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22, , Molecular taxonomy – Applications,, Limitations and future, Sandhya Sukumaran and A. Gopalakrishnan, Central Marine Fisheries Research Institute, Kochi-682 018, , Organisms are characterized by unique biological attributes, which enhance their fitness and survival to a particular, environment. The driving force for enhanced survival and, fitness is the genetic variation inherent in an individual as, well as in a population. The information regarding genetic, diversity and variation has wide application in research, on evolution, conservation and management of natural, populations. The advent of DNA cloning and sequencing, methods have contributed immensely to the development, of molecular taxonomy and population genetics over the last, 2 decades. These modern methods have revolutionized the, field of molecular taxonomy and population genetics with, improved analytical power and precision., Molecular markers can be characterized as Type I and Type II, markers; Type I markers are associated with genes of known, function and type II markers are associated with genes of, unknown function. Allozyme markers are type I markers as, the proteins they encode are associated with some functions., Microsatellites and other neutral markers are type II markers, unless they are associated with genes of some known, function., , Allozyme, Allozyme electrophoresis is a method which can identify, genetic variation at the level of enzymes that are directly, encoded by DNA. Protein variants called allozymes originates, from allelic variants and they will differ slightly in electric, charge. Allozymes are codominant markers having been, expressed in a heterozygous individual in a Mendelian way., Thus allozyme analysis provides us with data on single locus, genetic variation which can answer many questions about fish, and fish populations. To detect allozyme variation, the first step, is to extract allozymes from tissues using specific protocols., Then the variation is detected through electrophoresis in, an acrylamide or cellulose acetate gel. Individuals that are, homozygous show a single band whereas heterozygous, individuals show two bands. Allozymes are one of the most, , studied form of molecular variation due to their simplicity,, low cost and the requirement of little specialized equipment., Any kind of soluble protein is suitable for allozyme analysis. A, large number of loci can be screened at a time. The limitations, of this technique include requirement of a large amount of, tissue and consequently this method could not be applied, when the organisms are small (for eg; larval forms). The, tissue sampling method is invasive and so the fish needs to, be sacrificed and the tissue needs to be stored cryogenically., A point mutation in a nucleotide sequence may not result in a, change in amino acid at all and thus could not be detected by, protein electrophoresis. In addition to that, a change in DNA, that results in a change in amino acid will not result in the, overall charge of the protein and therefore is not detected. In, spite of their limitations, the use of allozyme analysis has been, widespread in fisheries mainly in fish systematics, population, structure, conservation genetics, mixed stock fishery analysis, and forensic analysis., , Mitochondrial DNA markers, Mitochondrial DNA is non- nuclear DNA in the cell having, located in within organelles in the cytoplasm called, mitochondria. Mitochondrial DNA is maternally inherited, with a haploid genome. The entire genome undergoes, transcription as one single unit. They are not subjected to, recombination and thus they are homologous markers. They, are selectively neutral occurring in multiple copies in each, cell. Mitochondrial DNA is physically separate from the rest, of cell’s DNA and so it is relatively easy to isolate from any, tissue or blood sample. Due to the maternal inheritance of, mitochondrial DNA, the effective population size is smaller, than nuclear DNA and so mitochondrial DNA variation is, more sensitive to population bottlenecks and hybridizations., The differences in the nucleotide sequence of DNA molecule, in the mitochondria can be determined directly or indirectly, by several methods. Many population genetic studies have, employed RFLP (Restriction Fragment Length Polymorphism), analysis of mitochondrial DNA for understanding population, 157

