This study aimed to investigate the quantitative relationships between four fish species from three genera based on molecular analyses (barcoding) of nine species from five genera utilizing the mitochondrial COI gene. Species within the same genus showed more transitional incompatibilities than transversional mismatches. The samples were divided into four main groups by a phylogenetic tree built from the sequencing data (cytochrome COI) of samples from the two populations using the neighbour-joining method. As dissimilar species were clustered under separate nodes and similar species were clustered under the same nodes, the neighbour-joining tree revealed various clusters corresponding to the taxonomic status of the species. In conclusion, the mitochondrial CO1 gene is a useful molecular marker for DNA barcoding.


Anisha S Menon

Manoj Mathews

Nandini N J

Valiyaparambil, Jisha Jacob

English

Christ University Bengaluru: Open Journal Systems

133Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 GeneMapana – Journal of Sciences2024, Vol. 23, No. 1, 133-147ISSN 0975-3303|https://doi.org/10.12723/mjs.68.7DNA Barcoding of Fish Fauna Using Mitochondrial CO1 GeneAnisha S Menon*, Nandini N.J.†, Manoj Mathews‡ and Jisha Jacob*Abstractbased on molecular analyses (barcoding) of nine species Species within the same genus showed more transitional incompatibilities than transversional mismatches. a phylogenetic tree built from the sequencing data (cytochrome COI) of samples from the two populations using the neighbour-joining method. As dissimilar species were clustered under separate nodes and similar species were clustered under the same nodes, the neighbour-joining tree revealed various clusters corresponding to the taxonomic status of the species. In conclusion, the mitochondrial CO1 gene is a useful molecular marker for DNA barcoding.Keywords: DNA barcoding, mitochondrial CO1 gene, molecular taxonomy, biodiversity assessment, DNA sequencing.* Molecular Microbial Ecology Lab, PG and Research Department of Zoology, St. Joseph’s College (Autonomous), Devagiri, University of Calicut 680 555, Kerala, India; anishasumith13@gmail.com, jishajacob@devagiricollege.orggmail.com‡ PG and Research Department of Chemistry, St. Joseph’s College (Autonomous), Devagiri, University of Calicut 680 555, Kerala, India; mathewsmanoj@gmail.com134ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1IntroductionFish is high in protein and micro- and macronutrient supplements, as well as vitamins A, B, and D. Omega-3 fatty acids, which are vital for the body and brain, are also abundant in it [1]. Fish are among the healthiest foods on the earth and are consumed in a broad variety feed. [3]. belonging to 5 genera: . Etroplusof the native cichlids is the Etroplus suratensis, often known as the freshwater environments in peninsular India, mostly in Kerala and Sri Lanka. [4] [5]. Orange chromide, or , is found in Kerala’s state’s seafood food security and is said to be an inexpensive treat that Keralites can add to their diets to increase their protein intake on India’s west coast is the oil sardine, or  [7].is found in fresh, brackish, and marine along Mumbai, Karnataka, and Kerala coasts [8]. as Bullet tuna, is found exclusively in tropical oceans, including the  albacares species, seen except in the Mediterranean [10]. Oreochromis species (  (Nile tilapia, cultured since 2012) (Wami tilapia) are used in aquaculture wherein utilizes their high adaptability, fecundity and ability to attain enormous size in a short time to culture and feed the rising population [11]. thought to be more phenotypically variable than most other vertebrates 135Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 Genemeasurement is frequently inconsistent [12]. In this work, an attempt the mitochondrial COI gene that is utilized as a "species barcode" is organisms can be evaluated by the construction of a phylogenetic tree existing within and between species in a population cannot be overemphasized as it has become a handy method used in tracing the origin and evolution of species from the sequences obtained from organisms [15].Materials and Methodsdistrict. A preliminary analysis of species of Etroplus, Sardinella, and  was done with the help etc, their morphological appearances were deeply studied.Molecular AnalysisElutionkit following the   manufacturer’s protocol using 25 mg abdominal muscle tissue from samplesForward primer, F1:5 136ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1Reverse primer, R1:5can be estimated with the help of a 1kb base pair DNA ladder. Gel Using the forward primer 5’3’ and the reverse primer 5’3’, the COI gene-containing region of the mitochondrial DNA genome was Sequencing of the Gene, Analysis, and Construction of Phylogenetic Treewere trimmed and multiple sequence alignment is done using Evolutionary Genetic Analysis) software by trimming the ends for the construction of a phylogenetic tree by the neighbour-joining method.Results and Discussion137Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 GeneFig 1- Table 1- 1. Etroplus suratensis 2. 3. 4. 5. 6. Auxis rochei7. 