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Review ArticleApplication of Microsatellite Markers inConservation Genetics and Fisheries ManagementRecent Advances in Population Structure Analysisand Conservation Strategies

P M Abdul-Muneer12

1 National Bureau of Fish Genetic Resources (NBFGR) Cochin Unit CMFRI Campus Cochin Kerala 682 018 India2 JFK Medical Center Edison NJ 08820 USA

Correspondence should be addressed to P M Abdul-Muneer pmamuneergmailcom

Received 27 January 2014 Revised 19 February 2014 Accepted 19 February 2014 Published 7 April 2014

Academic Editor Norman A Doggett

Copyright copy 2014 P M Abdul-MuneerThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microsatellites are the most popular and versatile genetic marker with myriads of applications in population genetics conservationbiology and evolutionary biology These are the arrays of DNA sequences consisting of tandemly repeating mono- di- tri- andtetranucleotide units which are distributed throughout the genomes of most eukaryotic species Microsatellites are codominantin nature highly polymorphic easily typed and Mendelian inherited all properties which make them very suitable for thestudy of population structure and pedigree analysis and capable of detecting differences among closely related species PCRfor microsatellites can be automated for identifying simple sequence repeat polymorphism Small amount of blood samples oralcohol preserved tissue is adequate for analyzing them Most of the microsatellites are noncoding and therefore variations areindependent of natural selectionThese propertiesmakemicrosatellites ideal geneticmarkers for conservation genetics and fisheriesmanagement This review addresses the applications of microsatellite markers in conservation genetics and recent advances inpopulation structure analysis in the context of fisheries management

1 Introduction

Organisms are incessantly undergoingmicro- andmacroevo-lutionary processes both at molecular and organismal levelsIn fact the process of evolution starts at the molecular levelmore precisely from a single base of the DNA molecule andends up in variations at the organismal level Genes are thefactors which determine the phenotypic characters of anyorganism Thus the variations that happen to the genes inturn produce individuals which are different either at themolecular level or at the organismal level These individualsmay form separate groups within the species itself and suchgroups are the fundamental genetic units of evolution Theseintraspecific groups were called as ldquostocksrdquo and fisherybiologists started using these stocks as a basis to managecommercially important marine organisms Shaklee et al[1] defined a stock as ldquoa panmictic population of related

individuals within a single species that is genetically distinctfrom other such populationsrdquoTherefore in any managementregime identification of discrete stocks becomes a criticalelement [2 3]

Genetic variation in populations became a subject ofscientific enquiry in the late nineteenth century prior even tothe rediscovery of Mendelrsquos paper in 1900 Genetic variationin the form of multiple alleles of many genes exists in mostnatural populations In most sexually reproducing popula-tions no two organisms (barring identical twins or othermultiple identical births) can be expected to have the samegenotype for all genes [4] In 1990s genetic markers becamemore popularized for the identification of stock structure andgenetic variation in a population The detection of geneticvariation among individuals is a requirement in all applica-tions of genetic markers in fisheries biology A geneticallyinherited variant in which the genotype can be inferred from

Hindawi Publishing CorporationGenetics Research InternationalVolume 2014 Article ID 691759 11 pageshttpdxdoiorg1011552014691759

2 Genetics Research International

the phenotype during genetic screening is known as a geneticmarkerThemost common use of geneticmarkers in fisheriesbiology is to determine if samples from culture facilities ornatural populations are genetically differentiated from eachother They are also used to identify different species in theevent of taxonomic disputes and to detect genetic introgres-sion in a species The detection of genetic differentiationwould imply that the source groups comprise different stocks[5] and should be treated as separate management units orstocks [6] A common objective ofmolecular genetic analysesis to find diagnostic differences among presumed stocksin either nuclear allelic types or mtDNA haplotypes [7]Polymorphic DNAmarkers can provide fisheries researcherswith new insights into the behavior ecology and geneticstructure of fish populations levels of inbreeding disassor-tative mating success of alternative reproductive strategiesand life histories and the intensity of natural and sexualselection [8] Microsatellites are one of the best suitablegenetic markers for analyzing pedigree population structuregenome variation evolutionary process and fingerprintingpurposes