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Molecular taxonomy – Applications, Limitations and future, genetic variation either by digesting the whole purified mtDNA, with restriction endonucleases or by DNA sequencing of small, segments of mtDNA molecule obtained by PCR amplification., These techniques with increased resolution and maximum, information have made mtDNA analysis very popular., The newly emerged sequencing technologies have enabled, direct sequencing of mitochondrial genes and several sets, of universal primers have been developed from conserved, sequence regions. Slow evolving gene regions are being used, for inter species comparisons and fast evolving gene regions, for population comparisons. The only non coding region of, mtDNA is D-loop region and this region is fast evolving and, mostly used for population comparisons. The cytochrome, b and ND-1 and ND-5/6 gene regions are also being used, widely. Mitochondrial Cytochrome C Oxidase I gene has, been identified as the universal barcode for species level, identification due to its conserved nature across a wide range, of taxa. DNA barcodes are segments of approximately 600, base pairs of the mitochondrial COI gene which is a fast,, efficient and inexpensive technique helpful in cataloguing the, biodiversity. During the last two decades, mitochondrial DNA, genes have found widespread application fish taxonomy,, biology and population genetics., , Arbitrary Nuclear DNA markers, Arbitrary markers are used when we target a segment of DNA, of unknown function. The widely used methods of amplifying, unknown regions are RAPD (Random Amplified Polymorphic, DNA) and AFLP (Amplified Fragment Length Polymorphism), DNA. RAPD uses an arbitrary primer which can amplify, anonymous loci. It is fast, cheap and shows very high, amount of polymorphism and this marker does not require, knowledge of the genetic makeup of the organism. The major, drawback with RAPD markers is the lack of reproducibility, and repeatability and the large number of products, generated. RAPD is a dominant marker and so homozygous, and heterozygous states cannot be differentiated and these, patterns are sensitive to slight changes in amplification, conditions. Amplified Fragment Length Polymorphism (AFLP), markers combine the benefits of both RFLP and RAPD. The, total genomic DNA is digested using two restriction enzymes., Double–stranded nucleotide adapters are ligated to the ends, of DNA fragments to serve as primer binding sites for PCR, amplification. Primers complementary to the adapter and, restriction site sequence, with additional nucleotides at the, 3’-end, are used as selective agents to amplify a subset, of ligated fragments. The presence of absence of DNA, fragments are detected on polyacrylamide gels and thus, polymorphisms are studied., , Specific Nuclear DNA markers, Variable Number of Tandem Repeat is a segment of DNA that, is repeated tens or even hundreds to thousands of times in, nuclear genome of eukaryotes. They repeat in tandem; vary, in number in different loci and differently in individuals. There, , are two main classes of repetitive and highly polymorphic, DNA; minisatellite DNA referring to genetic loci with repeats, of length 9-65 bp and microsatellite DNA with repeats of, 2-8 bp (1-6) long. Microsatellites are much more numerous, in the genome of vertebrates than mini satellites. They are, widely used in population genetics of fishes and aquatic, invertebrates. Minisatellites can be classified into multilocus, and single-locus minisatellites. Multilocus minisatellites are, composed of tandem repeats of 9-65 base pair and have a, total length ranging from 0.1 to 7kb. Minisatellite loci are, used mainly in parentage analysis. They are less useful for, population genetic analysis unless we use large sample, sizes. The complexity of mutation processes undergone by, minisatellite loci is also a limitation. Due to the difficulties in, the interpretation of multilocus fingerprints, the research work, were concentrated on single locus minisatellite probes and, this procedure required reasonable quantities of high-quality, DNA. These single locus minisatellite probes have been very, useful and successful in detecting genetic variations within, and between populations. It has also been used in fisheries, for forensics, parentage, genetic identity, estimating mating, success and confirming gynogenesis., , Microsatellites, A microsatellite is a simple DNA sequence which is repeated, several times across various points in the DNA of an organism., These repeats are highly variable and these loci can be used, as markers. Microsatellite occur once in every 10 kbp while, minisatellite loci occur once in every 1500 kbp in fishes and, due this, microsatellites are more useful in genome mapping, and population genetics studies. They are highly variable,, non-coding and selectively neutral and the basic assumption, while using microsatellite loci is that the predicted amount, of sequence divergence between units of interest is directly, related to length of time since separation. Microsatellites, are codominant markers which are inherited in a Mendelian, fashion and they are highly evolving with 10-3-10-4, mutation/generation. The