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DNA Barcoding of Selected Perciformes (Infra Class: Teleostei) Fishes from Indian Coast

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DNA Barcoding of Selected Perciformes (Infra Class: Teleostei) Fishes from Indian Coast Indian Journal of Biotechnology Vol 16, July 2017, pp 315-321 DNA barcoding of selected Perciformes (Infra Class: Teleostei) fishes from Indian coast N Sadurudeen, A Pavan-Kumar*, P Gireesh-Babu, A K Jaiswar, A Chaudhari, Gopal Krishna and W S Lakra ICAR-Central Institute of Fisheries Education (CIFE), Versova, Mumbai-400061, India Received 22 October 2014; revised 21 September 2015; accepted 6 October 2015 India has a rich aquatic biodiversity spreading across different ecosystems. Total 2358 endemic fish species have been reported from India comprising marine (1368) , brackish water (113) and freshwater fishes (877) . Marine fish identification is seldom difficult because of their high diversity and profound changes in appearance during their development stages. DNA barcoding technique has been successfully used to discriminate all animal taxa. In this study, DNA barcodes were generated for 32 species representing 13 families of the order perciformes. The average genetic divergence values for within species, genus and family were 0.42, 13.91 and 18.05%, respectively. The genetic divergence values increased several folds (15-20 times) from lower to higher taxa. Barcode gap analysis showed the absence of overlapping between intra and interspecific divergence values. Intraspecific variation has increased several folds (15-20 times) after including conspecific sequences from different geographical locations. Presence of allopatric lineages / cryptic species was observed in several fishes that have Indo-pacific region distribution. The neighbour joining (NJ) tree constructed based on mitochondrial cytochrome oxidase submit 1 kimura two parameter (COI K2P) values showed distinct clusters shared by congeneric species. Around 100 nucleotide diagnostic characters exclusive to Perciformes fish species were also identified. Keywords: Perciformes, DNA barcoding, cytochrome c oxidase subunit 1, allopatric divergence Introduction difficult taxonomic questions and diagnose species in India is one of the mega biodiverse countries of 34 cases where morphology alone has proved biodiversity global hotspots. Around 2,649 fish inadequate4. Mitochondrial partial cytochrome species were reported from India including freshwater c oxidase subunit I (COI) gene has been successfully (877) , brackish water (113), marine water (1368) and used as a standard gene for discriminating all animal exotic (291), which together accounts for 7.8% of the life because of its faster rate of evolution at the global finfish diversity1. Accurate identification of synonymous codon third base position5. In India, fish species is essential for documentation of diversity DNA barcoding technique has been successfully used and assessing the stock structure thereby formulating to document major groups of Indian fish diversity6-9. management and conservation measures. Perciformes is the largest order in fishes and Traditionally, fish species identification is relied upon comprises nearly 40% of bony fishes10. Previous external morphological features, including body studies on this group from India have not included shape, pattern of colors, scale size and count, fin much diversity of these fishes and have not addressed number and its relative positions, numbers and types COI gene divergence pattern as geographical distance of fin rays and various relative measurements of the increases between conspecific individuals6. With this body parts2. However, these methods are constrained background, the present study was carried to document by phenotypic plasticity, life-stage specific the commercially important Perciformes fishes through identification cues and the occurrence of cryptic DNA barcoding and to investigate the pattern and level species3. These limitations of traditional taxonomy led of divergence at COI locus between conspecific to the development of new methods such as DNA individuals collected from different locations. based taxonomy. DNA sequence data from mitochondrial genome have long been used to resolve Material and Methods Sample Collection —————— *Author for correspondence Around 86 specimens of order: Perciformes were Tel: 022-26361447 Ext. 452 collected from different landing centres of East and [email protected] West coast of India (Table 1). Wherever possible, 316 INDIAN J BIOTECHNOL, JULY 2017 more than two individuals per species were collected Genomic DNA isolation, PCR amplification and purification and the species were identified at the field by Total genomic DNA was isolated by salting out 12 observing morphological and meristic characters of method with slight modifications. Mitochondrial the specimens11. Voucher specimens