Genetic markers are basically two typesmdashprotein andDNA (molecular) In the beginning of 1960s the proteinssuch as haemoglobin and transferrin were involved in allstudies In protein markers allozyme markers are very pop-ular and most of the genetic variation studies have been con-ducted based on this marker [9ndash14] Molecular markers canbe categorized into two classes nuclear DNA and mitochon-drial DNA (mtDNA) markers based on their transmissionand evolutionary dynamics [15] Nuclear DNA markers suchas random amplified polymorphic DNA (RAPDs) amplifiedfragment length polymorphisms (AFLPs) variable number oftandem repeats loci (VNTRs minisatellites microsatellites)and single nucleotide polymorphisms (SNPs) are bipar-ently inherited Mitochondrial DNA markers are maternallyinherited exhibit high rates of mutation and are non-recombining such that they have one-quarter the geneticeffective population size (Ne) of nuclear markers [8] Usingrestriction enzymes mtDNA sequence can be cut at specificsites to generate restriction fragment length polymorphisms(RFLPs) or sequence analysis of different genes of mtDNAcan be used to detect phylogenetic relationships undertakepedigree analysis and assess population differentiation inmany species

Detection of polymorphisms at the nucleotide sequencelevel represents a new area for genetic studies especially astechnologies become available which allow routine appli-cation with relative ease and low cost From the 1990s anincreasing number of studies have been publishedmaking useof random parts of a genome With the advent of thermo-cyclers the amplification of small fragment of DNA throughpolymerase chain reaction (PCR) gained popularityThePCRtechnique was discovered in 1985 and the development ofDNA amplification using the PCR technique has opened thepossibility of examining genetic changes in fish populationsover the past 100 years or more using archive materials suchas scales [8] The advent of PCR coupled with automatedDNA sequencers made feasible major technological innova-tions such as minisatellite variant repeat mapping [16] and

assessment of the variations at microsatellite loci [17] ThePCR based techniques have the added attraction of requiringonly extremely small amounts of DNA that has led to wideusage of this technique in aquaculture and fisheries In thisreview we discuss the application of the most prevalentgenetic marker microsatellites in population genetic struc-ture and its usefulness in conservation of fish fauna

2 Microsatellites Markers

Recently attention has turned to another type of genetic vari-ation that of differences in the number of repeated copiesof a segment of DNA These sequences can be classifiedbased on decreasing sizes into satellites minisatellites andmicrosatellites [13] Satellites consist of units of severalthousand base pairs repeated thousands or millions of timesMinisatellites consist of DNA sequences of some 9ndash100 bp inlength that are repeated from 2 to several 100 times at a locusMinisatellites discovered in human insulin gene loci withrepeat unit lengths between 10 and 64 bp were also referredto as ldquovariable number of tandem repeatsrdquo (VNTRs) DNA[18] Microsatellites have a unique length of 1ndash6 bp repeatedup to about 100 times at each locus [19] They are also calledas ldquosimple sequence repeatrdquo (SSR) by Tautz [13] or ldquoshorttandem repeatrdquo (STR) DNA by Edwards et al [20] Jeffreyset al [21] and Weber [22] opined that length variations intandemly arrayed repetitiveDNA inmini- andmicrosatellitesare usually due to an increase or decrease in repeat unit copynumbers Differences in repeat numbers represent the basefor most DNA profiling techniques used today Later onlymicrosatellites became very common in population geneticsstudies

Microsatellites are short tandemly arrayed di- tri- ortetranucleotide repeat sequences with repeat size of 1ndash6 bprepeated several times flanked by regions of nonrepetitiveunique DNA sequences [13] Polymorphism at microsatelliteloci was first demonstrated by Tautz [13] and Weber andMay [17] Alleles at microsatellite loci can be amplified bythe polymerase chain reaction [23] from small samples ofgenomic DNA and the alleles separated and accurately sizedon a polyacrylamide gel as one or two bands and they areused for quantifying genetic variations within and betweenpopulations of species [24]The very high levels of variabilityassociated with microsatellites the speed of processing andthe potential to isolate large number of loci provide a markersystem capable of detecting differences among closely relatedpopulations Microsatellites that have been largely utilizedfor population studies are single locus ones in which boththe alleles in a heterozygote show codominant expression[25] Individual alleles at a locus differ in the number oftandem repeats and as such can be accurately differentiatedon the basis of electrophoresis (usually PAGE) according totheir size Different alleles at a locus are characterized bydifferent number of repeat units They give the same kind ofinformation as allozymes distinguishable loci with codomi-nant alleles but they are generally neutral and more variablethan allozymes [26] Like allozymesmicrosatellites alleles areinherited in a Mendelian fashion [27] Moreover the allelescan be scored consistently and compared unambiguously

Genetics Research International 3

TT G

Sample collection

Genomic DNA isolation

Restriction digestion of DNA

DNA fragment

Vector DNA

+

RecombinantDNA

Construction of recombinant DNA

Transform in bacteria and screenfor microsatellite repeat sequence

with radiolabelled probe

Selection of positive coloniesand sequencing

3998400

3998400

5998400

5998400

CG

G

T

CG

T

A

A GC

C

GC

TA

A

A

CG

CG C C

C

C

G G

3998400

5998400

3998400

5998400

AT

A

A

A

A A

T

T

T

Figure 1 Schematic representation of traditional method of devel-opment of species specific microsatellite markers

even across different gels An additional advantage is that theyallow the use of minute or degraded DNA [26]

