Tetraselmis is a genus of quadriflagellated single-celled green algae belonging to the Phylum Chlorophyta, commonly used in aquaculture with very promising biotechnological potential. The varied morphological characteristics, in some cases, have led to confusion in taxonomic identification. To solve this problem, new techniques based on molecular markers and restriction enzymes can ensure the identification of microalgae without sequencing. This study aimed to compare in silico modeling with an experimental restriction pattern based on the 18S rDNA gene for the identification of a microalgae strain. The strain grew in a culture medium, based on organic fertilizer. Theoretical analyses allowed the design of three primers based on the alignment of eight sequences obtained from NCBI, applying the Geneious Prime® 2019 and V1.3 and Oligo Calculator version 3.2. programs. The in silico restriction patterns was obtained with the NEBcutter v2.0 program. Experimental analyses began with the extraction of the DNA using the TENS protocol, then PCR amplification using PM-016F/PM-016R and PM-001F/PM-016R primers of 18S rDNA and finally the product was digested with BbvCI and Eco53kI; BstUI, RsaI and MspI enzymes. The DNA concentration extraction reached 3200 ng µl-1 and a purity of 2.0. The PCR amplified two products: 950 bp and 1400 bp, which brought us closer to identifying the microalgae. The in silico modeling and experimental restriction patterns showed similar fragments. In this way, the efficient response of restriction enzymes was demonstrated by confirming that the PM013 strain corresponds to the Tetraselmis genus. This method can be considered as a fast and safe alternative to identify wild microalgae in a basic molecular biology laboratory.
| Published in | International Journal of Genetics and Genomics (Volume 8, Issue 3) |
| DOI | 10.11648/j.ijgg.20200803.14 |
| Page(s) | 114-119 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2024. Published by Science Publishing Group |
Tetraselmis, Restriction Pattern, 18S rDNA, Molecular Identification, in silico Modeling
| [1] | Macías, P., Coello, D., & García, D. (2018). Floración de Tetraselmis sp., en la zona costera frente a las Provincias de Manabí y Santa Elena (Abril 2018). Guayaquil: Instituto Nacional de Pesca. |
| [2] | Úbeda, P., Chileh, T., Dautor, Y., García, F., & Alonso, D. (2015). Tools for microalgal biotechnology: Development of an optimized transformation method for an industrially promising microalga Tetraselmis chuii. J. Appl. Phycol, 1 (27), 223-232. |
| [3] | Guiry, M., & Guiry, G. (2018). AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Recuperado el 11 de Junio de 2019, de World-wide electronic publication, National University of Ireland, Galway: http://www.algaebase.org |
| [4] | Arora, Mani. (2016). Tetraselmis: an Introducción. The Botanica, 66. 155-175. |
| [5] | Juliet, B., & Lewis, J. (2007). Unravelling the algae. the past, present and future of algal systematics. London, UK: Systematics Association special volume series, 75. |
| [6] | Arora, M., Anil, A., Leliaert, F., Delany, J., & Mesbahi, E. (2013). Tetraselmis indica (Chlorodendrophyceae, Chlorophyta), a new species isolated from salt pans in Goa, India. European Journal of Phycology, 48 (1), 61–78. |
| [7] | Lee, H. & Hur, S., (2009). Genetic Relationships among Multiple Strains of the Genus Tetraselmis Based on Partial 18S rDNA Sequences. Algae, 24 (4), 205-212. |
| [8] | Olmos, J., Paniagua, J., & Contreras, R. (2000). Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene. Letters in Applied Microbiology, 80–84. |
| [9] | Zou, S., Fei, C., Wang, C., Gao, Z., Bao, Y., He, M., & Wang, C. (2016). How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae). Scientific reports, 6, 36822. 10.1038/srep36822. |
| [10] | Tragin, M., Lopes dos Santos, A., Christen, R., & Vaulot, D. (2016). Diversity and ecology of green microalgae in marine systems: an overview based on 18S rRNA gene sequences. Perspectives in Phycology, 3 (3), 141–154. |
