DNA Barcoding Markers and Next Generation Sequence Analysis of Rbcl in Intraspecies Genetic Variations of Three Varieties of Cissus quadrangularis L.

Cissus quadrangularis L. is a well-known plant used in Ayurveda. It is also called Asthisamharaka. The etymology of its name indicates its use in the healing of the fractured bone. It is a widely growing plant belonging to the family of vitaceae. C. quadrangularis (Family: Vitaceae) is commonly distributed throughout the hotter parts of India and Sri Lanka. C. quadrangularis is mentioned in classical literature like Bhava Prakash and Chakra Dutta. The present study design to compare three different varieties of C. quadrangularis which includes the species of quadrangular, round and flat types are used to study DNA barcoding marker and phylogenetic analysis. Whole-genome was isolated from the stem parts of three varieties of C. quadrangularis ; quadrangular, round and flat. The PCR amplification of DNA barcoding markers (rbcl, mat k and ITS) was analyzed by DNA electrophoresis. The DNA barcoding markers and next-generation sequencing are able to identify the intraspecies genetic variations among these closely related plant varieties of C. quadrangularis of quadrangular, round and flat.


Introduction
Asthisamharaka (C. quadrangularis) is a well known Ayurvedic drug for its effect on the reunion of a fractured bone. C. quadrangularis paste is also used in Vataja diseases (neuromuscular diseases), worm infestation, skin diseases, debility, arthritis, rheumatism etc 1  Accepted on: 26.04.2020 ReseaRch aRticle quadrangular, round and flat types are observed. Even studies denote that taxonomically quadrangular and flat variety do not have much difference 2 . Though there is a difference of opinions regarding the identification of flat and round varieties majority consider these as C. quadrangularis varieties and to be further confirmed and differentiated from C. repens and C. repanda 3 . The study is designed to evaluate the DNA barcoding markers of rbcl, mat k and ITS universal genes and next-generation sequencing to explore the intraspecies genetic variations among these closely related three varieties of C. quadrangularis.

Chemicals
The whole-genome kit was purchased from Favorogen, PCR master mix (2X) 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 mat k, rbcl, and ITS was synthesized by Eurofins Genomics India Pvt. Ltd, Bangalore, Karnataka, India. The chemicals and solvents were purchased from Himedia, India.

Extraction of Whole Genomic DNA
The stem parts from three varieties of C. quadrangularis viz quadrangular, round and flat, 100 mg each and leaf of round variety of C. quadrangularis were crushed with liquid nitrogen into a fine powder. Whole-genome was extracted from the plants using the Favorogen whole genomic isolation kit. The concentration of the genomic DNA was determined using a Nanodrop. The integrity of DNA was confirmed by visualization on 0.8% agarose gel using ethidium bromide staining.

PCR Amplification
The whole genome was used as a template in the PCR reaction. PCR amplification was conducted with PCR master mix (2X), 10 µM forward and reverse primer, nuclease-free water and 10 ng of template DNA ( Table 1). The PCR conditions were initial denaturation at 94 0 C for 5 minutes, 35 cycles of denaturation at 94 0 C for 30-sec annealing at 50 0 C for 1 minute, extension at 72 0 C for 2 minutes followed by a final extension at 72 0 C for 10 minutes. The amplified products were detected by agarose gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer and visualized with gel documentation system.

Amplification and Next Generation Sequence Analysis
PCR was performed using the rbcl gene region. The PCR was done in 25µL consisting of 2X PCR master mix, 0.25 µM forward and reverse primer and nucleasefree water and 10 ng of the whole genome. The PCR was incubated at 94 0 C for 1 minute and amplification was performed with the following 35 thermal cycles; denaturation for the 30s at 94 0 C, annealing for 40s at 53 0 C, an extension for 40s at 72 0 C, and the final extension for 5 min at 72 0 C. The PCR products were detected by agarose gel electrophoresis and visualized with a gel documentation system. PCR products were precipitated using propanol and then washed with ethanol. The products were air-dried and resuspended in denaturing buffer. The sequencing was done using Applied Biosystems DNA sequencer following standard protocol. Sequence analyzing was outsourced to Eurofins Genomics India Pvt. Ltd, India and was analyzed by Next Generation Sequencing (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
The phylogenetic analysis (MEGA) version 10, was used to construct a maximum likely hood tree for the obtained sequences to identify its intraspecies relationships.

Genomic DNA quantification
The extracted plant DNA was analyzed using Nanodrop Eppendorf. The obtained DNA concentration from the stem of quadrangular variety was 20 ng/µl, the stem of round variety was 23 ng/µl, the stem of flat variety was 23 ng/µl and the leaf of round variety was 22 ng/ µl respectively. Hence, due to the good quality of genomic DNA concentrations in all three varieties of C. quadrangularis was used as a DNA template for Rbcl, matk and ITS-4 and 5 for further molecular marker studies.

