DNA barcoding in molecular identification and phylogenetic relationship of beneficial wild Balinese red algae, Bulung sangu (Gracilaria sp.)

I Gede Putu Wirawan, Maria Malida Vernandes Sasadara , I Nyoman Wijaya, Anak Agung Keswari Krinandika

I Gede Putu Wirawan
Faculty of Agriculture, Universitas Udayana, Denpasar, Bali, Indonesia

Maria Malida Vernandes Sasadara
Department of Natural Medicine, Faculty of Pharmacy, Universitas Mahasaraswati, Denpasar, Bali, Indonesia ; Department of Natural Medicine, Faculty of Pharmacy, Universitas Mahasaraswati, Denpasar, Bali, Indonesia. Email: mmvsasadara@gmail.com

I Nyoman Wijaya
Faculty of Agriculture, Udayana University, Denpasar, Bali, Indonesia

Anak Agung Keswari Krinandika
Faculty of Agriculture, Udayana University, Denpasar, Bali, Indonesia
Online First: April 01, 2021 | Cite this Article
Wirawan, I., Sasadara, M., Wijaya, I., Krinandika, A. 2021. DNA barcoding in molecular identification and phylogenetic relationship of beneficial wild Balinese red algae, Bulung sangu (Gracilaria sp.). Bali Medical Journal 10(1): 82-88. DOI:10.15562/bmj.v10i1.2093

Background: Bulung sangu (Gracilaria sp). is wildly widespread Rhodophyta in Bali and usually consumed as vegetable. Bulung sangu is reported for its bioactive compound scientifically proven as antioxidant, anti-inflammatory, and anti-hypercholesterolemia. Likewise, Bulung sangu as Rhodophyta is a potential source for food, fertilizer, cosmetic and pharmaceutical industry. Bulung sangu production is highly fluctuating and unable to meet its demands. Proper and correct cultivation methods based on molecular information are expected to increase the availability of Bulung sangu in Bali. Complete taxonomy is required to design the proper cultivation method. Meanwhile, Bulung sangu taxonomy is limited to the genus level. More information on Bulung sangu strain is needed, either does its relatedness.

Methods: Fresh Bulung sangu was collected from Serangan coastal area, Bali. DNA extraction was applied followed by PCR amplification using six combinations of COI primer sequence. DNA sequences obtained was evaluated to determine pairwise distance, percent of similarity and phylogenetic relationship compared to Gracilaria species registered in GenBank.

Results: PCR amplification produced 730 base pairs amplicons. Genetic distance and percentage of similarity obtained exhibit relatedness to Gracilaria gracilis with 0.487 of pairwise distance and 49.04% of similarity. The phylogenetic tree produced seven clades in which Bulung sangu and Gracilaria gracilis were in the same clade.

Conclusion: Bulung sangu showed closest relatedness to Gracilaria gracilis.


Yong WTLY, Chin GJWL, Rodrigues KF. Genetic Identification and Mass Propagation of Economically Important Seaweeds. In: Picco S, Villegas L, Tonelli F, Merlo M, Rigau J, Diaz D, et al., editors. Algae - Organisms for Imminent Biotechnology. London, United Kingdom: Intech Open; 2016. p. 278–305.

Sasadara MMV, Wirawan IGP, Sritamin M, Suada IK, Adiartayasa. Antioxidant Activity of the Topical Preparation of Bulung. Int J Biosci Biotechnol. 2020;7(2):83–90.

Kolanjinathan K, Ganesh P, Saranraj P. Pharmacological Importance of Seaweeds: A Review. World J Fish Mar Sci. 2014;6(1):1–15.

Di T, Chen G, Sun Y, Ou S, Zeng X, Ye H. Antioxidant and immunostimulating activities in vitro of sulfated polysaccharides isolated from Gracilaria rubra. J Funct Foods. 2017;28:64–75.

de Almeida CLF, Falcão H de S, Lima GR d. M, Montenegro C de A, Lira NS, de Athayde-Filho PF, et al. Bioactivities from marine algae of the genus Gracilaria. Int J Mol Sci. 2011;12(7):4550–73.

