Skip to main navigation menu Skip to main content Skip to site footer
Plant Science Today (PST)

Research Articles

Vol. 12 No. 3 (2025)

In silico phylogenetic analysis reveals nuclear ribosomal DNA as the most effective marker for DNA barcoding of Cucumis L. species

DOI
https://doi.org/10.14719/pst.6289
Submitted
23 November 2024
Published
14-07-2025 — Updated on 24-07-2025
Versions

Abstract

Cucumis L. species represent a globally important crop genus with high economic value and genetic diversity, yet their wild relatives remain understudied and potentially threatened by habitat loss and genetic erosion. This knowledge gap hinders effective conservation strategies and limits the utilization of wild genetic resources for crop improvement. A deeper understanding of Cucumis diversity and evolution is especially critical for conserving wild relatives of cultivated species, which may harbor valuable traits for climate resilience and disease resistance. This study investigated the effectiveness of 3 DNA barcode markers (matK, rbcL and nrDNA) for phylogenetic analysis across 38 Cucumis species, addressing a key methodological gap in Cucurbitaceae systematics with direct applications in germplasm conservation and crop improvement programmes. Sequences were retrieved from NCBI and analyzed using MEGA 11, DnaSP v6 and PopArt. The hypothesis that nuclear ribosomal DNA (nrDNA) would outperform plastid markers (matK and then rbcL) in resolving species relationships was validated. The nrDNA displayed the highest genetic distance and clearest clade separation of intraspecific variation, making it particularly valuable for: (i) accurate identification of wild Cucumis germplasm in conservation efforts, (ii) precise selection of genetic resources for breeding programmes and (iii) resolution of taxonomic uncertainties in agricultural and ecological studies. matK provided moderate resolution, while rbcL performed poorly at the intraspecific level. These findings provide a robust molecular toolkit for biodiversity conservation and sustainable agricultural development, enabling more effective utilization and protection of Cucumis genetic resources worldwide.

