Physiology and Molecular Biology of Plants

, Volume 24, Issue 4, pp 631–641 | Cite as

Molecular insights into genetic diversity and population dynamics of five medicinal Eulophia species: a threatened orchid taxa of Africa

  • Paromik Bhattacharyya
  • Johannes van Staden
Research Article


Genetic diversity existing amongst five Eulophia orchid species were assessed using start codon targeted polymorphism (SCoT) and inter-retrotransposon amplified polymorphism (IRAP) markers. A total of 12 SCoT and 5 IRAP markers revealed an average of 63% genetic variability [SCoT = 63.87; IRAP = 64.95%] amongst the five Eulophia species investigated. The genetic similarities were assessed using both UPGMA and Bayesian approaches which indicated identical clustering patterns at a genetic similarity level of 50%. Analysis of molecular variance (AMOVA) revealed the presence of a significant degree of genetic variability, mostly compartmentalized within the species level. Amongst the five assessed Eulophia species, E. parviflora was the most genetically diverse representative whereas E. welwitschii was found to be least diverse based on a comparative assessment of various population genetic parameters like Nei’s gene diversity (h) and Shannon’s information index (I) with an overall gene flow value greater than 1. In order to evaluate the comparative marker efficiency, SCoT and IRAP marker data were subjected to various benchmark analyses like marker index, resolving power, polymorphic index content, multiplex ratio and effective multiplex ratio which revealed the robustness of both the marker techniques in assessment of genetic diversity. The present report provides the first molecular insights into the aspects of inter and intra specific genetic variability in medicinally as well as horticulturally important Eulophia species along with addressing their conservation concerns. In a nutshell, the present approach is simple, rapid and cost effective and can be extended for analysis of genetic diversity of other related plant species.


Gene targeted markers African medicinal orchids SCoT-PCR IRAP-PCR AMOVA 



Polymerase chain reaction


Polymorphic information content


Analysis of molecular variance


Resolving power



Paromik Bhattacharyya thanks the University of KwaZulu-Natal (UKZN), South Africa for support in the form of a Postdoctoral Fellowship.

Supplementary material

12298_2018_523_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 33 kb)


