The identification of microalgae based on their morphological features is always challenging due to their similar traits. DNA barcoding is an established technique for obtaining information about genomic sequences to identify different species. However, obtaining DNA from microalgae requires long cultivation times in order to produce enough cells and many steps are needed for the extraction of DNA, requiring significant time and money. In previous studies direct polymerase chain reaction (PCR) has been suggested as a feasible approach for some microalgae, but this protocol still requires pretreatment to deal with the specimens. In the current study, a direct PCR that avoids the DNA extraction step was tested in Chlorella, Scenedesmus, Dunaliella salina and diatoms living in freshwater and seawater. Our optimization involves the following three major aspects: (1) the use of different species, (2) protocol simplification, and (3) microalgae and DNA amount. We also detected DNA levels required for our direct PCR method. After the optimization, the improvement realized was our method’s simplicity, efficiency, and cost-effectiveness, since all the pretreatments were omitted. However, no alterations were observed in the results and equal quality PCR products were obtained compared with conventional PCR. Also, this technique has the same phylogenetic utility as the other PCR methods. To the best of our knowledge, this is the simplest and most direct way to perform PCR in both freshwater and marine microalgae. We also show that the optimized results can be used for routine multi-locus phylogenetic analysis and demonstrate high resolution of DNA barcoding gaps.
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An SM, Choi DH, Lee JH, Lee H, Noh JH (2017) Identification of benthic diatoms isolated from the eastern tidal flats of the Yellow Sea: comparison between morphological and molecular approaches. PLoS One 12:e0179422
Azanza RV, Brosnahan ML, Anderson DM, Hense I, Montresor M (2018) The role of life cycle characteristics in harmful algal bloom dynamics. In: Glibert PM, Berdalet E, Burford MA, Pitcher GC, Zhou M (eds) Global ecology and oceanography of harmful algal blooms. Springer, Cham, pp 133–161
Bellstedt DU, Pirie MD, Visser JC, de Villiers MJ, Gehrke B (2010) A rapid and inexpensive method for the direct PCR amplification of DNA from plants. Am J Bot 97:E65–E68
Bock C, Pröschold T, Krienitz L (2010) Two new Dictyosphaerium-morphotype lineages of the Chlorellaceae (Trebouxiophyceae): Heynigia gen. nov. and Hindakia gen. nov. Eur J Phycol 45:267–277
Cao M, Fu Y, Guo Y, Pan J (2009) Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma 235:107–110
Carroll E, Gallego R, Sewell M, Zeldis J, Ranjard L, Ross H, Tooman L, O’Rorke R, Newcomb R, Constantine R (2019) Multi-locus DNA metabarcoding of zooplankton communities and scat reveal trophic interactions of a generalist predator. Sci Rep 9:281
Chen Y, Bi C, Tong S, Gong Z, Hou H (2019) An improved and reliable method for microalgae direct PCR. J Appl Phycol 31:2411–2421
Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306
Clauson, C., Schärer, O. D., & Niedernhofer, L. (2013). Advances in understanding the complex mechanisms of DNA interstrand cross-link repair. Cold Spring Harbor Perspectives in Biology, 5(10): a012732
Fawley MW, Fawley KP (2004). A simple and rapid technique for the isolation of DNA from microalgae. J Phycol 40:223–225
Fama P, Wysor B, Kooistra WHCF, Zuccarello GC (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. Jf Phycol 38:1040–1050
Ghosh S, Love NG (2011) Application of rbcL based molecular diversity analysis to algae in wastewater treatment plants. Bioresour Technol 102:3619–3622
Ghosh A, Khanra S, Mondal M, Haider G, Tiwari ON, Saini S, Bhowmick TK, Gayen K (2016) Progress toward isolation of strains and genetically engineered strains of microalgae for production of biofuel and other value added chemicals: a review. Energy Convers Manag 113:104–118
Grevelding CG, Kampkotter A, Hollmann M, Schafer U, Kunz W (1996) Direct PCR on fruitflies and blood flukes without prior DNA isolation. Nucleic Acids Res 24:4100–4101
Guillard RR, Ryther JH (1962). Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239
Guillard RR, Bold HC, MacEntee FJ (1975). Four new unicellular chlorophycean algae from mixohaline habitats. Phycologia, 14:13–24
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biol 59:307–321
Hamilton PB, Lefebvre K, Bull R (2015) Single cell PCR amplification of diatoms using fresh and preserved samples. Front Microbiol 6:1084
Hebert PDN, deWaard JR, Landry J-F (2010) DNA barcodes for 1/1000 of the animal kingdom. Biol Lett 6:359–362
Hofmann MA, Brian DA (1991) Sequencing PCR DNA amplified directly from a bacterial colony. Biotechniques 11:30–31
Hollingsworth PM, Forrest LL, Spouge JL, Hajibabaei M, Ratnasingham S, van der Bank M, Chase MW, Cowan RS, Erickson DL, Fazekas AJ, Graham SW, James KE, Kim K-J, Kress WJ, Schneider H, van AlphenStahl J, Barrett SCH, van den Berg C, Bogarin D, Burgess KS, Cameron KM, Carine M, Chacon J, Clark A, Clarkson JJ, Conrad F, Devey DS, Ford CS, Hedderson TAJ, Hollingsworth ML, Husband BC, Kelly LJ, Kesanakurti PR, Kim JS, Kim Y-D, Lahaye R, Lee H-L, Long DG, Madrinan S, Maurin O, Meusnier I, Newmaster SG, Park C-W, Percy DM, Petersen G, Richardson JE, Salazar GA, Savolainen V, Seberg O, Wilkinson MJ, Yi D-K, Little DP, Grp CPW (2009) A DNA barcode for land plants. Proc Natl Acad Sci U S A 106:12794–12797