Generally microsatellite loci are abundant and dis-tributed throughout the eukaryotic genome [28] and eachlocus is characterized by known DNA sequence Thesesequences consist of both unique DNA (which defines thelocus) and repetitive DNA motifs (which may be sharedamong loci) The repetitive elements consist of tandemreiterations of simple sequence repeats (SSRs) and are typ-ically composed of two to four nucleotides such as (AC)n

T G

3998400

3998400

5998400

5998400

CG

T

CG

T

A

A GC

C

GC

TA

A

A

CG

AT

Collect microsatellite sequenceinformation from resource species

(closely related or from the same family)design primers

Genomic DNA isolation fromspecies of interest

Taqpolymerase

PrimerNucleotides

DNA

ATCG

PCR amplification of DNA usingresource primers

Gel electrophoresis of PCR product

Elute DNA from gel

Topoisomerase

Topoisomerase

Tyr-274P

P

O

O

OH

AAPCR product

Tyr-274

HO

Clone PCR product in a TA vector

Transformation and isolationof recombinant plasmid

Sequencing and confirmationof microsatellites

CCCTTGGGA

AGGGTTCCC

Figure 2 Schematic representation of development ofmicrosatellitemarkers by cross-species amplification

or (GATA)n where n lies between 5 and 50 [29] Withinvertebrates the dinucleotide repeats -GT and CA- arebelieved to be themost commonmicrosatellites [30] Study ofsingle locus microsatellites requires specific primers flankingthe repeat units whose sequences can be derived from (i)genomic DNA libraries or (ii) from available sequences inthe gene banks (Figures 1 and 2) These two methods aregenerally used for the development of microsatellite markersThe second method is extensively described in the comingsection In a review Zane et al [31] showed several methodsof development of microsatellite markers


Populationgenetics andconservation

biology Gene taggingand QTLanalysis

Hybridizationand breeding

Functionalgenomics

Forensic science

Genome mapping

Diversity andcultivar analysis

Diagnosisand identification

of humandiseases

Taxonomicand phylogenetic

studies

Pedigreeand gender

identification

Applications ofmicrosatellites

Figure 3 Applications of microsatellite markers in different areas

3 Advantages of Microsatellite Markers

The major advantages of microsatellite markers are codom-inant transmission (the heterozygotes can be distinguishedfrom homozygotes) locus-specific in nature highly poly-morphic and hypervariable high information content andproviding considerable pattern relative abundance with uni-form genome coverage higher mutation rate than standardand easy to sample preparation Advantages ofmicrosatellitessuch as short size range uninterrupted stretches of identicalrepeat units high proportion of polymorphisms insightsgained in understanding the mutational process which helpsin developing statistical procedures for interpopulation com-parisons their abundance in fish genomes the availability ofmethodologies for cloning of microsatellites have all resultedin their abundant use in fisheries research Tetranucleotidemicrosatellites are also very useful for paternity and forensicinvestigations in humans The advantageous properties ofmicrosatellites have led to modern developments such asdigital storage and automated detection and scoring systemssuch as automated DNA sequences and fluorescent-imagingdevices [27] Disadvantages of microsatellites include theappearance of shadow or stutter bands presence of nullalleles (existing alleles that are not observed using stan-dard assays) homoplasy and too many alleles at certainloci that would demand very high sample size for analysis[32] Also microsatellite flanking regions (MFRs) sometimescontain lengthmutationswhichmay produce identical lengthvariants that could compromise microsatellite populationlevel studies (and comparisons of levels of variation acrossspecies for homologous loci) and phylogenetic inferences as

these length variants in the flanking regions can potentiallyminimize allele length variation in the repeat region [30]