References

  1. 1. Pichot C, Djari A, Tran J, Verdenaud M, Marande W, Huneau C et al. Cantaloupe melon genome reveals 3D chromatin features and structural relationship with the ancestral Cucurbitaceae karyotype. iScience. 2021; 25:103696. https://doi.org/10.1016/j.isci.2021.103696
  2. 2. Christenhusz MJM, Byng JW. The number of known plant species in the world and its annual increase. Phytotaxa. 2016;261:201–17. https://doi.org/10.11646/phytotaxa.261.3.1
  3. 3. Ghebretinsae AG, Thulin M, Barber JC. Relationships of cucumbers and melons unraveled: molecular phylogenetics of Cucumis and related genera (Benincaseae, Cucurbitaceae). Am J Bot. 2007;94(7):1256–66. https://doi.org/10.3732/ajb.94.7.1256
  4. 4. Renner SS, Schaefer H, Kocyan A. Phylogenetics of Cucumis (Cucurbitaceae): Cucumber (C. sativus) belongs in an Asian/Australian clade far from melon (C. melo). Evol Biol. 2007;7:58. https://doi.org/10.1186/1471-2148-7-58
  5. 5. Rolnik A, Olas B. Vegetables from the Cucurbitaceae family and their products: positive effect on human health. Nutr. 2020;78:110788. https://doi.org/10.1016/j.nut.2020.110788
  6. 6. Martinez C, Jamilena M. To be a male or a female flower, a question of ethylene in cucurbits. Curr Opin Plant Biol. 2021;59:101981. https://doi.org/10.1016/j.pbi.2020.101981
  7. 7. Grumet R, McCreight JD, McGregor C, Weng Y, Mazourek M, Reitsma K et al. Genetic resources and vulnerabilities of major Cucurbit crops. Genes. 2021;12:1222. https://doi.org/10.3390/genes12081222
  8. 8. Yusuf AF, Wibowo WA, Subiastuti AS, Daryono BS. Morphological studies of stability and identity of melon (Cucumis melo L.) 'Hikapel' and comparative cultivars. In: AIP Conference Proceedings. 2020;2260(1). https://doi.org/10.1063/5.0017606
  9. 9. Yusuf AF, Daryono BS. Studies of genetic and morphological characteristics of Indonesian melon (Cucumis melo L. 'Hikapel') germplasm. Int J Adv Sci Eng Info Techno. 2021;11:2023–30. https://doi.org/10.18517/ijaseit.11.5.14047
  10. 10. Jin ZT, Hodel RGJ, Ma DK, Wang H, Liu GN, Ren C et al. Nightmare or delight: taxonomic circumscription meets reticulate evolution in the phylogenomic era. Mol Phylogenet Evol. 2023;189:107914. https://doi.org/10.1016/j.ympev.2023.107914
  11. 11. Hinchliff CE, Smith SA, Allman JF, Cranston KA, Hillis DM. Synthesis of phylogeny and taxonomy into a comprehensive tree of life. Proc Natl Acad Sci. 2015;112:12764–69. https://doi.org/10.1073/pnas.142304111
  12. 12. Chung SM, Staub JE, Chen JF. Molecular phylogeny of Cucumis species as revealed by consensus chloroplast SSR marker length and sequence variation. Genome. 2006;49:219–29. https://doi.org/10.1139/g05-101
  13. 13. Ling J, Xie X, Gu X, Zhao J, Ping X, Li Y et al. High-quality chromosome-level genomes of Cucumis metuliferus and Cucumis melo provide insight into Cucumis genome evolution. Plant J. 2021;107(1):136–48. https://doi.org/10.1111/tpj.15279
  14. 14. Antil S, Abraham JS, Sripoorna S, Maurya S, Dagar J, Makhija S et al. DNA barcoding, an effective tool for species identification: A review. Mol Biol Rep. 2023;50:761–75. https://doi.org/10.1007/s11033-022-08015-7
  15. 15. Letsiou S, Madesis P, Vasdekis E, Montemurro C, Grigoriou ME, Skavdis G et al. DNA barcoding as a plant identification method. Appl Sci. 2024;14(4):1415. https://doi.org/10.3390/app14041415
  16. 16. Mathew D. DNA barcoding and its applications in horticultural crops. In: Lim CT, Goh JCH, editors. Biotechnology in Horticulture: Methods and Applications; India. Dordrecht: New India Publishing Agency; 2013. p. 25–50. https://doi.org/10.5281/zenodo.836319
  17. 17. Kress WJ. Plant DNA barcodes: applications today and in the future. J Syst Evol. 2017;55:291–307. https://doi.org/10.1111/jse.12254