were prepared by partial cytochrome c oxidase subunit I (COI) gene preserving the specimen in absolute alcohol with was amplified using reported universal primers, proper labelling. Fins and muscle tissues were collected Fish F1: 5’-TCAACCAACCACAAAGACATTGGC under aseptic conditions and preserved in absolute AC-3’; Fish R1: 5’-TAGACT TCTGGGTGGCCAA alcohol in the field, then deeply frozen at -80° C in the AGAATCA-3’; Fish F2: 5’-TCGACTAATCATAAA laboratory for further analysis. In addition, reported GATATCGGCAC-3’ and Fish R2: 5’-ACTTCA mitochondrial cytochrome c oxidase subunit I (COI) GGGTGACCGAAGAATCAGAA-3’. PCR was gene sequences for selected conspecific individuals performed in 25 μl reaction volume containing 100 ng were downloaded from GenBank National Centre for template DNA, 10 pmol of each specific primer, Biotechnology Information (NCBI), to calculate 200 μM of each dNTPs, 1.0 unit of Taq DNA divergence values between populations. polymerase and 1X Taq buffer containing 1.5 mM Table 1 — List of species along with GenBank/BOLD accession numbers S. No Family Species (number of specimen) GenBank Acc. Number / BOLD BIN 1. Carangidae Atropus atropus (1) KJ920133 2. Megalaspis cordyla (2) KJ920111, KM079290 3. Selaroides leptolepis (2) KJ920124, KM079293 4. Gnathanodon speciosus (2) KJ920129, KM079289 5. Lethrinidae Lethrinus crocineus (3) KJ920118, KM079301-302 6. Lethrinus lentjan (2) KJ920116, KM079303 7. Lethrinus mahsena (3) KJ920117, KM079304-305 8. Lethrinus microdon (4) KJ920114, KM079306-308 9. Lethrinus nebulosus (3) KJ920115, KM079309-310 10. Lethrinus ornatus (4) KJ920113, KM079311-313 11. Serranidae Epinephelus diacanthus (3) KJ920101, KM079296-97 12. Epinephelus longispinis (2) KJ920103, KM079298 13. Epinephelus undulosus (3) KJ920104, KM079299-300 14. Pogonoperca ocellata (1) KJ920102 15. Lutjanidae Lutjanus lutjanus (2) KJ920132, KM079315 16. Lutjanus russellii (3) KJ920119, KM079316-17 17. Lutjanus sebae (2) KJ920120, KM079314 18. Mullidae Mulloidichthys vanicolensis (4) KJ920106, KM079318-320 19. Parupeneus heptacanthus (4) KJ920105, KM079321-323 20. Parupeneus indicus (4) KJ920109, KM079324, KM079339-40 21. Upeneus moluccensis (4) KJ920110, KJ920112, KM079325-326 22. Upeneus sulphureus (4) KJ920107, KM079327-329. 23. Upeneus tragula (3) KJ920108, KM079330-331 24. Terapontidae Terapon jarbua (3) KJ920134, KM079294-295 25. Terapon puta (1) KJ920126 26. Stromatidae Pampus argenteus (2) KJ920121, KM079333 27. Pomacanthidae Pomacanthus semicirculatus (1) KJ920131 28. Latidae Psammoperca waigiensis (02) KJ920125, KM079334 29. Scatophagidae Scatophagus argus (3) KJ920130, KM079335-36 30. Nemipteridae Scolopsis bimaculatus (3) KJ920127, KM079337-38 31. Scombridae Scomberomorus commerson (4) KJ920122, KJ920128, KM079291-92 32. Drepaneidae Drepane punctata (2) KJ920123, KM079332 SADURUDEEN et al :DNA BARCODING OF PERCIFORMES FISHES 317 MgCl2. The thermocycler was programmed for initial position. The number of transitional pairs (Si = 69) denaturation at 95°C for 5 min, followed by 35 cycles were more than transversional pairs (Sv = 46) with an of 94°C for 30 sec, 54°C for 30 sec, 72°C for 1 min average ratio of 1.50. for denaturation, annealing and extension, with final The average distance values (K2P) of COI gene was extension at 72°C for 10 min. The PCR amplification increased from lower taxa towards the higher products were purified using gel extraction kit taxonomic rank (i.e. within species, genera, family). (Qiagen, Germany) following manufacturer’s The average interspecific distance value (D = 13.91%) protocols. The purified PCR products were sequenced was ~33 times higher than the average intraspecific directly using PCR primers. For all the genes, distance (D = 0.42%). whereas, the mean divergence sequencing was performed in both directions for value among genera within families was 18.05% better accuracy using ABI Big DYE terminator (Table 2). DNA barcode gap analysis showed that all method (Eurofins lab, Bangalore, India) . species were distinct from their nearest neighbour Sequence Analysis (NN) species (Fig. 1). The COI partial gene sequences obtained for each The level and pattern of average barcode species were manually assembled using Gene Runner divergence for selected fish species (15) collected V 3.0 software. Assembled sequences were end- from different geographical locations showed less trimmed to a homologous region to avoid sequencing intraspecific divergence value (0.0 - 0.017) within errors. All the COI gene sequences were aligned using populations than between populations (0.007 - 0.192). ClustalW program14. Only those sequences with more Mantel test results also showed significant correlation than 550 bp in size were used for the analysis. between geographical and genetic distances (Table 3 Sequence divergence values within and between & Fig. 2). species were calculated using Kimura
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