4 Application of Microsatellites inPopulation Structure Analysis in Fisheriesand Aquaculture

The high variability ease and accuracy of assaying microsat-ellites make them the marker of choice for high-resolutionpopulation analysis [33] Microsatellites with only a fewalleles are well suited for population genetic studies while themore variable loci are ideal for genomemapping and pedigreeanalysis and the fixed or less polymorphic microsatellite lociare used to resolve taxonomic ambiguity in different taxa[5] Highly polymorphic microsatellite markers have greatpotential utility as genetic tags for use in aquaculture andfisheries biology They are powerful DNA markers for quan-tifying genetic variations within and between populationsof species [25] They may prove particularly valuable forstock discrimination and population genetics due to the highlevel of polymorphism comparedwith conventional allozymemarkers [34 35] Microsatellite DNAmarkers are among themost likely to conform to the assumption of neutrality andhave proven to be powerful in differentiating geographicallyisolated populations and sibling species and subspecies [30]The qualities of microsatellites make them very useful asgenetic markers for studies of population differentiation andstock identification [35 36] in kinship and parentage exclu-sion [37 38] and in genome mapping [39] Microsatellitesare also being used as genetic markers for identification of


population structure genome mapping pedigree analysisand to resolve taxonomic ambiguities in many other animalsbesides fishes [40ndash49] The broad areas of applications ofmicrosatellite markers are depicted in Figure 3

Various authors have reported microsatellite polymor-phisms and sequences in some marine and freshwater fishspecies for population genetic analysis [25 34 50ndash55]The development of polymorphic microsatellite markersto determine the population structure of the Patagoniantoothfish Dissostichus eleginoides has been reported by[56 57] Similarly Appleyard et al [58] examined sevenmicrosatellite loci in the same species of Patagonian toothfishfrom three locations in the Southern Ocean Microsatellitepolymorphisms have been used to provide evidence thatthe cod in the northwestern Atlantic belongs to geneticallydistinguishable populations and that genetic differences existbetween the northwestern and southeastern cod populations[59] Recently Larsen et al [60] showed differences in salinitytolerance and its gene expression in two populations ofAtlantic cod (Gadus morhua) Drinan et al [61] reported20 microsatellites for determining the patterns of populationgenetic variation in westlope cutthroat trout Oncorhynchusclarkia lewisii in 25 populations from four rivers Davies et al[62] identified 12 microsatellite loci in tuna species of genusThunnus and investigated genetic polymorphism at these lociin North Atlantic and Mediterranean Sea populations In acichlid Eretmodus cyanostictus Taylor et al [63] determinedfour polymorphic microsatellite loci for studying nine popu-lations in Lake Tanganyika In another study recently 7 poly-morphic microsatellite markers were identified in snakeheadmurrelChanna striata fromMalaysia [64] Similarly severalauthors reported population genetic structure of differentspecies of catfish few of them are in the farmed catfish fromTamaulipas Mexico [65] in neotropical catfish [66] in Pseu-doplatystoma reticulatum [67] OrsquoConnell et al [24] reportedthe investigation of five highly variable microsatellite locifor population structure in Pacific herring Clupea pallasicollected from6 sites in Kodiak Island Similarlymany othershave reported studies of polymorphic microsatellite loci toevaluate population structure of different fish species Thusmicrosatellite markers have wide range of applications inpopulation genetics and fisheries management

Salzburger et al [68] reported a case of introgressivehybridization between an ancient and genetically distinctcichlid species in Lake Tanganyika that led to the recognitionof a new species This is evidenced by the analysis of flankingregions of the single copy nuclear DNA locus (TmoM27) andby studying the parental lineages in six other microsatelliteloci Leclerc et al [69] had cloned and characterized a highlyrepetitive DNA sequence from the genome of the NorthAmerican Morone saxatilis that was used to distinguish thefour other species Neff et al [70] described 10 microsatelliteloci from blue gill (Lepomis macrochirus) and discussedtheir evolution within the family Centrarchidae Kellogget al[71] applied microsatellite-fingerprinting approach toaddress questions about paternity in cichlids The usefulnessof microsatellite markers for genetic mapping was deter-mined inOreochromis niloticus by Lee and Kocher [72] whileBrooker et al [73] reported the difference in organization

of microsatellite between mammals and cold water teleostfishes DeWoody and Avise [29] reported microsatellitevariation in marine fresh water and anadromous fishescompared with other animals Microsatellite DNA variationwas used for population structure in Oncorhynchus kisutch[74] Atlantic salmon [75] and in Brown Trout Salmo trutta[76] Microsatellite markers have been studied in a fewcyprinids also Naish and Skibinski [77] studied tetranu-cleotide (TCTA) repeat sequences in Indianmajor carpCatlacatla as potential DNA markers for stock identificationGopalakrishnan et al [51] and Das et al [78] carried outcharacterization of dinucleotide microsatellite repeats inLabeo rohita