high levels of polymorphism shown, by microsatellites have made them one of the most popular, genetic markers. Cross amplification with primers developed, in closely related species is also possible which minimizes the, cost associated with detecting microsatellite sequences in a, different species. The analysis of microsatellite loci involves, DNA extraction, amplification of the microsatellite loci using, specific primers in a PCR machine and examination of the, bands using poly acrylamide gel electrophoresis. The recent, introduction of automated genotyping machines has made, the analysis of size polymorphisms of microsatellite loci with, automated genotyping using labeled primers. The use of large, number of samples and loci is now possible due to automated, genotyping which has increased precision and speed with, microsatellite analysis. The constraints of using microsatellite, markers are the presence of null alleles and presence of, stutter bands. Null alleles are found when mutations occur, at primer binding sites of microsatellite locus. The presence, of null alleles reduce accuracy especially in parentage or, , Summer School on Recent Advances in Marine Biodiversity Conservation and Management, , 158

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Sandhya Sukumaran and A. Gopalakrishnan, relatedness analysis and assignment tests and the best, option is to discard loci showing null alleles. Stutter bands, occur when a ladder of bands differing between 1-2 bp is, seen and these occur due to slipped strands impairing during, PCR or incomplete denaturation of amplification products., Tri-nucleotide and tetra nucleotide repeats usually do not, show significant amounts of stuttering. Microsatellite markers, are used in fisheries and aquaculture for phylogenetics and, phylogeography, population genetic structure, biodiversity, conservation, stocking impacts and hybridization. It is, also being increasingly used for forensic identification of, individuals, genome mapping and determination of kinship, and behavioral patterns., , Single Nucleotide Polymorphisms, Single nucleotide polymorphisms arise due to single, nucleotide substitutions (transitions/transversions) or single, nucleotide insertions/deletions. These point mutations, give rise to different alleles with alternative bases at a, particular nucleotide position. SNP,s are the most abundant, polymorphisms in the genome (coding and non-coding) of any, organism. These single nucleotide variants can be detected, using PCR, microchip arrays or fluorescence technology. They, are considered as next generation markers in fisheries and, can be employed for population genetics studies, genomics, studies and for detection of diseases., , DNA microarrays or DNA chips, DNA microarray consists of small glass microscope slides,, silicon chip or nylon membranes with many immobilized, DNA fragments arranged in a standard pattern. A DNA, microarray can be utilized as a medium for matching, a reporter probe of known sequence against the DNA, isolated from the target sample which is of unknown origin., Species-specific DNA sequences could be incorporated to, a DNA microarray and this could be used for identification, purposes. DNA extracted from a target sample should be, labeled with a specific fluorescent molecule and hybridized, to the microarray DNA. When the hybridization is positive a, fluorescent signal is detected with appropriate fluorescence, scanning/imaging equipment., , Expressed Sequence Tags (ESTs), ESTs are single-pass sequences which were generated from, random sequencing of cDNA clones. ESTs can be used to, identify genes and analyze their expression by means of, expression analysis. Fast and reliable analysis can be made, for the genes expressed in particular tissue types under, specific physiological conditions or developmental stages., Differentially expressed genes could be identified using cDNA, microarrays in a systematic way. ESTs are most valuable for, linkage mapping., , Applications of molecular markers in, fisheries, Inter specific and intra specific variations, 16 February - 8 March 2015, , Molecular genetic markers can be used as a supplementary, marker system which will increase resolution in taxonomic, research. The molecular evolution among taxa is highly, variable and the extent of divergence in DNA or genes can, be taken as the basis for differentiation among species. The, morphological and ecological characters may diverge at a, faster rate compared to genetic differentiation at neutral, loci and so sometimes we may observe poor correlation, between morphological traits and gene divergence. Thus, molecular markers have to be interpreted critically among, different sets of traits. Molecular markers are most useful, when some species which occur in mixed catches