Evaluation of Genetic Diversity by DNA Barcoding of Local Lotus Populations from Thua Thien Hue Province

DOI: 10.18805/IJARe.A-564    | Article Id: A-564 | Page : 121-128
Citation :- Evaluation of Genetic Diversity by DNA Barcoding of Local Lotus Populations from Thua Thien Hue Province.Indian Journal Of Agricultural Research.2021.(55):121-128
Dang Thanh Long, Hoang Thi Kim Hong, Le Ly Thuy Tram, Nguyen Thi Quynh Trang dtlong@hueuni.edu.vn
Address : Institute of Biotechnology, Hue University, provincial road No. 10, Phu Vang, TTHue, Vietnam. 
Submitted Date : 16-04-2020
Accepted Date : 10-08-2020


Background: DNA barcoding is a relatively new method of identifying plant species using short sequences of chloroplast DNA. Although there is a large number of studies using barcoding on various plant species, there are no such studies in the genus Nelumbo
Methods: Three chloroplast DNA regions (rbcL, matK, trnH-psbA) were tested for their suitability as DNA barcoding regions of thirty three lotus samples which were collected in Thua Thien Hue province, Vietnam. Universal primers were used and sequenced products were analyzed using Minimum Evolution method  in the MEGA 7.0 program.
Result: We did not observe high variability in nucleotide sequences within the rbcL region (0.135%). White Nelumbo, while, the most encoding matK (8.013%) and variable trnH-psbA (with different number of repeating regions TAAAA) intergenic regions was the most useful for Nelumbo barcoding. Individual application of the studied regions did not provide the expected results. None of the regions used in the study allowed the division of white and pink lotus varieties of N. nucifera specie according to the adopted classification of the genus Nelumbo. The results confirm that the use of matK, rbcL and trnH-psbA or combine all three regions together is insufficient for DNA barcoding in white and pink lotus varieties of N. nucifera specie and better discrimination within the genus Nelumbo. Our results also indicate the necessity of using a different region. All of the new sequences have been deposited in GeneBank under the following accession numbers: rbcL (MN011708 to MN068956); matK (MN011719 to MN068978) and trnH-psbA (MN011730 to MN086252). 