  1. Bhattacharyya P, Kumaria S (2015) Molecular characterization of Dendrobium nobile Lindl., an endangered medicinal orchid, based on randomly amplified polymorphic DNA. Plant Syst Evol 301:201–210CrossRefGoogle Scholar
  2. Bhattacharyya P, Van Staden J (2016) Ansellia africana (Leopard orchid): a medicinal orchid species with untapped reserves of important biomolecules—a mini review. S Afr J Bot 106:181–185CrossRefGoogle Scholar
  3. Bhattacharyya P, Kumaria S, Kumar S, Tandon P (2013) Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl., an endangered medicinal orchid species. Gene 529:21–26CrossRefPubMedGoogle Scholar
  4. Bhattacharyya P, Kumaria S, Tandon P (2015) Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile, an endangered medicinal orchid. Phytochemistry 117:306–316CrossRefPubMedGoogle Scholar
  5. Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach for propagation of medicinally important orchid species. S Afr J Bot 104:232–243CrossRefGoogle Scholar
  6. Bhattacharyya P, Ghosh S, Sen Mandi S, Kumaria S, Tandon P (2017) Genetic variability and association of AFLP markers with some important biochemical traits in Dendrobium thyrsiflorum, a threatened medicinal orchid. S Afr J Bot 109:214–222CrossRefGoogle Scholar
  7. Cabo S, Ferreira L, Carvalho A, Martins-Lopes P, Martín A, Lima-Brito JE (2014) Potential of Start Codon Targeted (SCoT) markers for DNA fingerprinting of newly synthesized tritordeums and their respective parents. J Appl Genet 55:307–312CrossRefPubMedGoogle Scholar
  8. Chen L, Zhao L, Bai Y, Hu R, Si J (2009) Genetic relationship analysis of different provenances of Leonurus japonicus by ISSR marker. China J Chin Mater Med 34:1343–1345Google Scholar
  9. Chinsamy M, Finnie JF, Van Staden J (2011) The ethnobotany of South African medicinal orchids. S Afr J Bot 77:2–9CrossRefGoogle Scholar
  10. Chowdhery HJ (2001) Orchid diversity in North-east India. J Orchid Soc India 15:1–17Google Scholar
  11. Collard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93CrossRefGoogle Scholar
  12. Cunningham AB (1988) An investigation of the herbal medicine trade in Natal/KwaZulu. Institute of Natural Resources, University of Natal, PietermaritzburgGoogle Scholar
  13. Ellstrand NC, Elam DR (1993) Population genetic consequences of small population size: implications for plant conservation. Annu Rev Ecol Syst 24:217–242CrossRefGoogle Scholar
  14. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620CrossRefPubMedGoogle Scholar
  15. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47CrossRefGoogle Scholar
  16. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587PubMedPubMedCentralGoogle Scholar
  17. Falush D, Stephens M, Pritchard JK (2007) Inference of population structure using multilocus genotype data: dominant markers and null alleles. Mol Ecol Notes 7:574–578CrossRefPubMedPubMedCentralGoogle Scholar
  18. Feng S, He R, Yang S, Chen Z, Jiang M, Lu J, Wang H (2015) Start codon targeted (SCoT) and target region amplification polymorphism (TRAP) for evaluating the genetic relationship of Dendrobium species. Gene 567:182–188CrossRefPubMedGoogle Scholar
  19. Gepts P (2002) A comparison between crop domestication, classical plant breeding, and genetic engineering. Crop Sci 42:1780–1790CrossRefGoogle Scholar
  20. Gerstner J (1941) A preliminary check list of Zulu names of plants: with short notes. Bantu Stud 15:277–301CrossRefGoogle Scholar
  21. Graner A, Dehmer KJ, Thiel T, Börner A (2004) IV. Plant genetic resources: benefits and implications of using molecular markers. Evol Role Genebanks Fast-developing F Mol Genet Genet Resour No 11, 26 Aug 2004Google Scholar
  22. Gupta PK, Varshney RK, Prasad M (2002) Molecular markers: principles and methodology. In: Molecular techniques in crop improvement. Springer, pp 9–54Google Scholar
  23. Hamrick JL, Godt MJW (1996) Effects of life history traits on genetic diversity in plant species. Philos Trans R Soc Lond Ser B Biol Sci 351:1291–1298CrossRefGoogle Scholar
  24. Holsinger KE, Mason-Gamer RJ, Whitton J (1999) Genes, denies, and plant conservation. In: Landweber LF, Dobson AP (eds) Genetics and the extinction of species. Princeton University Press, Oxford, pp 23–46Google Scholar
  25. Hulme M (1954) Wild flowers of Natal. Shuter & Shooter, PietermaritzburgGoogle Scholar
  26. Hutchings A (1996) Zulu medicinal plants: an inventory. University of Natal Press, PietermaritzburgGoogle Scholar
  27. Jin WT, Yao SP (2006) Cultivation and appreciation of noble Spring Orchid cultivars. Guangdong Science and Technology Press, Guangzhou, ChinaGoogle Scholar
  28. Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat Protoc 1:2478–2484CrossRefPubMedGoogle Scholar
  29. Kalendar R, Grob T, Regina M, Suoniemi A, Schulman A (1999) IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor Appl Genet 98:704–711CrossRefGoogle Scholar
  30. Lande R (1999) Extinction risks from anthropogenic, ecological and genetic factors. In: Landweber LF, Dobson AP (eds) Genetics and the extinction of species. Princeton University Press, pp 1–22Google Scholar
  31. Landry BS, Li RQ, Cheung WY, Granger RL (1994) Phylogeny analysis of 25 apple rootstocks using RAPD markers and tactical gene tagging. Theor Appl Genet 89:847–852PubMedGoogle Scholar
  32. Li A, Ge S (2006) Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China. Plant Syst Evol 258:251–260CrossRefGoogle Scholar