5 Development of Microsatellite Markers byCross-Species Amplification

Although microsatellite DNA analysis via PCR is an idealtechnique for answering many population genetic questionsthe development of species-specific primers for PCR ampli-fication of alleles can be expensive and time-consumingas it involves construction of genomic libraries screen-ing of clones with microsatellite sequences and designingmicrosatellite primers However there are reports whichpoint to the fact that flanking sequences of some microsatel-lite loci are conserved among related taxa so that primersdeveloped for one species can be used to amplify homologousloci in related species The method of microsatellite markersdevelopment by cross-species amplification is shown inFigure 2 The conservation of flanking regions of microsatel-lite sequences among closely related species has beenreported by a number of groups [79ndash82] Such an approachcan circumvent extensive preliminary work necessary todevelop PCR primers for individual loci that continues tostand in theway of quick andwidespread application of singlelocus microsatellite markers Thus by using heterologousPCR primers the cost of developing similarmarkers in relatedspecies can be significantly reduced

Schlotterer et al [83] found that homologous loci can beamplified from a diverse range of toothed (Odontoceti) andbaleen (Mysticeti) whales with estimated divergence times of35ndash40 million years Moore et al [84] found that microsatel-lites flanking regions were conserved across species as diverseas primates artiodactyls and rodents Microsatellite primersdeveloped from foxtail millet (Setaria italica L) were used instudies of other millets and nonmillets species [85] Similarlyprimers developed for passerine birds were used in studies ofa variety a of other bird species [86]

A number of attempts have beenmade to study the cross-species amplification of microsatellite loci in fishes RecentlyGupta et al [87] developed polymorphicmicrosatellitemark-ers in featherback Notopterus notopterus by cross-speciesamplification of primers developed in 3 fish species of familiesnotopteridae and osteoglossidae Polymerase chain reaction(PCR) microsatellite multiplex assays were established forgenetic studies of the population structure hybridization andconservation status of European whitefish Coregonus lavare-tus L and cross-species amplification and rearrangementof the same loci analyzed in C albula L [88] Dubut et al


M 1 2 3 4 5 6 7 8 9 10 11 12M M

Figure 4 Cross-species amplification of microsatellite markers forthe population genetic structure from three river systems inHoraba-grus brachysoma (yellow catfish) from the primer (Cga06) developedin Clarias gariepinus (African catfish) The data of this figure hasbeen published by Abdul Muneer et al [25] M molecular weightmarker (pBR322 with MspI cut)

[89] have developed five multiplex PCR sets optimized toanalyze 41 cyprinid-specific polymorphic microsatellite loci(including 10 novel loci isolated from Chondrostoma nasusChondrostoma toxostoma and Leuciscus leuciscus) for theindividuals from other different European cyprinid species

We have developed several microsatellite markers indifferent fresh water species by cross-species amplificationIn Horabagrus brachysoma an endangered yellow catfishwe have developed eight microsatellite markers from othercatfish of order Siluriformes [25 52] Figure 4 shows thecross-species amplification microsatellites in Horabagrusbrachysoma from the primer developed in African catfishClarias gariepinus [25] In addition we developedmicrosatel-lite markers for differentiating two species of endangeredcatfishHorabagrus by using the primers of Siluriformes andOsteoglossiformes [79] May et al [90] reported microsatel-lite genetic variation through cross-species amplification insturgeons Acipenser and Scaphirhynchus Takagi et al [91]reported that microsatellite primers isolated from one tunamight be used to amplify microsatellite loci in other tunaspecies especially those of the genusThunnus Microsatellitesfrom rainbow trout Oncorhynchus mykiss have been used forthe genetic study of salmonids [75 92] Heterologous primershave been used to characterize bull trout by using three sets ofprimers from sockeye salmon rainbow trout and brook trout[93] for several Salvelinus species using primers of Salvelinusfontinalis for Brook charr [94] and Oreochromis shiranusand O shiranus chilwae by using primers of Nile tilapia[95]The cross-species amplification of 32Oreochromis niloti-cus microsatellite markers from 15 different African cichlidspecies was successfully tested and analyzed [96] There aresome reports in which the flanking sequences are conservedbetween families of the same order Primers of sticklebackand cod have been used in Merlangius merlangus (Gadidae)[97] that of rainbow trout (Family Salmonidae) in whitefishCoregonus nasus [98] and primers of goldfish Carassiusauratus in nine species of cyprinids [99] Yue et al [100]developed 15 polymorphicmicrosatellite loci in silver cruciancarp Carassius auratus gibelio and reported eleven out of 15

primer pairs cross-amplified in the genome of common carp(Cyprinus carpio) Zardoya et al [30] through a classical studydemonstrated that microsatellite flanking regions (MFRs)contain reliable phylogenic information and they were ableto recover with considerable confidence the phylogeneticrelationship within family Cichlidae and other families ofthe suborder Labroidei from different parts of the worldMohindra et al [32] have carried out cross-species amplifica-tion of C catla G1 primer in Catla catla from Gobind SagarLabeo dero L dyocheilus L rohita and Morulius calbasuand sequenced the loci in these species Das et al [78] alsocarried out characterization of dinucleotide microsatelliterepeats in Labeo rohita Recently we successfully developedpolymorphic microsatellite markers for Gonoproktopteruscurmuca through cross-species amplification of primers fromother cyprinid fishes [101 102] The development of 59polymorphic microsatellite markers in silver crucian carp(Carassius auratus gibelio) and its successful cross-speciesamplification have been reported in crucian carp (Carassiusauratus) [103]