Fang J, Zhu X, Wang C, Shangguan L. Applications of DNA Technologies in Agriculture. Curr Genomics. 2016;17:379–86.

Sulistyawati P, Widyatmoko A. Genetic relationship of Shorea gysbertsiana with other three Shorea species producing Tengkawang based on RAPD marker. J Pemuliaan Tanam Hutan. 2018;12(2):85–94.

Konzen ER. Towards conservation strategies for forest tree endangered species: the meaning of population genetic statistics. Adv For Sci Rev Adv For Sci Cuiabá. 2014;(11):45–51.

Saunders GW. Applying DNA barcoding to red macroalgae: A preliminary appraisal holds promise for future applications. Philos Trans R Soc B Biol Sci. 2005;360(1462):1879–88.

Letchuman S. Short Introduction of DNA Barcoding. Int J Res. 2018;5(4):673–86.

Imtiaz A, Mohd Nor SA, Md. Naim D. Progress and potential of DNA barcoding for species identification of fish species. Biodiversitas. 2017;18(4):1394–405.

Iwatsuki Y, Tanaka F, Allen G. Lutjanus xanthopinnis, a new species of snapper (Pisces: Lutjanidae) from the Indo-west Pacific, with a redescription of Lutjanus madras (Valenciennes 1831). J Ocean Sci Found. 2015;17:22–42.

Zamudio KR, Bell RC, Mason NA. Phenotypes in phylogeography: Species’ traits, environmental variation, and vertebrate diversification. Proc Natl Acad Sci U S A. 2016;113(29):8041–8.

Higashi R, Sakuma K, Chiba SN, Suzuki N, Chow S, Semba Y, et al. Species and lineage identification for yellowfin Thunnus albacares and bigeye T. obesus tunas using two independent multiplex PCR assays. Fish Sci. 2016;82(6):897–904.

Chiu TH, Kuo CW, Lin HC, Huang DS, Wu PL. Genetic diversity of ivory shell (Babylonia areolata) in Taiwan and identification of species using DNA-based assays. Food Control. 2015;48:108–16.

Damasceno JS, Siccha-Ramirez R, Oliveira C, Mendonça FF, Lima AC, Machado LF, et al. Molecular identification of Atlantic goliath grouper epinephelus itajara (Lichtenstein, 1822) (Perciformes: Epinephelidae) and related commercial species applying multiplex PCR. Neotrop Ichthyol. 2016;14(3).

Bączkiewicz A, Szczecińska M, Sawicki J, Stebel A, Buczkowska K. DNA barcoding, ecology and geography of the cryptic species of Aneura pinguis and their relationships with Aneura maxima and Aneura mirabilis (Metzgeriales, Marchantiophyta). PLoS One. 2017;12(12):1–21.

McHugh DJ. A Guide to the Seaweed Industry. A Guide to the Seaweed Industry. Rome: Food and Agriculture Organization; 2003.

Costa ES, Plastino EM, Petti R, Oliveira EC, Oliveira MC. The Gracilariaceae Germplasm Bank of the University of São Paulo, Brazil-a DNA barcoding approach. J Appl Phycol. 2012;24(6):1643–53.

Steentoft M, Irvlne LM, Farnham WF. Two terete species of Gracilaria and Gracilariopsis ( Gracilariales , Rhodophyta ) in Britain. Phycologia. 1995;34(2):113–27.

Capillo G, Savoca S, Costa R, Sanfilippo M, Rizzo C, Giudice A Lo, et al. New insights into the culture method and antibacterial potential of gracilaria gracilis. Mar Drugs. 2018;16(12):1–21.

Parsa M, Jalilzadeh H, Pazoki M, Ghasemzadeh R, Abduli M. Hydrothermal Liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production. Bioresour Technol. 2017;

Heffernan N, Smyth TJ, Fitzgerald RJ, Brunton NP. Phenolic content and antioxidant activity of fractions obtained from selected Irish macroalgae species ( Laminaria digitata , Fucus serratus , Gracilaria gracilis and Codium fragile ). J Appl Phycol. 2014;27(1):519-30.

