DNA barcoding and phylogenetic analysis of endemic Astragalus nezaketiae and Vicia alpestris subsp. hypoleuca (Fabaceae): Evidence from nrDNA ITS and cpDNA matK and rbcL sequences


  • Mehmet Y. PAKSOY Munzur University, Department of Medical Services and Techniques, Tunceli Vocational School, 62000 Tunceli (TR)
  • Emre SEVİNDİK Aydın Adnan Menderes University, Faculty of Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydin (TR)
  • İsa BAŞKÖSE Ankara University, Faculty of Science, Department of Biology, 06100 Ankara (TR)




Astragalus, barcoding, ITS, matK, rbcL, Vicia


In this study, we performed DNA barcoding and phylogenetic analysis using one nuclear (ITS) and two chloroplast DNA regions (matK and rbcL) of endemic Astragalus nezaketiae A. Duran & Aytaçand Vicia alpestris Stev. subsp. hypoleuca (Boiss.) Davis taxa in Turkey. PCR reactions were performed using universal primers. Sequences of the PCR products were edited using BioEdit and FinchTV software and contigs were obtained. All contigs were Blasted at NCBI and similarities were analysed. Using the MEGA 6.0 program, maximum likelihood trees were constructed including some sequences retrieved from NCBI. For Astragalus nezaketiae; in the ITS analysis, Astragalus nezaketiae appeared separately from other species, and for matK, Astragalus nezaketiae appeared together with Astragalus cicer L. However, rbcL tree was polytomic. For Vicia alpestris subsp. hypoleuca; in ITS, rbcL and matK results Vicia alpestris subsp. hypoleuca were found together with Vicia cracca L., Vicia benghalensis L. and Vicia villosa Roth species. Analysis of the combined data revealed similar results with all barcode regions for Vicia alpestris subsp. hypoleuca while different phylogenetic results were obtained for Astragalus nezaketiae.


Metrics Loading ...


Antonio-Domingues H, Corrêa AMDS, Queiroz RTD, Bitar NAB (2018). Pollen morphology of some Fabaceae species from Patos de Minas, Minas Gerais State, Brazil. Hoehnea 45:103-114. https://doi.org/10.1590/2236-8906-54/2017

Atasagun B, Aksoy A, Güllü IB, Albayrak S (2021). Reproductive Biology of Astragalus argaeus (Fabaceae), a critically endangered endemic species. Anais da Academia Brasileira de Ciências 93. https://doi.org/10.1590/0001-3765202120201613

Bafeel SO, Arif IA, Bakir MA, Khan HA, Al Farhan AH, Al Homaidan AA, ... Thomas J (2011). Comparative evaluation of PCR success with universal primers of maturase K (matK) and ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (rbcL) for barcoding of some arid plants. Plant Omics 4(4): 195-198.

Bozkurt M, Ertuğrul K, Uysal T (2013). The determination of genetic relationships among some Vicia L. (Vetch) taxa by using ISSR markers. Biological Diversity and Conservation 6(3):135-139.

CBOL Plant Working Group (2009). A DNA barcode for land plants. Proceedings of the National Academy of Sciences 106:12794-12797. https://doi.org/10.1073/pnas.090584510

Dastpak A, Osaloo SK, Maassoumi AA, Safar KN (2018). Molecular phylogeny of Astragalus sect. Ammodendron (Fabaceae) inferred from chloroplast ycf1 gene. Annales Botanici Fennici 55:75-82. https://doi.org/10.5735/085.055.0108

Dizkirici A, Ekici M, Kaya Z (2014). Comparative molecular phylogenetics of Astragalus L. sections from Turkey with New World Astragalus species using nrDNA ITS sequences. Plant Systematics and Evolution 300(1):163-175. https://doi.org/10.1007/s00606-013-0868-9

Dizkirici A, Koroglu Z (2018). Molecular analysis of five Centaurea species found in Van province and determination of taxonomic position of Centaurea depressa. Süleyman Demirel University Journal of Natural and Applied Sciences 22(1):226-231. https://doi.org/10.19113/sdufbed.39508

Dong W, Liu J, Yu J, Wang L, Zhou S (2012). Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding. PloS One 7(4):e35071. https://doi.org/10.1371/journal.pone.0035071

Duran A, Aytaç Z (2005). Astragalus nezaketae (Fabaceae), a new species from Turkey. Annales Botanici Fennici 42:381-385.

Endo Y, Choi B, Kakinuma D, Kenicer G, Zhu XY, Ohashi H (2010). Molecular phylogeny of Vicia sect. Amurense (Leguminosae). The Journal of Japanese Botany 85:337-349.

Felsenstein J (1985). Confidence limits on the phylogenies: an approach using the bootstrap. Evolution 39:783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x

Filiz E, Koç İ (2012). DNA Barcodes in Plants. Afyon Kocatepe University Journal of Science 12(1):53-57.

Hall TA (1999). Bioedit: a user-friendly biological sequence alignment editor and analyses program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95-98.