  18. 18. Saidon NA, Wagiran A, Samad AFA, Mohd Salleh F, Mohamed F, Jani J et al. DNA barcoding, phylogenetic analysis and secondary structure predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana. Genes. 2023;14(3):697. https://doi.org/10.3390/genes14030697
  19. 19. Linh NN, Trung LQ, Binh NQ, Ha LTT, Hang PLB, Hanh NP et al. DNA barcoding and phylogenetic analysis of the species in the genus Alpinia. Int J Agric Biol. 2023;29:227–34. https://doi.org/10.17957/IJAB/15.2024
  20. 20. Prasetya E, Lazuardi F, Harahap F, Rachmawati Y, Yusuf Y, Al Idrus SI et al. Region of nuclear ribosomal DNA (ITS2) and chloroplast DNA (rbcL and trnL-F) as a suitable DNA barcode for identification of Zingiber loerzingii Valeton from North Sumatera, Indonesia. J Trop Biodivers Biotech. 2023;08:1–12. https://doi.org/10.22146/jtbb.76956
  21. 21. Kiran KM, Sandeep BV. Phylogenetic study of mangrove associate grass Myriostachya wightiana (Nees ex Steud.) Hook. f. using rbcL gene sequence. Plant Sci Today. 2021;8:590–95. https://doi.org/10.14719/pst.2021.8.3.1133
  22. 22. Ho VT, Tran TKP, Vu TTT, Ho VT, Widiarsih S. Comparison of matK and rbcL DNA barcodes for genetic classification of jewel orchid accessions in Vietnam. J Genet Eng Biotechnol. 2021;19(1):93. https://doi.org/10.1186/s43141-021-00188-1
  23. 23. Duan H, Wang W, Zeng Y, Guo M, Zhou Y. The screening and identification of DNA barcode sequences for Rehmannia. Sci Rep. 2019;9:17295. https://doi.org/10.1038/s41598-019-53752-8
  24. 24. Kang Y, Deng Z, Zang R, Long W. DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Sci Rep. 2017;7:12564. https://doi.org/10.1038/s41598-017-13057-0
  25. 25. Talavera G, Lukhtanov V, Pierce NE, Vila R. DNA barcodes combined with multilocus data of representative taxa can generate reliable higher-level phylogenies. Syst Biol. 2022;71:382–95. https://doi.org/10.1093/sysbio/syab038
  26. 26. Retnaningati D. Intraspecies phylogenetic relationship of Cucumis melo L. based on DNA barcode gen matK. Biota. 2017;2:62–67. https://doi.org/10.24002/biota.v2i2.1658
  27. 27. Ho VT, Nguyen MP. An in silico approach for evaluation of rbcL and matK loci for DNA barcoding of Cucurbitaceae family. Biodiversitas. 2020;21:3879–3885. https://doi.org/10.13057/biodiv/d210858
  28. 28. Sholiha FU, Yuniastuti E, Hartati S. The matK gene analysis in five types of matoa (Pometia pinnata J.R. Forst. & G. Forst) based on the pericarp colour. J Appl Biol Biotechnol. 2024;12(3):54–58. https://doi.org/10.7324/JABB.2024.167495
  29. 29. Wu CT, Hsieh CC, Lin WC, Tang CY, Yang CH, Huang YC, Ko YJ. Internal transcribed spacer sequence-based identification and phylogenic relationship of I-Tiao-Gung originating from Flemingia and Glycine (Leguminosae) in Taiwan. J Food Drug Anal. 2013;21(4):356–62. https://doi.org/10.1016/j.jfda.2013.08.002
  30. 30. Omonhinmin CA, Olomukoro EE, Onuselogu CC, Popoola JO, Oyejide SO. Intra-specific genetic variability dataset on rbcL gene in Moringa oleifera Lam. (Moringaceae) in Nigeria. Data in Brief. 2023;50:109399. https://doi.org/10.1016/j.dib.2023.109399
  31. 31. Li Q, Li H, Huang Wu, Xu Y, Zhou Q, Wang S, Ruan J, Huang S, Zhang Z. A chromosome-scale genome assembly of cucumber (Cucumis sativus L.). GigaScience. 2019;8(6):giz072. https://doi.org/10.1093/gigascience/giz072
  32. 32. Johnson MS, Venkataram S, Kryazhimskiy S. Best practices in designing, sequencing and identifying random DNA barcodes. J Mol Evol. 2023;91:263–80. https://doi.org/10.1007/s00239-022-10083-z
  33. 33. Qin X, Zhang Z, Lou Q, Xia L, Li J, Li M, et al. Chromosome-scale genome assembly of Cucumis hystrix– A wild species interspecifically cross-compatible with cultivated cucumber. Hortic Res. 2021;8:40. https://doi.org/10.1038/s41438-021-00475-5