Internal Transcribed spacer (ITS) 4 and 5 gene amplification
ITS is situated between the small subunit 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 670 base pairs (bp) in size of agarose gel electrophoresis. The gene was amplified successfully in C. quadrangularis of quadrangular, flat and round varieties.

Next Generation Sequence Analysis
Homology of C. quadrangularis was detected using BLAST. The sequence length of Cissus quadrangularis isolate TMP 36 ribulose, Cissus quadrangularis isolate SBB 1201 was 609, 611 and 540 nucleotides match with Cissus quadrangularis of quadrangular, round and flat varieties respectively. rbcl sequence homology of all the three species was 99% identical ( Table 2). We observed that species substitution as marker nucleotides because they may be crucial for identifying each variety. In quadrangular variety there was a mismatch sequence that was observed at the position of 153(C,T), deletion and insertion were not observed. In C. quadrangularis round variety the mismatch sequences was observed at the position of 11(C,T), 527(G,T) and 531-532(TT,AC) nucleotide sequence and the insertion was observed at the position of 12-13(TG,--), 41481(T,-) and deletion was not observed. In C. quadrangularis of flat variety, we found that mismatch sequences at the position of 153(C,T) respectively and the insertion and deletion were not observed (Table 3).

Phylogenetic analysis
To study the phylogenetic relationships among the three species of C. quadrangularis i.e. quadrangular, round and flat. phylogenetic trees were constructed by applying the NJ method to the rbcl. As shown in the result of the phylogenetic tree.
Three DNA barcodes sequences were employed, C. quadrangularis round and C. quadrangularis quadrangular varieties were closer genetically when compared to Cissus quadrangularis flat (Figure 4). Therefore the rbcl barcode sequences provided advanced resolution for the identification of the clusters that constitute clades within the same species. From these phylogenetic trees, it is confirmed that the identification of the three species can be achieved using rbcl gene.

Discussion
C. quadrangularis is also called as Hadjod in Hindi and Asthisamharaka in Ayurveda 7 . It is used for the growth of bones and joints, bone mineral density; increases the rate of fracture healing and used to treat gout, internal bleeding, leucorrhea, and aphrodisiac action. It is used to improve men's stamina, vigor, and strength 8 . Many Cissus species are having medicinal values that are economically profitable; it is used as an ingredient of various Ayurvedic formulations and in fresh juice for bone fracture. There is a need for specific molecular markers to provide further rapid, automatable, and accurate species identification 9 . DNA barcodes can be used to verify accurate species identification. It is rapid and accurate identification of any plant species based on extracting short DNA sequences from a tiny sample of any organisms 10 .
The plant working group of consortium for the barcode of Life's (CBOL) 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 11 . 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 intraspecies genetic variations among these closely related three varieties of Cissus quadrangularis L.
Internal transcribed spacer (ITS) is a nonfunctional RNA situated between structural rRNA of a common precursor transcript 12 . ITS sequences were amplified from the genomic DNA of three varieties of Cissus species 13 . The amplified ITS region was approximately 670 bp in C. quadrangularis.
In our present study, we have found the amplification of the gene was successful in rbcl gene region, Mat K gene was not amplified when compared with rbcl gene. However, the mat k region has not been successful in the Cissus species studied and in the out-group 5 . In Cissus species, rbcL DNA barcode gene becomes suitable and can be used as a single region 14 .
Next-generation sequencing and Sanger sequencing methods are of great importance in unraveling the complexity of genomes 15 . The present study amplified rbcl gene region and conserved sequence in three species of C. quadrangularis. Sequence homology of the amplified gene was detected using BLAST 16 . In our present study, we have identified the rbcl gene sequence homology of all the three varieties of C. quadrangularis i.e quadrangular, round and flat was 99% related to Cissus quadrangularis isolate TMP 36 ribulose. This shows that these three varieties are only of same species varieties i.e of C. quadrangularis In phylogenetic tree analysis we have found that C. quadrangularis round variety was closely related with that of quadrangular variety and distance relationship with flat.

Conclusion
Thus, from the results obtained it is observed that the DNA barcoding markers of ITS-4 and 5, mat k and rbcl gene amplification and sequencing are used to identify the intraspecies genetic variations among these closely related three varieties of C. quadrangularis. The quadrangular, round and flat varieties were closely related with Cissus quadrangularis isolate TMP 36 ribulose (99%). Rbcl barcode sequences provided higher resolution for the recognition of the clusters that constitute clades within the same varieties. These three quadrangular, round and flat variants can be considered as varieties of the same species i.e. C. quadrangularis Phylogenetic analyses confirm that the identification of the three species of C. quadrangularis can be achieved using rbcl gene sequence analysis.

Acknowledgement
Authors express their sincere thanks to the CCRAS, New Delhi, India for providing institutional-level financial assistance and all necessary facilities.