Han S, Sebastin R, Lee KJ, Wang X, Shin MJ, Kim SH, ... Chung JW. (2021a). Interspecific variation of seed morphological and micro‐morphological traits in the genus Vicia (Fabaceae). Microscopy Research and Technique 84(2):337-357. https://doi.org/10.1002/jemt.23592

Han S, Sebastin R, Wang X, Lee KJ, Cho GT, Hyun DY, Chung JW (2021b). Identification of Vicia species native to South Korea using molecular and morphological characteristics. Frontiers in Plant Science 12:608559. https://doi.org/10.3389/fpls.2021.608559

Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences 270:313-321. https://doi.org/10.1098/rspb.2002.2218

Ilcim A, Behçet L (2016). Astragalus topalanense (Fabaceae), a new species from Turkey. Turkish Journal of Botany 40(1):74-80. https://doi.org/10.3906/bot-1409-22

Inal B, Karaca M (2019). Molecular classification of some plant taxa using matK and trnH-psbA barcode genes. Turkish Journal of Agricultural Research 6(1):87-93. https://doi.org/10.19159/tutad.488296

Ionkova I, Shkondrov A, Krasteva I, Ionkov T (2014). Recent progress in phytochemistry, pharmacology and biotechnology of Astragalus saponins. Phytochemistry Reviews 13(2):343-374. https://doi.org/10.1007/s11101-014-9347-3

Jo IH, Han S, Shim D, Ryu H, Hyun TK, Lee Y, Kim D, So YS, Chung JW (2022). Complete chloroplast genome of the ınverted repeat-lacking species Vicia bungei and development of polymorphic simple sequence repeat markers. Frontiers in Plant Science 13:891783. https://doi.org/10.3389/fpls.2022.891783

Kaplan A, Ertekin AS, Gündüzer E (2021a). Molecular phylogenetic analyses of Vicia L. (Fabaceae) taxa growing in the South-eastern Anatolia region based on chloroplast TrnL sequences. International Journal of Nature and Life Sciences 5(1):11-22. https://doi.org/10.47947/ijnls.840322

Kaplan A, Ertekin AS, Gündüzler E (2021b). Molecular phylogenetic analysis of Vicia L. (Fabaceae) taxa growing in the Southeastern Anatolia region of Turkey: based on internal transcribed spacer (ITS). Turkish Journal of Agriculture - Food Science and Technology 9(10):1831-1839. https://doi.org/10.24925/turjaf.v9i10.1831-1839.4226

Karaman Erkul S, Duman H, Ateş MA (2022). Astragalus oksutdagensis (Fabaceae), a new species from Turkey. Nordic Journal of Botany (3):1-12. https://doi.org/10.1111/njb.03237

Keskin E, Atar HH (2013). DNA barcoding: molecular identification using mitochondrial COI gene. Türk Bilimsel Derlemeler Dergisi 6(2):01-08.

Kim K, Park KR, Lim CE (2020). DNA barcoding of Euphorbiaceae in Korea. Journal of Species Research 9(4):413-426. https://doi.org/10.12651/JSR.2020.9.4.413

Koohdar F, Aram N, Sheidai M (2021). Biosystematics, fingerprinting and DNA barcoding study of the genus Lallemantia based on SCoT and REMAP markers. Caryologia 74(4):77-83. https://doi.org/10.36253/caryologia-1163

Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E (2009). Plant DNA barcodes and a community phylogeny of a tropical forest dynamic plot in Panama. PNAS 106:18621-18626. https://doi.org/10.1073/pnas.090982010

Levin RA, Wagner WL, Hoch PC, Nepokroeff M, Pires JC, Zimmer EA et al. (2003). Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data. American Journal of Botany 90:107-115. https://doi.org/10.3732/ajb.90.1.107

Lin JY, Lin BY, Chang C D, Liao SC, Liu YC, Wu WL, Chang CC (2015). Evaluation of chloroplast DNA markers for distinguishing Phalaenopsis species. Scientia Horticulturae 192:302-310. https://doi.org/10.1016/j.scienta.2015.06.019

Manzione MG, Herrera‐Bravo J, Sharifi‐Rad J, Kregiel D, Sevindik M, Sevindik E, ... Pezzani R (2022). Desmodium adscendens (Sw.) DC.: A magnificent plant with biological and pharmacological properties. Food Frontiers https://doi.org/10.1002/fft2.170

Martin E, Yıldız HK, Kahraman A, Binzat OK, Eroğlu HE (2018). Detailed chromosome measurements and karyotype asymmetry of some Vicia (Fabaceae) taxa from Turkey. Caryologia 71(3):224-232. https://doi.org/10.1080/00087114.2018.1460058

Molares S, Ladio A (2012). The usefulness of edible and medicinal Fabaceae in Argentine and Chilean Patagonia: environmental availability and other sources of supply. Evidence-Based Complementary and Alternative Medicine. https://doi.org/10.1155/2012/901918

Penjor T, Anai T, Nagano Y, Matsumoto R, Yamamoto M (2010). Phylogenetic relationships of Citrus and its relatives based on rbcL gene sequences. Tree Genet Genomes 6(6):931-939. https://doi.org/10.1007/s11295-010-0302-1