  34. 34. Hu J, Yao J, Lu J, Liu W, Zhao Z, Li Y, et al. The complete chloroplast genome sequences of nine melon varieties (Cucumis melo L.): lights into comparative analysis and phylogenetic relationships. Front Genet. 2024;15:1417266. https://doi.org/10.3389/fgene.2024.1417266
  35. 35. Pere K, Mburu K, Muge EK, Wagacha JM, Nyaboga EN. Molecular discrimination and phylogenetic relationships of Physalis species based on ITS2 and rbcL DNA barcode sequence. Crops. 2023;3:302–19. https://doi.org/10.3390/crops3040027
  36. 36. Carlin JL. Mutations are the raw materials of evolution. Nature Education Knowledge. 2011;3:10.
  37. 37. Mendez-Harclerode FM, Strauss RE, Fulhorst CF, Milazzo ML, Ruthven DC, Bradley RD. Molecular evidence for high levels of intrapopulation genetic diversity in woodrats (Neotoma micropus). J Mammal. 2007;88:360–70. https://doi.org/10.1644/05-MAMM-A-377R1.1
  38. 38. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol Biol and Evol. 2011;28(10):2731–39. https://doi.org/10.1093/molbev/msr121
  39. 39. Liu H, Ye H, Wang J, Chen S, Li M, Wang G, et al. Genome-wide identification and characterization of YABBY gene family in Juglans regia and Juglans mandshurica. Agron. 2022;12:1914. https://doi.org/10.3390/agronomy12081914
  40. 40. Wang J, Yuan M, Feng Y, Zhang Y, Bao S, Hao Y, et al. A common whole-genome paleotetraploidization in Cucurbitales. Plant Physiol. 2022;190(4):2430–48. https://doi.org/10.1093/plphys/kiac410
  41. 41. Endl J, Achigan-Dako EG, Pandey AK, Monforte AJ, Pico B, Schaefer H. Repeated domestication of melon (Cucumis melo) in Africa and Asia and a new close relative from India. Am J Bot. 2018;105(10):1662–71. https://doi.org/10.1002/ajb2.1172
  42. 42. Sebastian P, Schaefer H, Telford IRH, Renner SS. Cucumber (Cucumis sativus) and melon (C. melo) have numerous wild relatives in Asia and Australia and the sister species of melon is from Australia. Proc Natl Acad Sci USA. 2010;107(32):14269–73. https://doi.org/10.1073/pnas.1005338107
  43. 43. Ramesh GA, Mathew D, John KJ, Ravisankar V. Chloroplast gene matK holds the barcodes for identification of Momordica (Cucurbitaceae) species from Indian subcontinent. Hortic Plant J 2022;8:89–98. https://doi.org/10.1016/j.hpj.2021.04.001
  44. 44. Tabita FR, Hanson TE, Satagopan S, Witte BH, Kreel NE. Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philos Trans R Soc B: Biol Sci. 2008;363(1504):2629–40. https://doi.org/10.1098/rstb.2008.0023
  45. 45. Reddy BU. Cladistic analyses of a few members of Cucurbitaceae using rbcL nucleotide and amino acid sequences. Int J Bioinform Res. 2009;1:58–64. https://doi.org/10.9735/0975-3087.1.2.58-64
  46. 46. Yong WTL, Mustafa AA, Derise MR, Rodrigues KF. DNA barcoding using chloroplast matK and rbcL regions for the identification of bamboo species in Sabah. Adv Bamboo Sci. 2024;7:100073. https://doi.org/10.1016/j.bamboo.2024.100073
  47. 47. Liu ZF, Ma H, Zhang XY, Ci XQ, Li L, Hu JL, et al. Do taxon-specific DNA barcodes improve species discrimination relative to universal barcodes in Lauraceae? Bot J Linn Soc. 2022;199:741–53. https://doi.org/10.1093/botlinnean/boab089
  48. 48. Almontero CC, Lalusin AG, Diaz MGQ, Ocampo ETM, Gentallan RP Jr, Rojas SM. DNA barcoding of 11 endemic Vanda species of the Philippines based on rbcL, matK and ITS sequences. Philipp J Sci. 2024;153(5):1783–800.
  49. 49. He S, Xu B, Chen S, Li G, Zhang J, Xu J, et al. Sequence characteristics, genetic diversity and phylogenetic analysis of the Cucurbita ficifolia (Cucurbitaceae) chloroplasts genome. Genom. 2024;25:384. https://doi.org/10.1186/s12864-024-10278-2

Downloads

Download data is not yet available.