Jahn MT, Schmidt K, Mock T (2014) A novel cost effective and high-throughput isolation and identification method for marine microalgae. Plant Methods 10:26
John DE, Patterson SS, Paul JH (2007) Phytoplankton-group specific quantitative polymerase chain reaction assays for RuBisCO mRNA transcripts in seawater. Mar Biotechnol 9:747–759
Kimura K, Tomaru Y (2013) A unique method for culturing diatoms on agar plates. Plankton Benthos Res 8:46–48
Kranzfelder P, Ekrem T, Stur E (2016) Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae. Mol Ecol Resour 16:353–363
Li H, Xu H, Zhao C, Sulaiman Y, Wu C (2011) A PCR amplification method without DNA extraction. Electrophoresis 32:394–397
Liu J, Gerken H, Li Y (2014) Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae. J Appl Phycol 26:1719–1726
Maneeruttanarungroj C, Incharoensakdi A (2016) Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551. World J Microbiol Biotechnol 32:99
Menetrez MY (2012) An overview of algae biofuel production and potential environmental impact. Environ Sci Technol 46:7073–7085
Posada D (2008) jModelTest: Phylogenetic model averaging. Mol Biol Evol 25:1253–1256
Radha S, Fathima AA, Iyappan S, Ramya M (2013) Direct colony PCR for rapid identification of varied microalgae from freshwater environment. J Appl Phycol 25:609–613
Ratnasingham S, Hebert PDN (2007) BOLD: the barcode of life data system (www.barcodinglife.org). Mol Ecol Notes 7(3):355–364
Reid BN, Le M, McCord WP, Iverson JB, Georges A, Bergmann T, Amato G, Desalle R, Naro-Maciel E (2011) Comparing and combining distance-based and character-based approaches for barcoding turtles. Mol Ecol Resour 11:956–967
Rochlin I, Santoriello MP, Mayer RT, Campbell SR (2007) Improved high-throughput method for molecular identification of Culex mosquitoes. J Am Mosq Control Assoc 23:488–492
Ronquist F, Huelsenbeck JP.(2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574
Rambaut, A. (2009). FigTree v1. 3.1. http://tree. bio. ed. ac. uk/software/figtree/.
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171–205
Sun X, Wu X, Li X, Pei L (2009) Molecular identification of Chlorella strains based on sequence analysis of nuclear rDNA ITS and chloroplast rbcL gene. J Fish China 33: 565–571
Tamura K, Stecher G, Peterson D, Filipsk, A, Kumar S (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729
Verma D, Daniell H (2007) Chloroplast vector systems for biotechnology applications. Plant Physiol 145:1129–1143
Vidal R, Meneses I, Smith M (2002) Enhanced DNA extraction and PCR amplification of SSU ribosomal genes from crustose coralline algae. J Appl Phycol 14:223–227
Wan M, Rosenberg JN, Faruq J, Betenbaugh MJ, Xia J (2011) An improved colony PCR procedure for genetic screening of Chlorella and related microalgae. Biotechnol Lett 33:1615–1619
Wong WH, Tay YC, Puniamoorthy J, Balke M, Cranston PS, Meier R (2014) “Direct PCR” optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction. Mol Ecol Resour 14:1271–1280
Woodman ME, Savage CR, Arnold WK, Stevenson B (2016) Direct PCR of intact bacteria (colony PCR). Curr Protoc Microbiol 42:A. 3D. 1–A. 3D. 7
Wuyts J, De Rijk P, Van de Peer Y, Pison G, Rousseeuw P, De Wachter R (2000) Comparative analysis of more than 3000 sequences reveals the existence of two pseudoknots in area V4 of eukaryotic small subunit ribosomal RNA. Nucleic Acids Res 28:4698–4708
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, Whire TJ (eds.). PCR protocols: a guide to methods and applications. Academic Press, London pp 315-322
Zhan J, Zhang Q, Qin M, Hong Y (2016) Selection and characterization of eight freshwater green algae strains for synchronous water purification and lipid production. Front Environ Sci Eng 10:548–558
Zou S, Fei C, Wang C, Gao Z, Bao Y, He M, Wang C (2016a) How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae). Sci Rep 6:36822
Zou SM, Fei C, Song JM, Bao YC, He ML, Wang CH (2016b) Combining and comparing coalescent, distance and character-based approaches for barcoding microalgaes: a test with Chlorella-like species (Chlorophyta). PLoS One 11:e0153833
The financial support from the National Key R&D Program of China (NO. 2018YFD0901605), the National Natural Science Foundation of China (NO. 31770436), and the Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization and Co-Innovation Center for Jiangsu Marine Bio-Industry Technology is acknowledged. This work was partially supported by a NYU Abu Dhabi grant and by the NYU Abu Dhabi Core Technology Facilities. We thank Enago (http://www.enago.cn/) for their assistance in manuscript editing.
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Fei, C., Zou, S., Wang, T. et al. A quick method for obtaining high-quality DNA barcodes without DNA extraction in microalgae. J Appl Phycol (2020). https://doi.org/10.1007/s10811-019-01926-2
- DNA barcodes
- Direct PCR
- Multiple gene loci