| [11] | Kusumawati, L., Wahyudi, R., Pinontoan, R., Gorreti, M., & Panggabean, L. (2015). Sequence Analysis of 18S DNA of Melosira sp., Dunaliella sp., Isochrysis sp. and Porphyridium sp. The 3rd International Conference on Biological Science 2013, 2, 592–597. |
| [12] | Turmel, M., De Cambiaire, J., Otis, C., & Lemieux, C. (2016). Distinctive architecture of the chloroplast genome in the chlorodendrophycean green algae scherffelia dubia and Tetraselmis sp. CCMP 881. PLoS ONE, 11 (2), 1-22. |
| [13] | Chatzou, M., Cedrik, M., Jia Ming, C., Carsten, K., Giovanni, B., Ionas, E., & Cedric, N. (2016). Multiple sequence alignment modeling: methods and applications. Oxford University, 17 (6), 1009–1023. |
| [14] | Gunapati, O., A. Thadoi., K, Ojit Singh., O, Avijeet Singh., Th, Indrama., Laxmipriya Koijam., Wangkhem, Indira., Chungkham, Silvia., A, Sharma., Romi, Khangembam., Minerva, Shamjetshabam., Th, Bidyababy., K, Sarabati., O.N, Tiwari & G.D, Sharma (2015). Molecular characterization of selected diazotrophic cyanobacteria of North East India for phylogenetic study Int. J. of Adv. Res. 3 (8). 261-271. |
| [15] | Flores, F., Maldonado, S., & Ortiz, J. (2016). Identificación molecular de microalgas clorófitas del Ecuador. Research Gate, 1-2. |
| [16] | Benoiston, A.-S., Ibarbalz, F., Bittner, L., Guidi, L., Jahn, O., Dutkiewicz, S., & Bowler, C. (2017). The evolution of diatoms and their biogeochemical functions. Phil. Trans. R. Soc. B, 372 (20160397), 2-10. |
| [17] | Bérard, A., Dorigo, U., Humbert, J., & Martin, F. (2005). Microalgae community structure analysis based on 18S rDNA amplification from DNA extracted directly from soil as a potential soil bioindicator. Agronomy for Sustainable Development, Springer Verlag/EDP Sciences/INRA, 25 (2), 285-291. |
| [18] | Galarza, J., Gimpel, J., Rojas, V., Arredondo-Vega, B., & Henríquez, V. (2018). Over-accumulation of astaxanthin in Haematococcus pluvialis through chloroplast genetic engineering. Algal Research, 31, 291–297. |
| [19] | Pillacela, B., Galarza, J., & Tufiño, C. (2020). Reliability of in silico Modeling Based on Restriction Patterns for the Study of the Carotenogenic Gene pds of Haematococcus sp. International Journal of Microbiology and Biotechnology, 5 (1), 16-21. |
| [20] | František, M. (2005). Physical Mapping-Restriction Mapping (4th edition ed.). USA: Molecular Cell Biology. |
| [21] | Vincze, T., Posfai, J., & Roberts, R. (2003). NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res, 31, 3688-3691. |
| [22] | Parashar, D, Srivastava R, Chauhan D, Sharma V, Maharaj Singh, Mallika Lavania, Chauhan A, & Bhatia A. (2006). Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region. J Commun Dis, 38 (2): 263-268. |
| [23] | Garduño-Solórzano, G., Rodríguez, M., Martínez, M., Quintanar, R., Lozano, C., Campos, J., & Monsalvo, A. (2011). Cultivos de microalgas del Lago de Catemaco, Veracruz. Latinoam Biotecnol Amb Algal, 2 (2), 67-80. |
| [24] | Nagaraj, S., Arulmurugan, P., Rajaram, M., Sundararaj, R., & Rengasamy, R. 2012. Enhanced production of astaxanthin at different physico-chemical parameters in the green alga Haematococcus pluvialis Flotow. Phykos, 42 (1), 59–71. |
| [25] | Kibbe, W. (2007). Oligo Calc:Oligonucleotide Properties Calculator. Recuperado el 6 de Junio de 2019, de http://biotools.nubic.northwestern.edu/OligoCalc.html. |
| [26] | Green, M., & Sambrook, J. (2012). Molecular Cloning. New York: Cold Spring Harbor Laboratory Press. |
| [27] | Rocha, P. (2002). Teoría y práctica para la extracción y purificación del ADN de palma de aceite. Bogotá, Colombia: Laboratorio de Marcadores Moleculares, Área de Fisiología y Mejoramiento. |
| [28] | Yáñez, R. (2011). Categorización taxonómica en base a marcadores moleculares de microalgas extremófilas nativas del Norte de Chile. Chile: Facultad De Ciencias; Instituto De Biología. |