Microsatellites have become the geneticmarkers of choicefor studies of population differentiation and parentage deter-mination However several microsatellite loci are requiredfor such studies in order to obtain an appropriate amount ofgenetic polymorphism [9 104] Fortunately genotypic datacollection has become efficient through the development ofautomated DNA sizing technology using fluorescent-labelledDNA and coamplification of multiple loci in a single PCR[24 105]

6 Importance of Microsatellite Markers inConservation and Fisheries Management

The microsatellite markers study generate important infor-mation on the genetic variation and stock structure of fishspecies and it is a significant step towards realizing the goalof management of fishery and conservation of the speciesin their natural populations The differentiation of a speciesinto genetically distinct populations is a fundamental partof the process of evolution and it depends upon physicaland biological forces such as migration selection geneticdrift and geographic barriers Endangered species will havesmall andor declining populations so inbreeding and loss ofgenetic diversity are unavoidable in them Since inbreedingreduces reproduction and survival rates and loss of geneticdiversity reduces the ability of populations to evolve to copewith environmental changes Frankham [106] suggested thatthese genetic factors would contribute to extinction riskespecially in small populations of threatened species Withthe loss of a populationgenetic stock a species also loses itsmembers adapted and evolved to survive in particular habitatHence conservation and fishery management strategy needto be stock-specific

In population genetic analysis low genetic variabil-ity (heterozygote deficiency and deviation from Hardy-Weinberg equilibrium) coupled with inbreeding (positivevalue of 119865IS) show consequence of genetic bottleneck result-ing from overexploitation and habitat [107] As these factorswould lead to a reduction in reproductive fitness [108] efforts


to increase the genetic diversity of the fish species should begiven high priority for conservation of the species based ongenetic principles as mentioned below

(i) The effective population size (Ne) should be main-tained as large as possible to maximize the contribu-tion of a large number of adults for reproduction soas to maintain natural genetic variability

(ii) The causative factors that reduce the effective popula-tion size such as overexploitation should be controlledat the earliest

(iii) No artificial gene flow between distinct stocks shouldbe created by means of haphazard stocking andrehabilitation programs

(iv) The rehabilitation strategy should also include means(screening the population using genetic markers) tomonitor impact of such program

To attain these objectives it is essential (i) to protect thepopulations and habitat against anthropogenic stress and (ii)enhance the population through propagation assisted stock-specific rehabilitation programs

(i) regulation of human activities either self-imposed(public understanding and awareness through educa-tion) or state imposed (formulation and implementa-tion of suitable laws)

(ii) imposing ban on fishing practices particularly duringbreeding seasons

(iii) stock assessment of different rivers and imposingquota systems for maintaining the population size

(iv) banning the sale of undersized specimens(v) restricting the fishing gear for not catching small

and immature fish species and preventing the use ofexplosives and chemicals for fishing

(vi) maintaining minimum water level in the rivers (incase there are dams and weirs) and declaring certainstretches of rivers as sanctuaries

The natural populations of the endangered species canbe enhanced by ldquosupportive breedingrdquo In this program afraction of the wild parents are bred in captivity and theprogeny are released in natural waters

(1) Brood stock of fish species collected from differentrivers must be tagged and maintained in separateponds in the holding facility

(2) Effective breeding population size and sex ratioshould not be restricted To achieve this collection ofdifferent sizeyear classes at different time intervals isto be preferred over the same sizeyear class

(3) Use of cryopreserved milt collected from differentmales and pooled would be useful for increasing theeffective population size and recovery of endangeredpopulations of fish species In comparison to thecaptive breeding program the gene banking throughsperm cryopreservation is relatively cheaper easy to

maintain and less prone to risk due to system failureormortality due to diseasesTherefore it should serveas a useful adjunct to the captive breeding program

(4) Different genetic stocks should be bred separatelyand ranched in the same rivers from where they arecollected

(5) Stretches of rivers harbouring resident population orthat can serve as a potential sanctuarymay be selectedfor ranching of fish populations

(6) Assessing the impact of ranching throughmonitoringthe parameters like catch per unit effortarea throughexperimental fishing should be done

