Determining Genetic Variability and Taxonomy of Hibiscus rosa-sinensis through rbcL Molecular Marker: Genetic Variability and Taxonomy of Hibiscus rosa-sinensis

Aftab  Iqbal; Muhammad Zia  Ur Rehman

doi:10.54393/pbmj.v6i06.896

Authors

Aftab Iqbal Govt Graduate College of Science Wahdat Road, Lahore, Pakistan
Muhammad Zia Ur Rehman Govt Graduate College of Science Wahdat Road, Lahore, Pakistan

DOI:

https://doi.org/10.54393/pbmj.v6i06.896

Keywords:

Hibiscus rosa-sinensis, rbcL Marker, Taxonomy, Genetic Variability

Abstract

Medicinal plants have been used in traditional medicine for a long time. These plants contain phytochemicals that have a variety of medicinal properties. However, accurate identification and authentication of medicinal plant species ensured their safety and efficacy. DNA barcoding using molecular markers has proven to be a useful method for plant species identification. The rbcL molecular marker was used for detailed characterization, amplification, and phylogenetic studies of Hibiscus rosa-sinensis. Objective: To evaluate the therapeutic properties and potential applications of Hibiscus rosa-sinensis. Methods: Samples of H. rosa-sinensis were collected, and DNA was isolated by the Doyle and Doyle method. The presence of DNA was confirmed by gel electrophoresis, and specific primers were used for PCR amplification. The PCR results were sequenced using next-generation sequencing techniques. After that, a neighbor-joining technique was used for phylogenetic analysis and to obtain pairwise nucleotide distances. Gel electrophoresis confirmed the presence of DNA in plant samples, and PCR amplification using rbcL primers generates successful amplification results. Results: The obtained sequence was 99.7% identical to the previously reported rbcL gene sequence from H. rosa-sinensis. Based on phylogenetic research, H. rosa-sinensis was discovered as a closely related species. Conclusions: The rbcL gene has been found as a viable molecular marker for H. rosa-sinensis identification and phylogenetic analysis. The results of this study demonstrated the therapeutic potential of H. rosa-sinensis and the importance of species identification in herbal medicine. DNA barcoding proved a reliable authentication and quality control technology in the herbal medicine business.

References

Sumner J. The natural history of medicinal plants. Timber Press; 2000.

Zhang M, Wang C, Zhang R, Chen Y, Zhang C, Heidi H, et al. Comparison of the guidelines on good agricultural and collection practices in herbal medicine of the European Union, China, the WHO, and the United States of America. Pharmacological Research. 2021 May; 167: 105533. DOI: https://doi.org/10.1016/j.phrs.2021.105533

Carrubba A and Scalenghe R. The scent of Mare Nostrum: medicinal and aromatic plants in Mediterranean soils. Journal of the Science of Food and Agriculture. 2012 Apr; 92(6): 1150-70. DOI: https://doi.org/10.1002/jsfa.5630

Leung PC. From Ayurveda to Chinese Medicine. World Scientific; 2017. DOI: https://doi.org/10.1142/10287

Zubairi SI, Jaies NS. Daun Hibiscus rosa sinensis: Analisis proksimat, aktiviti antioksidan dan kandungan bahan inorganik. Malaysian Journal of Analytical Sciences. 2014;18(2):260-70.

Udo IJ, Ben MG, Etuk CU, Tiomthy AI. Phytochemical, proximate and antibacterial properties of Hibiscus rosa-sinensis L. Leaf. Journal of Medicinal Plants Studies. 2016;4(5):193-5.

FU YM, JIANG WM, FU CX. Identification of species within Tetrastigma (Miq.) Planch.(Vitaceae) based on DNA barcoding techniques. Journal of Systematics and Evolution. 2011 May;49(3):237-45.

Vickers NJ. Animal communication: when i’m calling you, will you answer too?. Current biology. 2017 Jul 24;27(14):R713-5.

Abat JK, Kumar S, Mohanty A. Ethnomedicinal, phytochemical and ethnopharmacological aspects of four medicinal plants of Malvaceae used in Indian traditional medicines: A review. Medicines. 2017 Oct 18;4(4):75.

Lim TK, Lim TK. Echinacea purpurea. Edible Medicinal And Non-Medicinal Plants: Volume 7, Flowers. 2014:340-71. DOI: https://doi.org/10.1007/978-94-007-7395-0_23

Jadhav VM, Thorat RM, Kadam VJ, Sathe NS. Hibiscus rosa sinensis Linn–‘‘Rudrapuspa’’: a review. J Pharm Res. 2009 Jul;2(7):1168-73.