Rahman AHMM, Parvin MIA (2014). Study of medicinal uses on Fabaceae family at Rajshahi, Bangladesh. Research in Plant Sciences 2(1):6-8. https://doi:10.12691/plant-2-1-2

Raveendar S, Lee JR, Shim D, Lee GA, Jeon YA, Cho GT, ... Chung JW (2017). Comparative efficacy of four candidate DNA barcode regions for identification of Vicia species. Plant Genetic Resources 15(4):286-295. https://doi.org/10.1017/S1479262115000623

Rios JL, Waterman PG (1997). A review of the pharmacology and toxicology of Astragalus. Phytotherapy Research: An International Journal Devoted to Medical and Scientific Research on Plants and Plant Products 11(6):411-418. https://doi.org/10.1002/(SICI)1099-1573(199709)11:6<411::AID-PTR132>3.0.CO;2-6

Sun WH, Wu FF, Cong L, Jin MY, Wang XG (2022). Assessment of genetic diversity and population structure of the genus Vicia (Vicia L.) using simple sequence repeat markers. Grassland Science 68:205-213. https://doi.org/10.1111/grs.12356

Sun YL, Lee HB, Kim NY, Park WG, Hong SK (2012). Genetic diversity of Kalopanax pictus populations in Korea based on the nrDNA ITS sequence. Journal of Plant Biotechnology 39(1):75-80. https://doi.org/10.5010/JPB.2012.39.1.075

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30(12):2725-2729. https://doi.org/10.1093/molbev/mst197

Techen N, Parveen I, Pan Z, Khan IA (2014). DNA barcoding of medicinal plant material for identification. Current Opinion in Biotechnology 25:103-110. https://doi.org/10.1016/j.copbio.2013.09.010

Tekpinar A, Erkul SK, Aytac Z, Kaya Z (2016). Phylogenetic relationships between Oxytropis DC. and Astragalus L. species native to an Old World diversity center inferred from nuclear ribosomal ITS and plastid matK gene sequences. Turkish Journal of Biology 40(1):250-263. https://doi.org/10.3906/biy-1502-5

Toksoy S, Şeker M, Sağıroğlu M (2015). Phylogenetic and cladistic analyses of the enigmatic genera Bituminaria and Cullen (Fabaceae) in Turkey. Turkish Journal of Botany 39(1):60-69. https://doi:10.3906/bot-1312-4

Wang M, Zhao HX, Wang L, Wang T, Yang RW, Wang XL, ... Zhang L (2013). Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences. Gene 528(2):206-215. https://doi.org/10.1016/j.gene.2013.07.009

White TJ, Bruns T, Lee S, Taylor J (1990). Amplifications and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications. Innis M, Gelfand D, Sninsky J, White T (Eds). Academic Press, San Diego, California, USA, pp 315-322.

Wu FF, Gao Q, Liu F, Wang Z, Wang JL, Wang XG (2020). DNA barcoding evaluation of Vicia (Fabaceae): Comparative efficacy of six universal barcode loci on abundant species. Journal of Systematics and Evolution 58(1):77-88. https://doi.org/10.1111/jse.12474

Wu FF, Sun W, Liu F, Gao Q, Jin M, Liu B, Wang XG (2021). Phylogenetic relationships in Vicia subgenus Vicilla (Fabaceae) based on combined evidence from DNA sequences. Legume Research: An International Journal 44(8).

Yang JB, Yang HQ, Li DZ, Wong KM, Yang YM (2010). Phylogeny of Bambusa and its allies (Poaceae: Bambusoideae) inferred from nuclear GBSSI gene and plastid psbA-trnH, rpl32-trnL and rps16 intron DNA sequences. Taxon 59(4):1102-1110. https://doi.org/10.1002/tax.594010

Yu J, Xue JH, Zhou SL (2011). New universal matK primers for DNA barcoding angiosperms. Journal of Systematics and Evolution 49:176-181. https://doi.org/10.1111/j.1759-6831.2011.00134.x

Zengin G, Guler GO, Aktumsek A, Ceylan R, Picot CMN, Mahomoodally MF (2015). Enzyme inhibitory properties, antioxidant activities, and phytochemical profile of three medicinal plants from Turkey. Advances in Pharmacological Sciences. https://doi.org/10.1155/2015/410675

Zhu S, Liu Q, He J, Nakajima N, Samarakoon SP, Swe S, ... Komatsu K (2021). Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods. Journal of Ethnopharmacology 274:113909. https://doi.org/10.1016/j.jep.2021.113909



How to Cite

PAKSOY, M. Y., SEVİNDİK, E., & BAŞKÖSE, İsa. (2022). DNA barcoding and phylogenetic analysis of endemic Astragalus nezaketiae and Vicia alpestris subsp. hypoleuca (Fabaceae): Evidence from nrDNA ITS and cpDNA matK and rbcL sequences. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 50(3), 12900. https://doi.org/10.15835/nbha50312900



Research Articles
DOI: 10.15835/nbha50312900

Most read articles by the same author(s)