(7) Changes in genetic variation that is allele frequen-cies especially the occurrence of rare alleles over acourse of time [19 24] should be done It will be usefulto keep base genetic profile of representative samplesof fish stocked in the holding facility and those usedfor ranching Microsatellite markers and the baselinedata generated in this study can be helpful in furtherassessing the impact of genetic variation

7 Conclusion

Microsatellites are very powerful genetic markers for identi-fying fish stock structure and pedigree analysis and to studythe genetic variation of closely related species Microsatellitemarkers analysis provides essential information for formulat-ingmeaningful conservation strategies for fisheries and aqua-culture management This along with the other technologieslike captive breeding and sperm cryopreservation can beintegrated into a package for conserving genetic diversity andrehabilitation of the natural populations of fish species

Conflict of Interests

The author declares that he has no conflict of interests

Acknowledgments

The author is thankful to Dr VS Basheer Dr WS Lakra andDr AG Ponniah for encouragement support and guidance

References

[1] J B Shaklee F W Allendorf D C Morizot and G SWhitt ldquoGene nomenclature for protein-coding loci in FishrdquoTransactions of American Fisheries Society vol 119 pp 2ndash151990

[2] P E Ihssen H E Booke J M Casselman J M McGlade NR Payne and F M Utter ldquoStock identification materials andmethodsrdquo Canadian Journal of Fisheries and Aquatic Sciencesvol 38 pp 7838ndash7855 1981

[3] C M Fetterolf Jr ldquoForeword to the stock concept symposiumrdquoCanadian Journal of Fisheries and Aquatic Sciences vol 38 pp4ndash5 1981

[4] D L Hartl and A G Clark Principles of Population GeneticsSinauer Associates Sunderland Mass USA 2nd edition 1989


[5] G R Carvalho and LHauser ldquoMolecular genetics and the stockconcept in fisheriesrdquo Reviews in Fish Biology and Fisheries vol4 no 3 pp 326ndash350 1994

[6] C Moritz ldquoDefining ldquoevolutionarily significant unitsrdquo for con-servationrdquo Trends in Ecology and Evolution vol 9 no 10 pp373ndash375 1994

[7] R G Danzmann and P E Ihssen ldquoA phylogeographic survey ofbrook charr (Salvelinus fontinalis) in Algonquin Park Ontariobased upon mitochondrial DNA variationrdquo Molecular Ecologyvol 4 no 6 pp 681ndash697 1995

[8] MM Ferguson and R G Danzmann ldquoRole of genetic markersin fisheries and aquaculture useful tools or stamp collectingrdquoCanadian Journal of Fisheries and Aquatic Sciences vol 55 no7 pp 1553ndash1563 1998

[9] A Ferguson J B Taggart P A Prodohl et al ldquoThe applicationof molecular markers to the study and conservation of fishpopulations with special reference to Salmordquo Journal of FishBiology vol 47 supplement A pp 103ndash126 1995

[10] G A Penner A Bush R Wise et al ldquoReproducibility ofrandom amplified polymorphic DNA (RAPD) analysis amonglaboratoriesrdquo in PCR Methods and Applications pp 347ndash345Cold Spring Harbor Laboratory Press 1993

[11] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[12] J Welsh and M McClelland ldquoFingerprinting genomes usingPCR with arbitrary primersrdquoNucleic Acids Research vol 18 no24 pp 7213ndash7218 1990

[13] D Tautz ldquoHypervariability of simple sequences as a generalsource for polymorphic DNAmarkersrdquo Nucleic Acids Researchvol 17 no 16 pp 6463ndash6471 1989

[14] A J Jeffreys V Wilson and S L Thein ldquoHypervariableldquominisatelliterdquo regions in human DNArdquo Nature vol 314 no6006 pp 67ndash73 1985

[15] L K Park and P Moran ldquoDevelopments in molecular genetictechniques in fisheriesrdquo Reviews in Fish Biology and Fisheriesvol 4 no 3 pp 272ndash299 1994

[16] A J Jeffreys A MacLeod K Tamaki D L Neil and DMonckton ldquoMinisatellite repeat coding as a digital approach toDNA typingrdquo Nature vol 354 no 6350 pp 204ndash209 1991

[17] J L Weber and P E May ldquoAbundant class of human DNApolymorphisms which can be typed using the polymerase chainreactionrdquoThe American Journal of Human Genetics vol 44 no3 pp 388ndash396 1989

[18] Y Nakamura M Leppert and P OrsquoConnell ldquoVariable numberof tandem repeat (VNTR) markers for human gene mappingrdquoScience vol 235 no 4796 pp 1616ndash1622 1987