| [29] | Morrison, D., & Ellis, J. (1997). Effects of Nucleotide Sequence Alignment on Phylogeny Estimation: A Case study of 18S rDNAs of apicomplexa. Mol. Biol. Evol, 14 (4), 428-441. |
| [30] | Molins, A., Moya, P., García, F., Reig, J., & Barreno, E. (2018). A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations. The Lichenologist, 50 (1), 123–138. |
| [31] | Fawley, M. & Fawley, K. (2004). A simple and rapid technique for the isolation of DNA from microalgae. Phycological Society of America, Volumen 40, pp. 223-225. |
| [32] | Cruz, J. (2012). Comparación De Genes De Arn Ribosomal 18s De Tres Cepas Del Género Euglena: E. Pailasensis, E. Mutabilis Cpcc293 y Una Euglena Encontrada En Un Riachuelo Ácido Del Parque Nacional Volcán Rincón De La Vieja. Cartago: Instituto Tecnológico De Costa Rica. |
| [33] | Baskara, A., Jayakumar, T., Ganasan, M., Mohan, N., Senthil, C., Nagaraj, S., Rengasamy, R., Manubolu, M., Sheu, J., & Chang, C. (2018). Mass cultivation of new algae Tetraselmis straiata BBRR1 under open raceway ponds for biodiesel and biocrude production. Tamil Nadu, India: Preprints. |
| [34] | Chin, W., Teoh, P., Anton, A. & Kumar, S., (2018). Molecular Characterization And Identification Of Ribosomal Dna Sequences Of A Harmful Algal Bloom Species, Pyrodinium Bahamense Var. Compressum. Biotechnology Research Institute, 1 (1), pp. 1-8. |
| [35] | Montes, J., & Pulido, M. (2012). Obtención de protocolos para el aislamiento, cultivo y extracción de ADN de Chlorella vulgaris Beyerinck. Investigación y Ciencia del Gimnasio Campestre (45), 47-54. |
| [36] | Galarza, J., Delgado, N., & Henríquez, V. (2016). Cisgenesis and intragenesis in microalgae: promising advancements towards sustainable metabolites production. Publisher, Springer International Publishing. Applied Microbiology and Biotechnology, 100 (24), 10225–10235. |
| [37] | Cagney, G., Amiri, S., Premawaradena, T., Lindo, M., & Emili, A. (2003). In silico proteome analysis to facilitate proteomics experiments using mass spectrometry. Proteome Science, 1 (1), 5. |
| [38] | Jiménez, J., & Chaparro, A. (2016). In silico design and functional assessment of semisynthetic genes that confer tolerance to phosphinothricin. Revista Colombiana de Biotecnología, XVIII (2). |
| [39] | Cienfuegos, A., Conn, J., Gomez, G., & Correa, M. (2008). Diseño y evaluación de metodologías basadas en PCR-RFLP de ITS2 para la identificación molecular de mosquitos Anopheles spp (Diptera: Culicidae) de la Costa Pacífica de Colombia. Rev Biomed, 19 (1), 35–44. |
APA Style
Janeth Galarza, Kevin Crespín, Carolina Tufiño. (2020). Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene. International Journal of Genetics and Genomics, 8(3), 114-119. https://doi.org/10.11648/j.ijgg.20200803.14
ACS Style
Janeth Galarza; Kevin Crespín; Carolina Tufiño. Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene. Int. J. Genet. Genomics 2020, 8(3), 114-119. doi: 10.11648/j.ijgg.20200803.14
AMA Style
Janeth Galarza, Kevin Crespín, Carolina Tufiño. Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene. Int J Genet Genomics. 2020;8(3):114-119. doi: 10.11648/j.ijgg.20200803.14
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Download@article{10.11648/j.ijgg.20200803.14,
author = {Janeth Galarza and Kevin Crespín and Carolina Tufiño},
title = {Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene},
journal = {International Journal of Genetics and Genomics},
volume = {8},
number = {3},
pages = {114-119},
doi = {10.11648/j.ijgg.20200803.14},
url = {https://doi.org/10.11648/j.ijgg.20200803.14},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijgg.20200803.14},
