Condiciones contenidas en el otorgamiento de aprobación

data with taxonomic and specimen information or NCBI GenBank. The BOLD is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm (fig 3,4,5). BOLD is freely available to any researcher with interest in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is

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also well positioned to support projects that involve broad research alliances (Ratnasingham and Hebert, 2007).

Barcoding Elective: Other genes

If COI-5’ is not sufficient for species discrimination, other rapidly evolving gene(s) may need to be analyzed as potential barcoding targets. Possible supplementary sequences include the complete COI gene, other mitochondrial genes (e.g. 16S rRNA, cytochrome b), and/or ITS (internal transcribed spacer), which is a nuclear DNA segment located between rRNA genes. Small subunit nuclear ribosomal RNA (SSU rRNA) also referred to as 18S rRNA, is a slowly evolving gene useful for deeper phylogenetic analysis. In addition to the analysis of COI-5’, it will be ideal to determine SSU rRNA sequences from specimens. SSU rRNA is the basis for the Tree of Life and other comprehensive examinations of evolution of life.

Partial sequences of other nuclear genes useful in DNA Barcoding of animals are RAG-2 (Recombination Activation gene2, associated with the immune system); Rhodopsin (460 bp; Sevilla et al., 2007); an anonymous DNA fragment in fish (TMO M27) and aldolase gene fragment The introns-less fish rhodopsin gene provides quantitatively equal interspecies identification labels of targeted nuclear polymerase chain reaction (PCR) amplification products throughout its coding sequence. This gene has been successfully used in vertebrate phylogenetic studies. The utility of two nuclear protein-coding genes, phosphoenolpyruvate carboxykinase (PEPCK) and sodium–potassium ATPase a- subunit (NaK), as molecular markers for phylogenetics of decapods, insects and bilateral metazoans has been demonstrated (Maa et al., 2009). These two genes participate in fundamental cellular functions in the animal kingdom and are well-conserved throughout evolution. Presumably, these genes only exist as single-copy in most of the crustaceans. DNA Barcoding of Plants:

Those involved in initiating efforts in plant barcoding have focused on the search for a candidate locus for identifying species. These efforts are inspired by the success of the mitochondrial gene Cytochrome c oxidase I (COI) as the core of the global bioidentification system for animals. An optimal barcoding locus for plants will naturally have similar characteristics: sufficient variation between species such that species level discrimination can be achieved, but minimal variation within species. Unlike barcoding in animals, however, the mitochondrial COI gene is not good candidate for land plants as plant mitochondrial genes typically exhibit lower nucleotide substitution rates than plastid or nuclear genes (meaning that there is often no sequence variation among species within a genus) and often exchange of DNA has been reported between mitochondrial and nuclear genome. The substitution rate in plastid genes, however, is also not great, with rates about one quarter the rate observed in nuclear DNA and 10- to 20-fold less than mammalian mitochondrial DNA.

Even if the "perfect" plant barcoding locus were to be found, it is recognized that reliance on a single (usually) maternally inherited gene will be problematic in groups that exhibit hybridization and introgression. In species complexes that exhibit extensive introgression, incorporation of multiple nuclear regions will be a necessity to make confident identifications .The suggestion to include multiple loci in barcoding systems was welcomed by critics of barcoding. While the use of multiple loci is a straightforward response to the challenges for barcoding plants, the system must retain a minimal complement of loci for it to remain a fast and efficient tool. One obvious choice for evaluation as a potential standard core coding region is rbcL (ribulose bisphosphate

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carboxylase – large subunit) of the chloroplast DNA, given its universality and ease of amplification and alignment. While rbcL, seems to be a reasonable candidate for a first tier barcoding locus, there are other regions that may prove to be greater utility. Regions in cpDNA especially matK (maturase K gene- involved in group II intron splicing and usually located within the intron of another plastid gene – lysine coding tRNA - trnK) andtrH-psbA spacer, rpoC1, rpoB, accD and YCF5 are identified as the most promising regions in the cpDNA for DNA barcoding in plants and universal primer pairs for these regions have been developed ([email protected]). These loci meets the criteria necessary for a barcode locus: (i) significant species level genetic variability and divergence, (ii) an appropriately short sequence length so as to facilitate DNA extraction and amplification, and (iii) the presence of conserved flanking sites for developing universal primers. The plastid gene matK, for examples, has a substitution rate that is 2-3 times greater than rbcL in angiosperms, however there is only a small amount of data available for the bryophytes or ferns, precluding a quick evaluation. The plastid trnH- psbA spacer has also been identified as suitable single copy gene locus for DNA barcoding, but falls short of an additional criterion namely, ease of alignment and analysis. While ease of alignment is not a strictly necessary criterion for DNA identification, it is a critical requirement for developing bioinformatics tool. The presence of multiple indels that overlap (as in trnH-psbA)makes homology assessment and therefore accurate alignment difficult or impossible (Newmaster

et al., 2006).

