Molecular identification and phylogenetic analysis of Chlorella isolates from Indonesia using rbcL gene
DOI:
https://doi.org/10.22302/iribb.jur.mp.v89i1.408Keywords:
green microalgae, molecular identification, phylogenetic tree, rbcLAbstract
Identifying the newly isolated species is crucial to establishing a reliable algal database with successful commercial applications for different biotechnological applications. Morphological identification does not give sufficient description, especially for tiny unicellular microalgae. The rbcL gene encodes the large unit of ribulose-1, 5-bisphosphate carboxylase /oxygenase (Rubisco) has been widely known for barcoding in plants and developed for microalgae molecular identification. In this study, we examined the local strains of green microalgae from Indonesia using the rbcL partial gene sequence to identify the strains. Green microalgae isolates originated from Yogyakarta, Serayu, Gondol, Ancol, Cilegon, and Teluk Jakarta were cultured in f/2 media and harvested for DNA extraction. The DNA extracted was proceeded to PCR using 1AB_rbcL primer pair to amplify the sequences of rbcL gene with target band located at 582 bp, followed by the sequencing of the PCR product was conducted. Molecular identification of local green microalgae isolates was successfully carried out using primers 1AB_rbcL with a genetic similarity of 99% toward identified species in the NCBI database. Among six isolates, TJ, G, S, C, and A isolates were identified as C. pyrenoidosa. Only CP isolate from Yogyakarta identified as C. sorokiniana. Nannochloropsis gaditana rbcL sequence was selected as an outgroup. The phylogenetic analysis indicated that the five isolates of Chlorella belong to one clade and clearly distinguished from C. sorokiniana isolate from Yogyakarta.
Downloads
References
An SS, T Friedl, & E Hegewald (1999). Phylogenetic relationships of Scenedesmus and Scenedesmus-like coccoid green algae as inferred from ITS-2 rDNA sequence comparisons. Plant Biol 1(4), 418–428.
Ashour M, ME Elshobary, R El-Shenody, AW Kamil & AEF Abomohra (2019). Evaluation of a native oleaginous marine microalga Nannochloropsis oceanica for dual use in biodiesel production and aquaculture feed. Biomass Bioenergy 120, 439–447.
Basith A (2015). Peluang gen rbcL sebagai DNA barcode berbasis DNA kloroplas untuk mengungkap keanekaragaman genetik padi beras hitam (Oryza sativa L.) lokal Indonesia. Seminar Nasional XII Pendidikan Biologi FKIP UNS, 938–941.
Budiani A, RA Putranto, H Minarsih, I Riyadi, Sumaryono & B Abbas (2016). Kloning dan karakterisasi daerah promoter gen penyandi ADP glucose pyrophosphorylase dari Metroxylon sagu rendemen patitinggi dan rendah. Menara Perkebunan 84(1), 1–12.
Carneiro C, DM Moura, F Brambach, K Jair, H Bado, KV Krutovsky, H Kreft, SS Tjitrosoedirdjo, IZ Siregar & O Gailing (2019). Integrating DNA barcoding and traditional taxonomy for the identification of Dipterocarps in remnant lowland forests of Sumatra. Plants 8(461), 1–14.
Carpinelli EC, A Telatin, N Vitulo, C Forcato, M D’Angelo, R Schiavon, A Vezzi, GM Giacometti, T Morosinotto & G Valle (2014). Chromosome scale genome assembly and transcriptome profiling of Nannochloropsis gaditana in nitrogen depletion. Mol Plant 7(2), 323–335.
Fawley KP & MW Fawley (2007). Observations on the diversity and ecology of freshwater Nannochloropsis (Eustigmatophyceae), with descriptions of new taxa. Protist 158(3), 325–336.
Fawley MW, I Jameson & KP Fawley (2015). The phylogeny of the genus Nannochloropsis (Monodopsidaceae, Eustigmatophyceae), with descriptions of N. australis sp. Nov. and Microchloropsis gen. Nov. Phycologia 54(5), 545–552.
Fei C, S Zou, T Wang, C Wang, ND Kemuma, M He, SA Amin, & C Wang (2020). A quick method for obtaining high-quality DNA barcodes without DNA extraction in microalgae. J Appl Phycol 32(2), 1165–1175.
Ghosh S & NG Love (2011). Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants. Bioresour Technol 102(3), 3619–3622.
Guillard RR & JH Ryther (1962). Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8(1140), 229–239.
Hadi SIIA, H Santana, PPM Brunale, TG Gomes, MD Oliveira, A Matthiensen, MEC Oliveira, FCP Silva & BSAF Brasil (2016). DNA barcoding green microalgae isolated from neotropical inland waters. PLoS ONE 11(2), 1–18.