Genetic diversity analysis Lotus MatK Nelumbo nucifera RbcL TrnH-psbA


  1. Alam, A., Chadha, N.K., Kumar A.P., Chakraborty, S.K., Joshi, K.D., Sawant, P.B., Das, S.C.S., Kumar, J., Kumar, T. (2020). DNA Barcoding and Biometric Investigation on the Invasive Oreochromis niloticus (Linnaeus, 1758) from the River Yamuna of Uttar Pradesh. Indian Journal of Animal Research. 54: 856-863.
  2. Bieniek, W., Mizianty, M., Szklarczyk, M. (2015). Sequence variation at the three chloroplast loci (matK, rbcL, trnH-psbA) in the Triticeae tribe (Poaceae): comments on the relationships and utility in DNA barcoding of selected species. Plant Systematics and Evolution. 301: 1275-1286.
  3. Bolson, M., de Camargo, S.E., Brotto, M.A., Silva-Pereira V. (2015). ITS and trnH-psbA as Efficient DNA Barcodes to Identify Threatened Commercial Woody Angiosperms from Southern Brazilian Atlantic Rainforests. PLoS One. 10(12): e0143049.
  4. CBOL Plant Working Group, (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America. 106: 12794-12797.
  5. Chase, M.W., Cowan, R.S., Hollingsworth, P.M., van den Berg, C., Madriñán, S., Petersen, G., Wilkinson, M. (2007). A proposal for a standardized protocol to barcode all land plants. Taxon. 56: 295-299. 
  6. DeMattia, F., Bruni, I., Galimberti, A., Cattaneo, F., Casiraghi, M., Labra, M. (2011). A comparative study of different DNA barcoding markers for the identification of some members of Lamiaceae. Food Research International. 44: 693-702.
  7. Sharma, K., Mishra, A.K., Misra, R.S. (2008). A simple and efficient method for extraction of genomic DNA from tropical tuber crops. African Journal of Biotechnology. 7(8): 1018-1022.
  8. Fu, Y.X., Li, W.H. (1993). Statistical tests for neutrality of mutations. Genetics. 133: 693-709.
  9. Gamache, J., Sun, G. (2015). Phylogenetic analysis of the genus Pseudoroegneria and the Triticeae tribe using the rbcL gene. Biochemical Systematics and Ecology. 62: 73-81.
  10. Hebert, P.D.N, Cywinska, A., Ball, S.L., de Waard, J.R. (2003). Biological identification through DNA barcodes. Proceedings of the Royal Society of London. 270: 313-322.
  11. Kress, W.J., Erickson, D.L. (2007) .A Two-Locus Global DNA Barcode for land plants: The Coding rbcL Gene Complements the Non-Coding trnH-psbA Spacer Region. Plos One. 2e508.
  12. Kress, W.J., Erickson, D.L., Jones, F.A., Swenson, N.G., Perez, R., Sanjur, O., Bermingham, E. (2009). Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences USA. 106: 18621-18626.
  13. Kumar, S., Stecher,G., Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. 33: 1870-1874.
  14. Long, D.T., Hong, H.T.K., Tram, L.L.T., Trang, N.T.Q., Tien, N.P.T. and Hanh, N.T.N. (2020). Study of the Procedure for Bath Ultrasound-Assisted Extraction of Total Flavonoid from Lotus Seeds and Testing Some Biological Activities. Indian Journal of Agricultural Research. DOI: 10.18805/IJARe.A-525.
  15. Maqbool, S., Ullah, N., Zaman, A., Akbar, A., Saeed, S., Nawaz, H., Samad, N., Ullah, R., Bari, A. and Ali, S.S. (2019). Phytochemical Screening, In-vitro and In-vivo Anti-diabetic Activity of Nelumbo nucifera Leaves Against Alloxan-Induced Diabetic Rabbits. Indian Journal of Animal Research. 54(4): 1-6.
  16. Newmaster, S.G., Ragupathy, S. (2009). Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae). Molecular Ecology Resources. 9: 172-180.
  17. Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E., Sánchez-Gracia, A. (2017). DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution. 34(12): 3299-3302.
  18. Saitou, N., Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4: 406-425.
  19. Seberg, O., Petersen, G. (2009). How many loci does it take to DNA barcode a crocus. PLoS ONE. 4 e4598.
  20. Singh, H.K., Parveen, I., Raghuvanshi, S., Babbar, S.B.(2012). The loci recommended as universal barcodes for plants on the basis of floristic studies may not work with congeneric species as exemplified by DNA barcoding of Dendrobium species. BMC Research Notes. 5: 42. 
  21. Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 123: 585-595.
  22. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution. 30: 2725-2729.
  23. Theodoridis, S., Stefanaki, A., Tezcan, M., Aki, C., Kokkin, S., Vlachonasios, K.E. (2012). DNA barcoding in native plants of the Labiatae (Lamiaceae) family from Chios Island (Greece) and the adjacent Çesme-Karaburun Peninsula (Turkey). Molecular Ecology Resources. 12: 620-633.
  24. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. (1997). The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 24: 4876-4882.
  25. Tripathi, A.M., Tyagi, A., Kumar. A,, Singh, A., Singh, S., Chaudhary, L.B., Roy, S. (2013). The Internal Transcribed Spacer (ITS) region and trnH-psbA are suitable candidate loci for DNA barcoding of tropical tree species of India. PLoS One. 8: e57934.
  26. Yan, H.F., Hao, G., Hu, C.M., Ge, X.J. (2011). DNA Barcoding in closely related species: A case study of Primula L. sect. Proliferae Pax (Primulaceae) in China. Journal of Systematics and Evolution. 49: 225-236.
  27. Zimmermann, T., Bocksberger, G., Brüggemann, G., Berberich, W. (2013). Phylogenetic relationship and molecular taxonomy of African grasses of the genus Panicum inferred from four chloroplast DNA-barcodes and nuclear gene sequences. Journal of Plant Research. 126(3): 363-371.

Global Footprints