DNA Barcoding of Indian Fishes

India is blessed with huge inland water resources with 29,000 km of rivers, 0.3 million ha of estuaries, 0.9 million ha of backwaters and lagoons, 3.15 million ha of reservoirs, 0.2 million ha of floodplain wetlands and 0.72 million ha of upland lakes. It has been estimated that about 0.8 million tonnes of inland fish is contributed by different types of inland open water systems. The 14 major rivers, 44 medium rivers and innumerable small rivers of the country provide one of the richest fish faunistic resources (765 species of finfishes) of the world. The bounty of marine biodiversity, which is exploited from 2.02 million sq. km of the exclusive economic zone (EEZ) constitutes one of the largest heritage resources of India. There are nearly 1650 species of finfishes known from our seas. However, taxonomic ambiguity exists in several groups of Indian finfishes and shellfishes and many are insufficiently identified. Indian seas also have many unexplored habitats like the mesopelegic zone and deep waters that may harbour many species of finfishes yet to be documented. Although much of the finfish research at present depends upon species diagnoses based on morphological characters and meristic counts, taxonomic expertise has been collapsing in recent years. The limitations inherent in morphology based identification system and dwindling pool of taxonomists signal the need for new approach to document Indian marine finfish diversity. DNA based approaches to taxon identification which exploit diversity among DNA sequences, can be used to identity marine fishes and resolve taxonomic ambiguity including discovery of new / cryptic species.

The National Bureau of Fish Genetic Resources (NBFGR), Lucknow has been identified as the lead center in South Asia to generate DNA Barcodes of marine and freshwater finfish and shellfish species of the region in collaboration with the International

Consortium for the Barcode of Life (iBOL) – Fish BOL. The NBFGR has initiated a mega

programme on DNA Barcoding of Indian marine and freshwater fishes and 2066 samples have been collected covering 553 marine and freshwater finfish and shellfish species from the mainland and island ecosystems. The DNA Barcodes (DNA sequence profile of 655 bp fragment of cytochrome c oxidase I) of 550 species has been completed for the first time in India and taxonomic ambiguity of many species has been resolved (Silas et al., 2005; Lakra

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et al., 2009, 2010, 2011; Divya et al., 2009, 2010; Singh et al., 2011). This could be of great

utility in sustainable exploitation, management and conservation of Indian marine and freshwater fish species.

Forensic identification of meat of endangered aquatic species:

Whale shark (Rhincodon typus) is the largest shark in the ocean, reaching lengths of 20 meters and a weight of 20 tonnes. With very few defences, it has become susceptible to exploitation and has a global conservation status of ‘vulnerable to extinction’ as listed by World Conservation Union in the Red list of threatened species. To enable trade in whale shark products to be adequately regulated, Rhincodon typus was nominated in Appendix ІІ of Convention on International Trade in Endangered Species (CITES) in April 2000. To conserve the species in Indian waters, it is enlisted as one of the protected species and its fishing prohibited under Schedule  of the Indian Wildlife Protection Act, 1972, according to the Order No.1-2/2001 WL1 dated 28.05.2001, Govt. of India. Flesh suspected as that of whale shark was seized from fishermen by the Forest Range Officer (Govt. of Kerala), Kannur, Kerala. A case was filed and the Judicial First Class Magistrate, Thalassery, Kannur, Kerala approached NBFGR Cochin Unit (Case No. R.P.330/08, dated 29. Sept. 2008) to analyze the meat sample for confirmation of the species using DNA markers. Based on DNA sequencing of COI (660bp), 16SrRNA (595bp), cyt B(601bp) and RAG2 (981bp) genes and comparing with the sequences earlier generated by NBFGR (NCBI Genbank DNA sequence accession numbers FJ375724, FJ375725, FJ375726; DNA sample collected from a stranded whale shark along North Kerala on 30 October 2006), the seized meat sample was confirmed as that of endangered whale shark (Rhincodon typus) and the result was communicated to the court. This was the first case in India in which scientific evidence was sought to identify the meat of a fish enlisted in the Wildlife Protection Act, 1972 and the DNA markers reiterated their ability to reliably identify product/meat sample of a species, thus helping in curtailing illegal trade of the endangered organisms (Sajeela et al., 2010). In another case, identity of cooked fish (pomfret - Pampus chinensis) from a restaurant in Mumbai was also confirmed at NBFGR through DNA barcoding.

Perspective:

Despite these technical and conceptual challenges, molecular species identification in food products and forensic samples is likely to increase exponentially. Indeed, DNA barcoding has already produced significant and interesting results, for example in gourmet food (such as species identification in canned tuna or cooked pomfret) and on forensic samples made from endangered species (e.g., whale shark). Molecular identification has already proven useful in court [Sajeela et al., 2010]. However, reliable reference barcodes are yet to be developed for many other commercially important aquatic groups and this calls for concerted, joint efforts of molecular biologists and traditional taxonomists to generate accurate baseline information. The protocol/software to convert the DNA sequence information into digital vertical barcodes is still not available in public domain and this can lead to innovations in bioinformatics, electronics and devices such as handheld barcoders. Another option is to utilize the DNA barcode data for identification of species- specific SNPs that can lead to development of DNA chip for much faster and precise identification of many species and hybrids and detection of fish product adulteration.

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Indian Fisheries Paradigms: A Policy Outlook.

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Fig 12.1 Various steps of DNA Barcoding

Fig 12. 2. Two prototypes of hand-held DNA Barcoder (by 2015?).

(Source: http://www.barcodinglife.org/views/login.php)

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(Source: http://www.barcodinglife.org/views/login.php)

Fig 12.4 Specimen page for an individual

of Macroglossus mininus (Chiroptera).

1, voucher information; 2, full taxonomy; 3, collection location;

4, collection site maps; 5, specimen images.

(Source:http://www.barcodinglife.org/views/login.ph p)

Shyam S. Salim and R.Narayanakumar, (2012). Manual on World Trade Agreements and

Indian Fisheries Paradigms: A Policy Outlook..

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