Hall JD, K Fučíková, C Lo, LA Lewis & KG Karol (2010). An assessment of proposed DNA barcodes in freshwater green algae. Cryptogamie, Algologie 31(4), 529–555.
Huijbers MME, W Zhang, F Tonin & F Hollmann (2018). Light-Driven enzymatic decarboxylation of fatty acids. Angew Chem Int 57, 13648–13651.
Khan MI, JH Shin & JD Kim (2018). The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb Cell Fact 17(36), 1–21.
Leaché AD (2013). phylogenetic trees made easy: a how-to manual by Barry G. Hall. Q Rev Biol 88(1), 47–48.
Leliaert F, DR Smith, H Moreau, MD Herron, H Verbruggen, CF Delwiche & O De Clerck (2012). Phylogeny and molecular evolution of the green algae. CRC Crit Rev Plant Sci 31(1), 1–46.
Lorenz TC (2012). Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies. J Vis Exp 63, 1–15.
Meiklejohn KA, N Damaso & JM Robertson (2019). Assessment of BOLD and GenBank – Their accuracy and reliability for the identification of biological materials. PLoS ONE 14(6), 1–14.
NanoDrop Inc. (2007). 260/280 and 260/230 Ratios NanoDrop® ND-1000 and ND-8000 8-Sample Spectrophotometers - Technical Support Bulletin T009. 8–9. http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
Pasha F, S Saggu & MF Albalawi (2016). Future perspectives of DNA barcoding in marine zooplanktons and invertebrates. In: S Trivedi, A A Ansari, SK Ghosh & H Rehman (eds.), DNA Barcoding in Marine Perspectives: Assessment and Conservation of Biodiversity. Switzerland, Springer International Publishing p. 169–179.
Pawlowski J, S Audic, S Adl, D Bass, L Belbahri, C Berney, SS Bowser, I Cepicka, J Decelle, M Dunthorn, AM Fiore-Donno, GH Gile, M Holzmann, R Jahn, M Jirků, PJ Keeling, M Kostka, A Kudryavtsev, E Lara, J Lukes, DG Mann, EAD Mitchel, F Nitsche, M Romeralo, GW Saunders, AGB Simpson, AV Smirnov, JL Spouge, RF Stern, T Stoeck, J Zimmermann, D Schindel, & C de Vargas (2012). CBOL protist working group: barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms. PLoS Biol 10(11), 1–5.
Prehadi, A Sembiring, EM Kurniasih, Rahmad, D Arafat, B Subhan & HH Madduppa (2015). DNA barcoding and phylogenetic reconstruction of shark species landed in muncar fisheries landing site in comparison with southern Java fishing port. Biodiversitas 16(1), 55–61.
Rahman DY, S Praharyawan, S Raharjo, F Fadiyah & D Susilaningsih (2019). Morphology and molecular characterization of newly isolated microalgae strain Chlorella volutis LIPI13-WKT066 from Wakatobi Islands and its potential use. Ann Bogor 23(1), 13–19.
Richterich P (1998). Estimation of errors in “raw” DNA sequences: a validation study. Genome Res 8(3), 251–259.
Saputro TB, KI Purwani, D Ermavitalini & AF Saifulloh (2019). Isolation of high lipid content microalgae from wonorejo river, Surabaya, Indonesia and its identification using rbcL marker gene. Biodiversitas 20(5), 1380–1388.
Shi H, W Li & X Xu (2016). Learning the comparing and converting method of sequence phred quality score. In: Proceedings of the 6th International Conference on Management, Education, Information and Control (MEICI 2016). Shenyang, China. p, 260–263.
Suda S, M Atsumi & H Miyashita (2002). Taxonomic characterization of a marine Nannochloropsis species, N. oceanica sp. nov. (Eustigmatophyceae). Phycologia 41(3), 273–279.
Sundari N & N Papuangan (2019). Amplification and analysis of Rbcl gene (Ribulose-1,5-Bisphosphate Carboxylase) of clove in Ternate island. IOP Conf. Series: Earth and Environmental Science, 276, 1–7.
Tamura K & M Nei (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10(3), 512–526.
Wei L, Y Xin, D Wang, X Jing, Q Zhou, X Su, J Jia, K Ning, F Chen, Q Hu & J Xu (2013). Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae. BMC Genomics 14,1–17.
Yanuhar U, NR Caesar & M Musa (2019). Identification of local isolate of microalgae Chlorella vulgaris using Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase large subunit (rbcL) Gene. IOP Conf. Series: Materials Science and Engineering 546, 1–6.
Downloads
Submitted
Accepted
Published
How to Cite
Issue
Section
License
Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