abstract = {Tetraselmis is a genus of quadriflagellated single-celled green algae belonging to the Phylum Chlorophyta, commonly used in aquaculture with very promising biotechnological potential. The varied morphological characteristics, in some cases, have led to confusion in taxonomic identification. To solve this problem, new techniques based on molecular markers and restriction enzymes can ensure the identification of microalgae without sequencing. This study aimed to compare in silico modeling with an experimental restriction pattern based on the 18S rDNA gene for the identification of a microalgae strain. The strain grew in a culture medium, based on organic fertilizer. Theoretical analyses allowed the design of three primers based on the alignment of eight sequences obtained from NCBI, applying the Geneious Prime® 2019 and V1.3 and Oligo Calculator version 3.2. programs. The in silico restriction patterns was obtained with the NEBcutter v2.0 program. Experimental analyses began with the extraction of the DNA using the TENS protocol, then PCR amplification using PM-016F/PM-016R and PM-001F/PM-016R primers of 18S rDNA and finally the product was digested with BbvCI and Eco53kI; BstUI, RsaI and MspI enzymes. The DNA concentration extraction reached 3200 ng µl-1 and a purity of 2.0. The PCR amplified two products: 950 bp and 1400 bp, which brought us closer to identifying the microalgae. The in silico modeling and experimental restriction patterns showed similar fragments. In this way, the efficient response of restriction enzymes was demonstrated by confirming that the PM013 strain corresponds to the Tetraselmis genus. This method can be considered as a fast and safe alternative to identify wild microalgae in a basic molecular biology laboratory.},
year = {2020}
}
TY - JOUR T1 - Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene AU - Janeth Galarza AU - Kevin Crespín AU - Carolina Tufiño Y1 - 2020/09/24 PY - 2020 N1 - https://doi.org/10.11648/j.ijgg.20200803.14 DO - 10.11648/j.ijgg.20200803.14 T2 - International Journal of Genetics and Genomics JF - International Journal of Genetics and Genomics JO - International Journal of Genetics and Genomics SP - 114 EP - 119 PB - Science Publishing Group SN - 2376-7359 UR - https://doi.org/10.11648/j.ijgg.20200803.14 AB - Tetraselmis is a genus of quadriflagellated single-celled green algae belonging to the Phylum Chlorophyta, commonly used in aquaculture with very promising biotechnological potential. The varied morphological characteristics, in some cases, have led to confusion in taxonomic identification. To solve this problem, new techniques based on molecular markers and restriction enzymes can ensure the identification of microalgae without sequencing. This study aimed to compare in silico modeling with an experimental restriction pattern based on the 18S rDNA gene for the identification of a microalgae strain. The strain grew in a culture medium, based on organic fertilizer. Theoretical analyses allowed the design of three primers based on the alignment of eight sequences obtained from NCBI, applying the Geneious Prime® 2019 and V1.3 and Oligo Calculator version 3.2. programs. The in silico restriction patterns was obtained with the NEBcutter v2.0 program. Experimental analyses began with the extraction of the DNA using the TENS protocol, then PCR amplification using PM-016F/PM-016R and PM-001F/PM-016R primers of 18S rDNA and finally the product was digested with BbvCI and Eco53kI; BstUI, RsaI and MspI enzymes. The DNA concentration extraction reached 3200 ng µl-1 and a purity of 2.0. The PCR amplified two products: 950 bp and 1400 bp, which brought us closer to identifying the microalgae. The in silico modeling and experimental restriction patterns showed similar fragments. In this way, the efficient response of restriction enzymes was demonstrated by confirming that the PM013 strain corresponds to the Tetraselmis genus. This method can be considered as a fast and safe alternative to identify wild microalgae in a basic molecular biology laboratory. VL - 8 IS - 3 ER -
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