is to be, identified and where morphological identification is very, difficult. Processed fish products (filleted, smoked or salted), and early life stages of fishes like planktonic eggs and larval, forms cannot be identified using morphological characters, and molecular markers can be employed in such instances., Endangered and threatened whales, sharks and dolphins, which are dead and stranded can also be identified using, these methods as in most cases morphological identification, is not possible. Within a species, genetic differences are, more than between populations and so identification is, possible even when sample size is small (3-5). Molecular, markers have also been used in sub-species identification., Some of the high evolving loci will show more divergence, within species and so these loci can be used for finding out, intra-specific variations., , Phylogenetic and Phylogeograhical studies, Phylogenetic studies assess the historical processes which, affect relationships and phylogeographic studies assess the, geographical distributions. Phylogenetic and phylogeographic, studies started with the introduction of mtDNA markers in, population genetic analyses. The evolutionary history of, groups of fishes could be reconstructed which will give vital, information regarding historical demography. Information, regarding conservation units and ecological patterns could, also be derived using phylogenetic studies. Mitochondrial, DNA analysis has been used widely as a powerful tool, for intraspecific phylogenetic patterns inference in many, animal species. The high levels of mutation rate, smaller, levels of effective population size and predominantly, maternal inheritance of mtDNA will provide greater power, to identify population structure. The lack of recombination, and low efficiency of repair mechanisms induces high rate of, evolution in mtDNA which makes this molecule highly useful, in phylogenetic analyses. MtDNA has been used to resolve, relationships among species that had diverged as much as, 8-10 million years., , Structure of populations: between and, within populations, Identification of stock structure is a very important issue, in fisheries management and conservation programmes., Stocks are groups within species which are reproductively, isolated with different physiological and behavioural, , Central Marine Fisheries Research Institute, , 159

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Molecular taxonomy – Applications, Limitations and future, patterns. Morphological and meristic features have, genetic and environmentally influenced components and, so morphological and meristic data should be used in, conjunction with genetic data. Thus when a combination of, these methods is used, we get information regarding actual, genetic differences and important environmental effects on, phenotype. Microsatellite DNA is the most favored DNA, marker for stock structure studies due to their high rate, of evolution. The non-coding region of mtDNA (D-loop), has also been used for stock structure studies due to their, high rate of evolution. Mixed fisheries are comprised of, subunits of different populations, different life stages or, individuals from different stocks. Data from microsatellite, markers, allozymes or mitochondrial DNA markers is useful, for identifying the origin of stock components from mixed, stock fisheries., , Genetic tagging/marking, There are instances when we need to mark individual fish, for various purposes like tracking movement or migration,, estimating population size or evaluating contributions, of individual stock to mixed fishery. Physical tags are not, heritable and so cannot be employed for generations., Genetic marking by finding a rare allele in an individual, or populations and following them over generations will, provide information regarding the contribution of hatchery, programme on harvest, proportion of stocked individuals, on growth of targeted population and identifying migrants, from source populations., , Forensic investigation, In samples where morphological identification is not possible, like dead or stranded fishes, preserved or canned fish flesh, and fish fillets, molecular markers could be employed for, identification and certification. Forensics uses scientific, methods to draw inferences about past events and it is, being increasingly used in certification of fishery products, and detection of illegal trading in fish and fisheries products., Deliberate or accidental release into natural waters also could, be monitored using molecular tools., , and other unusual sources of DNA is now possible with, several methods. These types of studies have improved our, understanding regarding the evolutionary relationships, among different taxa., , Applications in aquaculture, Molecular markers have