telangana, India
3 Assistant Director In-charge, Regional Ayurveda Institute for Fundamental Research, Pune, Maharashtra, India
4 Research Officer (Pharmacognosy), Regional Ayurveda Institute for Fundamental Research, Pune, Maharashtra, India
5 Assistant Director (Pharmacognosy), Central Council for Research in Ayurvedic Sciences, New Delhi, India
6 Deputy Director General, Central Council for Research in Ayurvedic Sciences, New Delhi, India
|Date of Submission||01-Feb-2020|
|Date of Decision||29-May-2020|
|Date of Acceptance||06-Jul-2020|
|Date of Web Publication||30-Nov-2020|
Goli Penchala Prasad
Regional Ayurveda Institute for Fundamental Research, Kothrud, Pune - 411 038, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Abrus precatorius is also called as Gunja in Ayurveda. It is a commonly grown plant belonging to the family of Fabaceae. It is characterized under the Upavisha (semi-poisonous drugs) and used widely in various Ayurvedic formulations with great beneficial significance. It is used in the treatment of various diseases such as alopecia, edema, helminthiasis, skin diseases, itching, and urinary disorders after being passed through specific purification procedures. Objectives: The present study aims to compare three different varieties of A. precatorius which includes the species of white, black, and red which are used to study DNA barcoding marker and phylogenetic analysis. Materials and Methods: Whole genomic isolated DNA from three varieties (white, black, and red) of A. precatorius leaves and subjected to analysis of the polymerase chain reaction of rbcL, maturase K, and internal transcribed spacer 4,5 using 0.8% agarose gel electrophoresis. Results: The DNA barcoding markers and next-generation sequencing can identify the intraspecies genetic variations among these closely related plant varieties of A. precatorius of white, black, and red. Conclusion: The intraspecies genetic variations among these three varieties of A. precatorius white, black, and red are closely related with A. precatorius isolate TMP 144 Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit gene 99.81%.
Keywords: Abrus precatorius, ayurveda, internal transcribed spacer 4, maturase K, medicinal plants, next-generation sequencing, Rbcl
|How to cite this article:|
Marimuthu S, Pratap G P, Prasad GP, Prasad SB, Mangal AK, Srikanth N. DNA barcoding markers to identify intraspecies genetic variations in three ecotypes of Abrus Precatorius L. Phcog Mag 2020;16, Suppl S2:540-5
|How to cite this URL:|
Marimuthu S, Pratap G P, Prasad GP, Prasad SB, Mangal AK, Srikanth N. DNA barcoding markers to identify intraspecies genetic variations in three ecotypes of Abrus Precatorius L. Phcog Mag [serial online] 2020 [cited 2022 Jan 21];16, Suppl S2:540-5. Available from: http://www.phcog.com/text.asp?2020/16/5/540/301880
- Abrus precatorius is also called as Gunja in Ayurveda
- The study is designed to evaluate the molecular identification of genetic variations among these closely related three varieties of Abrus precatorius to understand the molecular similarities
- We have performed molecular markers such as rbcL, maturase K, internal transcribed spacer, and next-generation sequencing
- The intraspecies genetic variations among these closely three varieties of A. precatorius white, black, and red are closely related with A. precatorius isolate TMP 144 ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene of 99.81%.
Abbreviations used: rbcL: Ribulose bisphosphate carboxylase/oxygenase form I gene; matK: Maturase K; ITS: Internal transcribed spacer; PCR: Polymerase chain reactions; TAE: Tris-Acetate-EDTA buffer; BLAST: Basic Local Alignment Tool; MEGA: Molecular Evolutionary Genetics Analysis; rRNA: Ribosomal RNA.
| Introduction|| |
Abrus precatorius L. is a glabrous wiry climber. It is grown in tropical and subtropical regions. Gunja is the Visha Dravya mentioned in the ancient classical literature like Samhita and other Ayurvedic texts. In Samhita, it is mentioned under Sthavara Visha and in the texts of Rasashastra, it is classified under Upavishas. Aacharya Charaka mentioned this drug in Vajikarana Adhyaya and Aacharya Sushruta classified Gunja under MoolaVisha (root poison). In Bhavaprakasha Nighantu, it is mentioned under Guduchyadi Varga (group starting with Guduchi-Tinospora sinensis (Lour.) Merr.), Yogaratnakara and other Rasashashtra texts mentioned it under Upavisha (semi poisonous group). The two characteristics of Gunja are mentioned, i.e., Shweta Gunja and Rakta Gunja. Out of this, Shweta Gunja (white variety) is considered as highly toxic. Several synonyms are mentioned in Ayurvedic and other existing texts. The Rakta Gunja synonyms are Gunja, Chudamani, Tamrika, Raktika, Rakta, and Shitapaki, whereas the synonyms of Shweta Gunja are Shweta kambhoji, Kakapilu, Kakadani, Bhirintika, and Vaktrashalya. The common names of Gunja are rosary pea, Indian bead, and Buddhist rosary bead. There is a difference of opinion among the Aacharya's concerning the identification of the plant Gunja based on synonyms. Some controversial synonyms are Uchchata, Kakadani, Chudamani, and Shweta kambhoji. Although in classical literature, black variety is not mentioned. In Andhra Pradesh forest regions, a black variety of Gunja is also available. The vegetative parts and total habit of these three varieties appear similar, but flowering and stage-wise fruiting gradually change their color, as shown in [Figure 1]. Red-seeded variety shows light pink color flowering, white-seeded variety shows flowering in creamy white color, and black-seeded variety shows a dark pink color. The seeds in immature pods of red-seeded variety appear in pink and white color, and in the mature seeds, this pink color gradually changes into black and white color turns into red color. In the white variety, the immature and mature pods appear in white only. In the black-seeded variety, the immature pods have only pink color and this pink color gradually changed into complete black color in mature pods. These findings provoked us to conduct the present study to find out genetic modifications at the DNA level to make it clear they are intraspecific changes for the formation of ecotypes. In this study, we comparatively analyzed the three DNA barcode regions, internal transcribed spacer (ITS), maturase K (matK), and rbcL, and next-generation sequencing is used to evaluate their ability to identify the intraspecies genetic variation among these closely related three varieties of A. precatorius.