Linares E and Bye Jr RA. A study of four medicinal plant complexes of Mexico and adjacent United States. Journal of Ethnopharmacology. 1987 Mar; 19(2): 153-83. DOI: https://doi.org/10.1016/0378-8741(87)90039-0

Bennett BC and Balick MJ. Does the name really matter? The importance of botanical nomenclature and plant taxonomy in biomedical research. Journal of Ethnopharmacology. 2014 Mar; 152(3): 387-92. DOI: https://doi.org/10.1016/j.jep.2013.11.042

Kress WJ and Erickson DL. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS One. 2007 Jun; 2(6): e508. DOI: https://doi.org/10.1371/journal.pone.0000508

Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, et al. Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences. 2009 Nov; 106(44): 18621-6. DOI: https://doi.org/10.1073/pnas.0909820106

Bellemain E, Davey ML, Kauserud H, Epp LS, Boessenkool S, Coissac E, et al. Fungal palaeodiversity revealed using high‐throughput metabarcoding of ancient DNA from arctic permafrost. Environmental Microbiology. 2013 Apr; 15(4): 1176-89. DOI: https://doi.org/10.1111/1462-2920.12020

Hebert PD and Gregory TR. The promise of DNA barcoding for taxonomy. Systematic Biology. 2005 Oct; 54(5): 852-9. DOI: https://doi.org/10.1080/10635150500354886

van Velzen R, Weitschek E, Felici G, Bakker FT. DNA barcoding of recently diverged species: relative performance of matching methods. PloS One. 2012 Jan; 7(1):e30490. DOI: https://doi.org/10.1371/journal.pone.0030490

Thompson KA, Newmaster SG. Molecular taxonomic tools provide more accurate estimates of species richness at less cost than traditional morphology-based taxonomic practices in a vegetation survey. Biodiversity and Conservation. 2014 Jun;23:1411-24. DOI: https://doi.org/10.1007/s10531-014-0672-z

Asahina H, Shinozaki J, Masuda K, Morimitsu Y, Satake M. Identification of medicinal Dendrobium species by phylogenetic analyses using matK and rbcL sequences. Journal of natural medicines. 2010 Apr;64:133-8. DOI: https://doi.org/10.1007/s11418-009-0379-8

Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one. 2010 Jan 7;5(1):e8613. DOI: https://doi.org/10.1371/journal.pone.0008613

Saunders GW. Routine DNA barcoding of Canadian Gracilariales (Rhodophyta) reveals the invasive species Gracilaria vermiculophylla in British Columbia. Molecular ecology resources. 2009 May;9:140-50. DOI: https://doi.org/10.1111/j.1755-0998.2009.02639.x

Chase MW, Cowan RS, Hollingsworth PM, Van Den Berg C, Madriñán S, Petersen G, Seberg O, Jørgsensen T, Cameron KM, Carine M, Pedersen N. A proposal for a standardised protocol to barcode all land plants. Taxon. 2007 May;56(2):295-9. DOI: https://doi.org/10.1002/tax.562004

Tutin TG. 3. Diapensiaceae to Myoporaceae. Flora Europaea.. 1972. DOI: https://doi.org/10.2307/1218149

Doyle J. DNA protocols for plants. InMolecular techniques in taxonomy 1991 (pp. 283-293). Berlin, Heidelberg: Springer Berlin Heidelberg. DOI: https://doi.org/10.1007/978-3-642-83962-7_18

Lee JH, Park Y, Choi JR, Lee EK, Kim HS. Comparisons of three automated systems for genomic DNA extraction in a clinical diagnostic laboratory. Yonsei medical journal. 2010 Jan 31;51(1):104-10. DOI: https://doi.org/10.3349/ymj.2010.51.1.104

Southern E. [9] Gel electrophoresis of restriction fragments. InMethods in enzymology 1979 Jan 1 (Vol. 68, pp. 152-176). Academic Press. DOI: https://doi.org/10.1016/0076-6879(79)68011-4

Sudmoon R, Chaveerach A, Sanubol A, Monkheang P, Kwanda N, Aungkapattamagul S, Tanee T, Noikotr K, Chuachan C, Kaewdoungdee N. Identifying efficiency in herbal medicine Cinnamomum species (Lauraceae) using banding patterns and sequence alignments of rpoB, rbcL and matK regions. J. Med. Case Rep. 2014 Oct 1;41:1094-108.