  33. Manners V, Kumaria S, Tandon P (2013) SPAR methods revealed high genetic diversity within populations and high gene flow of Vanda coerulea Griff ex Lindl (Blue Vanda), an endangered orchid species. Gene 519:91–97CrossRefPubMedGoogle Scholar
  34. Martos F, Johnson SD, Peter CI, Bytebier B (2014) A molecular phylogeny reveals paraphyly of the large genus Eulophia (Orchidaceae): a case for the reinstatement of Orthochilus. Taxon 63:9–23CrossRefGoogle Scholar
  35. Maunder M, Culham A, Alden B, Zizka G, Orliac C, Lobin W, Bordeu A, Ramirez JM, Glissmann-Gough S (2000) Conservation of the Toromiro tree: case study in the management of a plant extinct in the wild. Conserv Biol 14:1341–1350CrossRefGoogle Scholar
  36. Mehra PN, Khosla PK (1973) Cytological studies of Himalayan Rutaceae. Silvae Genet 22:182–188Google Scholar
  37. Miller MP (1998) AMOVA-PREP 1.01: a program for the preparation of AMOVA input files from dominant-markers raw data. Comput Softw Distrib 22:927–934Google Scholar
  38. Milligan BG, Leebens-Mack J, Strand AE (1994) Conservation genetics: beyond the maintenance of marker diversity. Mol Ecol 3:423–435CrossRefGoogle Scholar
  39. Mulpuri S, Muddanuru T, Francis G, Sujatha M, Tarakeswari M, Francis G (2013) Start codon targeted (SCoT) polymorphism in toxic and non-toxic accessions of Jatropha curcas L and development of a codominant SCAR marker. Plant Sci 207:117–127CrossRefPubMedGoogle Scholar
  40. Muñoz M, Warner J, Albertazzi FJ (2010) Genetic diversity analysis of the endangered slipper orchid Phragmipedium longifolium in Costa Rica. Plant Syst Evol 290:217–223CrossRefGoogle Scholar
  41. Nekrutenko A, Makova KD, Baker RJ (2000) Isolation of binary species-specific PCR-based markers and their value for diagnostic applications. Gene 249:47–51CrossRefPubMedGoogle Scholar
  42. Nybom H (2004) Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol Ecol 13:1143–1155CrossRefPubMedGoogle Scholar
  43. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238CrossRefGoogle Scholar
  44. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  45. Rohlf JF (1998) NTSYS pc, numerical taxonomy and multivariate analysis system version 2.0 user guide. SetauketGoogle Scholar
  46. Sharma SK, Kumaria S, Tandon P, Rao SR (2011) Single primer amplification reaction (SPAR) reveals inter-and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae). Gene 483:54–62CrossRefPubMedGoogle Scholar
  47. Shimada T, Hayama H, Haji T, Yamaguchi M, Yoshida M (1999) Genetic diversity of plums characterized by random amplified polymorphic DNA (RAPD) analysis. Euphytica 109:143–147CrossRefGoogle Scholar
  48. Slatkin M (1987) Gene flow and the geographic structure of natural. Science 236:787–792CrossRefPubMedGoogle Scholar
  49. Slatkin M, Barton NH (1989) A comparison of three indirect methods for estimating average levels of gene flow. Evolution (NY) 43:1349–1368CrossRefGoogle Scholar
  50. Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, Senior ML, Mitchell SE, Kresovich S, Ziegle J (1997) An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theor Appl Genet 95:163–173CrossRefGoogle Scholar
  51. Sneller CH, Miles JW, Hoyt JM (1997) Agronomic performance of soybean plant introductions and their genetic similarity to elite lines. Crop Sci 37:1595–1600CrossRefGoogle Scholar
  52. Soorni A, Nazeri V, Fattahi R, Khadivi-Khub A (2013) DNA fingerprinting of Leonurus cardiaca L. germplasm in Iran using amplified fragment length polymorphism and inter-retrotransposon amplified polymorphism. Biochem Syst Ecol 50:438–447CrossRefGoogle Scholar
  53. Swarts ND, Dixon KW (2009a) Terrestrial orchid conservation in the age of extinction. Ann Bot 104:543–556CrossRefPubMedPubMedCentralGoogle Scholar
  54. Swarts ND, Dixon KW (2009b) Perspectives on orchid conservation in botanic gardens. Trends Plant Sci 14:590–598CrossRefPubMedGoogle Scholar
  55. Swarts ND, Sinclair EA, Krauss SL, Dixon KW (2009) Genetic diversity in fragmented populations of the critically endangered spider orchid Caladenia huegelii: implications for conservation. Conserv Genet 10:1199–1208CrossRefGoogle Scholar
  56. Tong-Jian S, Liu-Qin Z, Xin S (1991) A marker-coupled method for site-directed mutagenesis. Gene 103:73–77CrossRefGoogle Scholar
  57. Wang HZ, Wu ZX, Lu JJ, Shi NN, Zhao Y, Zhang ZT, Liu JJ, Zhang ZT, Liu JJ (2009) Molecular diversity and relationships among Cymbidium goeringii cultivars based on inter-simple sequence repeat (ISSR) markers. Genetica 136:391–399CrossRefPubMedGoogle Scholar
  58. Waugh R, McLean K, Flavell AJ, Pearce SR, Kumar A, Thomas BBT, Powell W (1997) Genetic distribution of Bare–1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet MGG 253:687–694CrossRefPubMedGoogle Scholar
  59. WCSP (2016) World Checklist of Selected Plant Families. Facilitated by the Royal Botanic Gardens, Kew. Accessed 20 June 2016
  60. Xiong F, Zhong R, Han Z, Jiang J, He L, Zhuang W, Tang R (2011) Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (Arachis hypogaea L.) genotypes. Mol Biol Rep 38:3487–3494CrossRefPubMedGoogle Scholar
  61. Yeh FC, Yang R-C, Boyle T (1999) PopGene version 1.31: Microsoft window-based freeware for population genetic analysis. University of Alberta, EdmontonGoogle Scholar
  62. Yu Q, Sheng XX, Sheng YF et al (2009) AFLP Analysis of genetic diversity of Leonurus japonicus germplasm resources. Chin Tradit Herb Drugs 40:1296–1299Google Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2018

Authors and Affiliations

  1. 1.Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal, PietermaritzburgScottsvilleSouth Africa

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