| Materials and Methods|| |
Plant DNA isolation kit was purchased from Favorogen, Polymerase chain reactions (PCR) Master Mix (×2) from Genetix Biotech Asia Pvt. Ltd., New Delhi, India. Agarose, 6X gel loading dye, 100 bp, and 1 kb DNA ladder were procured from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Primers such as matK, rbcL, and ITS-4 and 5 were synthesized by Eurofins Genomics India Pvt. Ltd., Bangalore, Karnataka, India.
Fresh leaves of A. precatorius of white, black, and red were collected from Talakona forest, Mamandur, Chittor district, Andhra Pradesh, and nearby forest areas from Pune, Maharashtra state. These three varieties are identified at Regional Ayurveda Institute for Fundamental Research (RAIFR), Kothrud, Pune, Maharashtra, India. Live plants are being maintained in the herbarium/garden of RAIFR, Pune, Maharashtra, India, with a herbarium accession number of white, black, and red being SVUBS1215, SVUBS1672, and SVUBS738.
Extraction of whole genomic DNA
The leaves of A. precatorius varieties of white, black and red, 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 Favorogen 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 (×2), 10 μM forward and reverse primer (2 μl each), 3.5 μl nuclease-free wate, r 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-sec annealing at 50°C for 1 min, and 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, matK, and rbcL genomic regions are shown in [Table 1].
|Table 1: Primers used for amplification of ITS-5 and 4, mat k and rbcl genome regions|
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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 (×2), 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 2 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 next-generation sequencing. The nucleotide sequence for rbcL gene sequence was subjected to sequence alignment using the Basic Local Alignment Search Tool (BLAST). The number of hits with homologous sequences is inferred based on similarity and alignment.
Molecular Evolutionary Genetics Analysis version 10 (The Pennsylvania State University, University Park, PA, USA) was used to construct a maximum likely hood tree for the obtained sequences to identify its intraspecies relationships.
| Results|| |
Genomic DNA quantification
The extracted plant DNA was analyzed using Nanodrop Eppendorf. The obtained DNA concentration from the A. precatorius of white was 20 ng/μl, A. precatorius of black was 23 ng/μl, and A. precatorius of red was 25 ng/μl. Good quality of genomic DNA concentration were obtained in three varieties of A. precatorius which were subjected to amplification of DNA templates for rbcl, matk, ITS-4 and 5 for further molecular marker analysis.
Internal transcribed spacer and gene amplification
ITS of nuclear ribosomal DNA is a small nonfunctional RNA situated between structural ribosomal RNAs (rRNA) of a common precursor transcript, which is mainly useful for elucidating relationships among closely related generic species. [Figure 2] shows the changes in the ITS-4 and 5 universal gene amplification to generate the PCR product approximately 670 base pairs (bps) in size of agarose gel electrophoresis. The gene was amplified successfully in A. precatorius of white, black, and red varieties.
|Figure 2: M represents marker 1 kb DNA ladder and Lanes L1 – Abrus precatorius white of Internal transcribed spacer, L2 – Abrus precatorius black of Internal transcribed spacer, and L3 – Abrus precatorius red of Internal transcribed spacer|
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Rbcl and maturase K gene amplification
[Figure 3] shows the changes in the rbcL and matK gene amplification to generate the PCR product of agarose gel electrophoresis. The results of DNA barcode markers of mat k and rbcl gene regions was amplified in the three varieties of A. precatorius. The amplification of the gene was successful in rbcL gene region, the fragment sizes are about 550 bp. Mat k gene expression was amplified successfully in all varieties of white, black, and red A. precatorius when compared with rbcL gene, the bp size of the amplified gene was around 889 bp. All three varieties of A. precatorius rbcL gene amplification are similar when compared with matK gene.