8. Molecular AnalysisAccession numbers for the COI sequences of the investigated species. Denoted by GenBank Accession numberEtroplus suratensis D3 MG923359D4 AP009505D1 MG251979Auxis rochei MH638691LC487084MF509598MK210574 Etroplus suratensis COI sequences and with D4  sequence shows 83 mismatches 138ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1 CO I sequences and with D2  sequence shows dissimilarities transversions found among those mismatches.and Auxis rochei COI sequences   COI sequence gave 33 mismatches including 29 transitions and 4 transversions. Sequences  COI sequence gave 31 mismatches and within them, there  COI sequence had 25 mismatches and out of them 21 were transition and 4 were transversion. So this pairwise alignment shows the molecular discrimination of different species based on the COI gene.Sequence Analysisthe database.Fig.2- niloticus139Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 GeneFig.3 - niloticusFig.4 with database sequences LC487084140ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1Fig.5- urolepisFig. 6- Fig. 7with database sequence of MF509598141Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 Gene Fig.8-142ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1Fig.10- sequence MK210574Fig.11 COI sequences with 143Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 GeneFig.12 COI sequences with COI sequences with 144ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1Construction of Phylogenetic TreeFig 14 - Phylogenetic tree constructed using the neighbour-joining method.according to population using the phylogenetic tree created from the sequence data (cytochrome COI) of samples from the two populations.common ancestor, four branches formed represent two descendant groups;  species (Clade 1) Etroplus species (clade 2)  species (Clade 3), and  species (Clade 4). Clade 1 is further branched suggesting that niloticus has diverged from the sister taxa  and . Similar species were clustered under the same nodes in the phylogenetic tree, whereas dissimilar species were clustered under distinct nodes, revealing an identical evolutionary link among the species.Conclusioninto and . Our result revealed that these 9 species can be differentiated by COI sequence analysis and the relationship can be suggested by the construction of a phylogenetic tree. From the results obtained, the 145Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 Genemitochondrial COI gene can be named as an ideal region for “species this tool is used for rapid analysis for commercial purposes especially in its biochemical contents [16]. In these experimental studies, we measured protein; carbohydrates, and lipids content in our sample. revealed that  have higher protein, carbohydrate, and lipid compared with the other 3 species of Etroplus suratensis, and available in tins, cans, etc. So, knowing the quality of the by-products design the internal primer from the already analyzed sequences. CO1 gene can also provide valuable information about evolutionary References[1] F. Jim et al., “Comparative analysis of nutritional value of  (Linnaeus), Nile tilapia, meat from three different ecosystems,” J. Food Qual., pp. 5-10, 2017.[2] Rust. BM, , 2003, pp. 367-452.contribution. Ecological and economic impacts and contributions , vol. 2, no. 11, pp. 958-963, 2001 [doi:10.1093/embo-reports/kve236].[4] K. G. Padmakumar et al., “Etroplus suratensis (Bloch), the State Fish of Kerala,” J. Biosci., vol. 37, no. 6, pp. 925-931, 2012 [doi:10.1007/s12038-012-9271-x].146ISSN 0975-3303 Mapana - Journal of Sciences, Vol. 23, No.1[5] Gunawickrama and K. B. Suneetha, “Morphological heterogeneity and population differentiation in the Green chromide in Sri Lanka,” Ruhuna J. Sci., vol. 2, pp. 70-81, 2007.[6] Shyam et al., “Sardine economy of Kerala: Paradigms and Perspectives,” , vol. 2, no. 6, pp. 351-356, 2015.sardine Sardinella longiceps Valenciennes,” Indian J. Fish., vol. 11a, no. 1, pp. 345-370, 1964.[8] R. Froese and D. Pauly, 2008, “FishBase. World Wide Web electronic publication,” . Available at: http://[9] E. G. Silas and P. P. Pillai, , 1982, pp. 10-15.[10] R. Froese, 2003, “FishBase. World Wide Web electronic publication,” . Available at: http://www.[11] C. D. Webster and C. Lim, , 2006, pp. 5-6.[12] V. Sachithanandam et al., “DNA bar coding, phylogenetic Pooranankuppam brackish water, Puducherry,” Rev., vol. 04, pp. 33-42, 2012., vol. 13, no. 6, pp. e0198109, 2018 [doi:10.1371/journal.pone.0198109].[14] J. Zhang and R. Hanner, “DNA barcoding is a useful tool for the , vol. 39, no. 1, p. 31-42, 2011 [doi:10.1016/j.bse.2010.12.017].[15] J. K. Chambers et al., “AG protein-coupled receptor for UDP-glucose,” , vol. 275, no. 15, p. 10767-10771, 2000 [doi:10.1074/jbc.275.15.10767].[16] R. Nagabhushanam and U. H. Mane, “Seasonal variation in the biochemical composition of Mytilus edulis at Ratnagiri on the west coast of India,” , vol. 57, no. 1, pp. 69-72, 1978 [doi:10.1007/BF00018630].147Menon et al.                             DNA Barcoding of Fish Fauna Using Mitochondrial CO1 Gene[17] Gopalsamy., Idayachandiran., Arumugam., Muthukumar., Nutritional Value of Marine Bivalve, Donax cuneatus (Linnaeus, 1758) from Cuddalore Coastal Waters, Southeast Coast of India. Invention. 1-10.

DNA Barcoding of Fish Fauna using Mitochondrial CO1 Gene

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DNA Barcoding of Fish Fauna using Mitochondrial CO1 Gene

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