Phang S, Yeong H, Ganzon-fortes ET, Lewmanomont K, Hau LN, Gerung GS, et al. Marine algae of the South China Sea bordered by Indonesia , Malaysia ,. Raffles Bull Zool. 2016;7600(34):13–59.

Rasyid A, Ardiansyah A, Pangestuti R. Nutrient Composition of Dried Seaweed Gracilaria gracilis. Indones J Mar Sci. 2019;24(1):1–6.

Istiqomawati, Kusdarwati R. Technique of seaweeds culture (Gracilaria verrucosa) at brackish water aqua culture development center situbondo of east java. J Ilm Perikan dan Kelaut. 2010;2(1):77–85.

Francavilla M, Franchi M, Monteleone M, Caroppo C. The red seaweed gracilaria gracilis as a multi products source. Mar Drugs. 2013;11(10):3754–76.

Natarajan S, Shanmugiahthevar KP. Cholinesterase inhibitors from Sargassum and Gracilaria gracilis : Seaweeds inhabiting South Indian coastal areas (Hare Island , Gulf of Mannar ). Nat Prod Res Former Nat Prod Lett. 2009;23(4):355–69.

Tuney I, Çadirci BH, Ünal D, Sukatar A. Antimicrobial Activities of the Extracts of Marine Algae from the Coast of Urla (Izmir,Turkey). Turk J Biol. 2006;30:171–5.

Ebrahimzadeh MA, Khalili M, Dehpour AA. Antioxidant activity of ethyl acetate and methanolic extracts of two marine algae , Nannochloropsis oculata and Gracilaria gracilis – an in vitro assay. Brazilian J Pharm Sci. 2017;54(1).

Park G, Jin W, Jang S, Kook J, Choi JA, Park GC, et al. Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis. Microbiol Immunol. 2015;59:285–98.

Hebert PDN, Cywinska A, Ball SL, DeWaard JR. Biological identifications through DNA barcodes. Proc R Soc B Biol Sci. 2003;270(1512):313–21.

Presting GG. Identification of conserved regions in the plastid genome : implications for DNA barcoding and biological function. Can H Bot. 2006;84:1434–43.

Kogame K, Uwai S, Anderson RJ, Choi H, Bolton JJ. South African Journal of Botany DNA barcoding of South African geniculate coralline red algae ( Corallinales , Rhodophyta ). South African J Bot. 2016;

Siddiqui ZH, Zahid A, Fundo UDP, Hakeem KR, Ilah A. DNA Barcoding and Molecular Phylogeny. DNA Barcoding Mol Phylogeny. 2018;

Peters AF, Couceiro L, Tsiamis K, Frithjof C, Valero M, Cryptogamie S. Barcoding of Cryptic Stages of Marine Brown Algae Isolated from Incubated Substratum Reveals High Diversity in Barcoding of cryptic stages of marine brown algae isolated from incubated substratum reveals high diversity in Acinetosporaceae ( Ectocarpales ,. Cryptogam Algol. 2015;36(1):3–29.

Bartolo AG, Zammit G, Peters AF. The current state of DNA barcoding of macroalgae in the Mediterranean Sea : presently lacking but urgently required. Bot Mar. 2020;63(3):253–72.

Hadi SIIA, Santana H, Brunale PPM, Gomes TG, Oliveira MD, Matthiensen A, et al. DNA barcoding green microalgae isolated from neotropical inland waters. PLoS One. 2016;11(2):1–18.

Clarkston BE, Saunders GW. A comparison of two DNA barcode markers for species discrimination in the red algal family Kallymeniaceae (Gigartinales, Florideophyceae), with a description of Euthora timburtonii sp.nov. Botany. 2010;88(2):119–31.

Freshwater DW, Tudor K, O’shaughnessy K, Wysor B. DNA barcoding in the red algal order Gelidiales: Comparison of COI with rbcL and verification of the “Barcoding gap.” Cryptogam Algol. 2010;31(4):435–49.

Saunders GW, Moore TE. Refinements for the amplification and sequencing of red algal DNA barcode and RedToL phylogenetic markers: A summary of current primers, profiles and strategies. Algae. 2013;28(1):31–43.

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