Ford CS, Ayres KL, Toomey N, Haider N, Van Alphen Stahl J, Kelly LJ, Wikström N, Hollingsworth PM, Duff RJ, Hoot SB, Cowan RS. Selection of candidate coding DNA barcoding regions for use on land plants. Botanical Journal of the Linnean Society. 2009 Jan 1;159(1):1-1. DOI: https://doi.org/10.1111/j.1095-8339.2008.00938.x

Asmussen CB, Chase MW. Coding and noncoding plastid DNA in palm systematics. American journal of botany. 2001 Jun;88(6):1103-17. DOI: https://doi.org/10.2307/2657094

Stanford AM, Harden R, Parks CR. Phylogeny and biogeography of Juglans (Juglandaceae) based on matK and ITS sequence data. American Journal of Botany. 2000 Jun;87(6):872-82. DOI: https://doi.org/10.2307/2656895

Tate JA, Simpson BB. Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Systematic Botany. 2003 Oct;28(4):723-37.

Levy SE, Myers RM. Advancements in next-generation sequencing. Annual review of genomics and human genetics. 2016 Aug 31;17:95-115. DOI: https://doi.org/10.1146/annurev-genom-083115-022413

Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution. 1987 Jul 1;4(4):406-25.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution. 2013 Dec 1;30(12):2725-9. DOI: https://doi.org/10.1093/molbev/mst197

Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Avila JA, López-Guerrero JA, Aguilar-Quesada R. DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and biobanking. 2016 Aug 1;14(4):264-70. DOI: https://doi.org/10.1089/bio.2015.0064

Karaca DE. Molecular mapping of the V locus in pea (Pisum sativum L.) (Doctoral dissertation, Washington State University).

Xu Q, Xiong G, Li P, He F, Huang Y, Wang K, Li Z, Hua J. Analysis of complete nucleotide sequences of 12 Gossypium chloroplast genomes: origin and evolution of allotetraploids.

Vickers NJ. Animal communication: when i’m calling you, will you answer too?. Current biology. 2017 Jul 24;27(14):R713-5.

Barbosa-Silva RG, Coutinho TS, Vasconcelos S, da Silva DF, Oliveira G, Zappi DC. Preliminary placement and new records of an overlooked Amazonian tree, Christiana mennegae (Malvaceae). PeerJ. 2021 Nov 15;9:e12244. DOI: https://doi.org/10.7717/peerj.12244

FU YM, JIANG WM, FU CX. Identification of species within Tetrastigma (Miq.) Planch.(Vitaceae) based on DNA barcoding techniques. Journal of Systematics and Evolution. 2011 May;49(3):237-45. DOI: https://doi.org/10.1111/j.1759-6831.2011.00126.x

Vickers NJ. Animal communication: when i’m calling you, will you answer too?. Current biology. 2017 Jul 24;27(14):R713-5. DOI: https://doi.org/10.1016/j.cub.2017.05.064

Abat JK, Kumar S, Mohanty A. Ethnomedicinal, phytochemical and ethnopharmacological aspects of four medicinal plants of Malvaceae used in Indian traditional medicines: A review. Medicines. 2017 Oct 18;4(4):75. DOI: https://doi.org/10.3390/medicines4040075

Hebert PD, Stoeckle MY, Zemlak TS, Francis CM. Identification of birds through DNA barcodes. PLoS biology. 2004 Oct;2(10):e312. DOI: https://doi.org/10.1371/journal.pbio.0020312

Habib S, Amrao L, Ahmed MZ, Ahmad M, Naz S, Zeshan MA, Asadullah HM, Hamza AM, Ghuffar S. Identification of beta satellite component in (Hibiscus rosa-sinensis L.) through PCR-RFLP from Faisalabad, Pakistan. Journal of Bioscience and Agriculture Research.;20(01):1664-70. DOI: https://doi.org/10.18801/jbar.200119.202

Lahaye R, Van der Bank M, Bogarin D, Warner J, Pupulin F, Gigot G, Maurin O, Duthoit S, Barraclough TG, Savolainen V. DNA barcoding the floras of biodiversity hotspots. Proceedings of the National Academy of Sciences. 2008 Feb 26;105(8):2923-8. DOI: https://doi.org/10.1073/pnas.0709936105

de Vere N, Rich TC, Ford CR, Trinder SA, Long C, Moore CW, Satterthwaite D, Davies H, Allainguillaume J, Ronca S, Tatarinova T. DNA barcoding the native flowering plants and conifers of Wales. PloS one. 2012 Jun 6;7(6):e37945. DOI: https://doi.org/10.1371/journal.pone.0037945

YANG JB, WANG YP, Moeller M, GAO LM, Wu D. Applying plant DNA barcodes to identify species of Parnassia (Parnassiaceae). Molecular Ecology Resources. 2012 Mar;12(2):267-75. DOI: https://doi.org/10.1111/j.1755-0998.2011.03095.x