wide range of applications in, aquaculture mainly; in genetic identification and discrimination, of hatchery stocks, finding out inbreeding events, assignment, of of progeny to parents using genetic tags, finding out, quantitative trait loci, marker assisted selection for selective, breeding trials and assessment of the effect of polyploidy, induction and gynogenesis. Genetic variability can also be, assessed between and within stocks using molecular markers., They are also useful in determining the contribution of, possible parents in mass spawning events. Genome mapping, and identification of quantitative trait loci (QTL), (the locations, of commercially important quantitative traits) is also possible., QTLs are important in breeding programmes and they are, detected by analyzing phenotypes with linked marker maps, and identification of markers related to QTLs can provide, information on relatedness between strains and families and, this knowledge is useful in marker-assisted programmes to, improve production related traits. Disease diagnosis is another, major area where the benefits of molecular markers can be, successfully harnessed. PCR assays for different pathogens, have become inexpensive, safe and user friendly in many, diagnostic laboratories. Several PCR based disease diagnosis, methods have been developed for pathogens like white spot, syndrome virus (WSSV), channel catfish virus (CCV), infectious, hematopoietic necrosis virus (IHNV), infectious pancreatic, necrosis virus (IPNV), viral hemorrhagic septicemia virus, (VHSV), viral nervous necrosis virus (VNNV) and several other, diseases. Genes that are resistant to pathogens like Major, Histo Compatibility genes (MHC) can be identified and used, for selection programmes to produce disease resistant strains, of fin or shell fishes., , Conclusion, , The most essential component of any ecological study is, determining trophic relationships within an ecosystem and, data on diet composition is very vital. Identifying the diet, components of species level is very difficult as most the, morphological features might get lost due to partial or, complete digestion. In many cases solid remains do not, exist inside the gut and this makes identification difficult., Molecular methods could be used for diet analysis studies, as it is possible to extract DNA from partially digested, samples., , Molecular markers find wide range of application fisheries, and aquaculture and their application has revolutionized the, field of fish genetics. The choice of a marker type should be, made cautiously in each case so as to maximize the quality, of output. No single molecular marker is superior to any, other and a combination of markers is always suitable. Of, late, markers developed and screened using next generation, sequencing technologies are increasingly being used in fish, genetics. There is an increasing global demand for aquaculture, products and modern molecular methods and molecular, genetics could play a major role in bringing out quality and, sustainability to aquaculture., , Analysis of ancient DNA, , Suggested reading, , Retrieval of DNA sequence information from preserved, samples in museums, fossil remains, archaeological finds, , Okumus, I. and Ciftci, Y. 2003. Fish population genetics and molecular markers:, II- Molecular markers and their applications in fisheries and aquaculture., Turkish Journal of Fisheries and Aquatic Sciences 3: 51-79., , Studying the trophic relationships, , Summer School on Recent Advances in Marine Biodiversity Conservation and Management, , 160

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Sandhya Sukumaran and A. Gopalakrishnan, Chauhan, T and Rajiv. K. 2010. Molecular markers and their application is fisheries, and aquaculture. Advances in Bioscience and Biotechnology 1: 281-291., Liu, Z.J. and Cordes, J.F. 2004. DNA marker technologies and their applications in, aquaculture genetics. Aquaculture 1-37., Presti, R.L., Lisa,C. and Stasio, L.D. 2009. Molecular genetics in aquaculture., Italian Journal of Animal Science 8: 299-313., Teletchea, F. 2009. Molecular identification methods of fish species: reassessment, and possible applications. Reviews in Fish Biology and Fisheries 19: 265293., Askari, G.H., Shabani, H. and Miandare, H.K. Application of molecular markers in, fisheries and aquaculture. Scientific Journal of Animal Science, 2(4): 82-88., Hallerman, E.M. 2006. Use of molecular tools for research and improvement of, aquaculture stocks. The Israeli Journal of Aquaculture – Bamidgeh, 58(4):, 286-296., Davis, G.P. and Hetzel, D.J.S. 2000. Integrating molecular genetic technology with, traditional approaches for genetic improvement in aquaculture species., Aquaculture Research, 31: 3-10., , 16 February - 8 March 2015, , Central Marine Fisheries Research Institute, , 161