|Figure 3: M represents marker 100 bp DNA ladder and Lanes L1 – Forward primer control of rbcL gene, L2 – Reverse primer control of rbcL gene, L3 – Forward primer control of Maturase K gene, L4 – Forward reverse control of Maturase K gene, L5 – Abrus precatorius white of rbcL gene, L6 – Abrus precatorius black of rbcL gene, L7 – Abrus precatorius red of rbcL gene, L8 – Abrus precatorius white of Maturase K gene, L9 – Abrus precatorius black of Maturase K gene, L10 – Abrus precatorius red of Maturase K gene|
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Next-generation sequence analysis
[Table 2] shows statistical simulation of BLAST sequence homology of A. precatorius of white, black, and red varieties with rbcL primers. Sequence homology of the amplified sequence was detected using BLAST. The sequence length of A. precatorius of rbcL was 614, 610, and 610 nucleotides match with A. precatorius of white, black, and red varieties, respectively. The interspecies genetic similarity among three varieties of A. precatorius are 99.81%.
|Table 2: Statistical simulation of basic local alignment tool sequence homology of Abrus precatorius of white, black, and red varieties with rbcL primers|
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[Table 3] shows identification of single-gene mutation of three varieties of A. precatorius of white, black, and red varieties with rbcL primers. We observed species-specific substitution as marker nucleotides because they may be crucial for identifying each species. In A. precatorius of white, there is a mismatch sequence that was observed at the position of 24(G-A), 586(C, G), 590(T, G), and 593(A, G) and deletion was observed at the position of 415 (---, A). In A. precatorius of the black variety, the mismatch sequences were observed at the position of 13(C, A), 15(C, A), 22(G, A), 589(T, G), and 592(A, G) nucleotide sequence and the deletion was not observed. In A. precatorius of the red variety, we found that mismatch sequences were observed at the position of 22(G, A), 589(T, G), and 592(A, G) and the deletion was not observed.
|Table 3: Identification of single-gene mutation of three varieties of Abrus precatorius of white, black, and red|
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To evaluate the phylogenetic relationships among the three varieties of A. precatorius of white, black, and red, phylogenetic trees were constructed by applying the Neighbor-Joining method to the rbcL sequences. As shown in the result of the phylogenetic tree [Figure 4] when all the three DNA barcodes sequences were employed, A. precatorius of red and A. precatorius of white were closer genetically and similar when compared to A. precatorius of black. 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 the rbcL universal gene.
|Figure 4: Phylogenetic tree showing the common linkage between Abrus precatorius white, black, and red|
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| Discussion|| |
Traditional herbs have been used in Ayurvedic medical systems since ancient times. According to the World Health Organization guidelines, authenticity, purity, and safety are important aspects of standardization and evaluation of traditional medicines. DNA barcoding has many applications in various fields like phylogenetic analysis, authentication, interspecies and intra-species diversity and identification of medicinal plants etc. The universal barcode loci for plants such as rbcL, matK, trnH-psbA, ITS, and 18S rRNA candidate regions are being used as DNA barcodes in plants. In this present study, we assessed the DNA barcoding markers of rbcL, ITS-4 and 5, and Mat-k, and next-generation sequences are used to identify the intragenetic variations among these closely related three varieties of A. precatorius. The plastid DNA sequences have been used for the focus of DNA barcodes for plants. The Plant Working Group of the Consortium for the Barcode of Life recommends using a combination of rbcL and matK regions as a universal DNA barcode Plantae. The results of PCR amplification revealed that the three molecular markers of ITS, rbcL, and Mat k genes are amplified successfully and there is no difference among three varieties of A. precatorius. These DNA barcode regions have been used to identify the intraspecies genetic variation among these closely related three varieties of A. precatorius, the intraspecies distance of rDNA-ITS was greater than those of matK and rbcL. Our results have shown that intraspecific distances are closely related to ITS locus followed by matK and rbcL genes in the three varieties of A. precatorius. In this study, we amplified rbcL gene region and sequenced the conserved regions in three varieties of A. precatorius white, black, and red varieties. Sequence homology of the amplified sequences was detected using BLAST. In this study, we have identified the rbcL gene sequence homology of all the three varieties of A. precatorius white, black, and red varieties. A. precatorius white was 99.81% homology related to A. precatorius isolate TMP 144 ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene. A. precatorius black was 99.81% homology related to A. precatorius voucher J. R. Abbott 24803 (FLAS) ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene. A. precatorius red was 99.81% homology related to A. precatorius isolate shawpc0583 L ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene.
| Conclusion|| |
Thus, from the results obtained we observed that the DNA barcoding markers of ITS, matK, and rbcL gene amplification and sequencing are used to identify the intraspecies genetic variations among these closely related three varieties of A. precatorius white, black, and red. These three were closely related with A. precatorius isolate TMP 144 ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene (99.81%). Phylogenetic analyses confirm that the identification of the three varieties of A. precatorius is three ecotypes with intraspecies genetic variations.
Financial support and sponsorship
The authors express their sincere thanks to the Central Council for Research in Ayurvedic Sciences, New Delhi, India, for providing institutional-level financial assistance and all necessary facilities.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]