[19] M Litt and J A Luty ldquoA hypervariable microsatellite revealedby in vitro amplification of a dinucleotide repeat within thecardiac muscle actin generdquo The American Journal of HumanGenetics vol 44 no 3 pp 397ndash401 1989

[20] A Edwards A Civitello H A Hammond and C T CaskeyldquoDNA typing and geneticmappingwith trimeric and tetramerictandem repeatsrdquoThe American Journal of Human Genetics vol49 no 4 pp 746ndash756 1991

[21] A J Jeffreys N J Royle VWilson and ZWong ldquoSpontaneousmutation rates to new length alleles at tandem-repetitive hyper-variable loci in humanDNArdquoNature vol 332 no 6161 pp 278ndash281 1988

[22] J LWeber ldquoInformativeness of human (dC-dA)(n)sdot(dG-dT)(n)polymorphismsrdquo Genomics vol 7 no 4 pp 524ndash530 1990

[23] R K Saiki D H Gelfand S Stoffel et al ldquoPrimer-directedenzymatic amplification of DNA with a thermostable DNApolymeraserdquo Science vol 239 no 4839 pp 487ndash491 1988

[24] M OrsquoConnell R G Danzmann J-M Cornuet J M Wrightand M M Ferguson ldquoDifferentiation of rainbow trout(Oncorhynchus mykiss) populations in Lake Ontario and theevaluation of the stepwise mutation and infinite allele mutationmodels using microsatellite variabilityrdquo Canadian Journal ofFisheries and Aquatic Sciences vol 54 no 6 pp 1391ndash1399 1997

[25] P M Abdul Muneer A Gopalakrishnan K K Musam-milu et al ldquoGenetic variation and population structure ofendemic yellow catfish Horabagrus brachysoma (Bagridae)among three populations of Western Ghat region using RAPDand microsatellite markersrdquoMolecular Biology Reports vol 36no 7 pp 1779ndash1791 2009

[26] D C Queller J E Strassmann and C R Hughes ldquoMicrosatel-lites and kinshiprdquo Trends in Ecology and Evolution vol 8 no 8pp 285ndash288 1993

[27] M OrsquoConnell and J M Wright ldquoMicrosatellite DNA in fishesrdquoReviews in Fish Biology and Fisheries vol 7 no 3 pp 331ndash3631997

[28] D Tautz and M Renz ldquoSimple sequences are ubiquitousrepetitive components of eukaryotic genomesrdquo Nucleic AcidsResearch vol 12 no 10 pp 4127ndash4138 1984

[29] J A DeWoody and J C Avise ldquoMicrosatellite variation inmarine freshwater and anadromous fishes comparedwith otheranimalsrdquo Journal of Fish Biology vol 56 no 3 pp 461ndash4732000

[30] R Zardoya DM Vollmer C Craddock J T Streelman S Karland A Meyer ldquoEvolutionary conservation of microsatelliteflanking regions and their use in resolving the phylogeny ofcichlid fishes (Pisces Perciformes)rdquo Proceedings of the RoyalSociety B Biological Sciences vol 263 no 1376 pp 1589ndash15981996

[31] L Zane L Bargelloni and T Patarnello ldquoStrategies formicrosatellite isolation a reviewrdquoMolecular Ecology vol 11 no1 pp 1ndash16 2002

[32] V Mohindra A Mishra M Palanichamy and A G PonniahldquoCross-species amplification of Catla catla microsatellite locusin Labeo rohitardquo Indian Journal of Fisheries vol 48 no 1 pp103ndash108 2001

[33] A Estoup P Presa F Krieg D Vaiman and R Guyomardldquo(CT)n and (GT)n microsatellites a new class of geneticmarkers for Salmo trutta L (brown trout)rdquoHeredity vol 71 no5 pp 488ndash496 1993

[34] P M Abdul Muneer A Gopalakrishnan K K Musammilu etal ldquoComparative assessment of genetic variability in the pop-ulations of endemic and endangered Yellow Catfish Horaba-grus brachysoma (Teleostei Horabagridae) based on allozymeRAPD and microsatellite markersrdquo Biochemical Genetics vol50 pp 192ndash212 2012

[35] J M Wright and P Bentzen ldquoMicrosatellites genetic markersfor the futurerdquo Reviews in Fish Biology and Fisheries vol 4 no3 pp 384ndash388 1994

[36] Z J Liu and J F Cordes ldquoDNA marker technologies and theirapplications in aquaculture geneticsrdquo Aquaculture vol 238 no1ndash4 pp 1ndash37 2004

[37] M SWebster andL Reichart ldquoUse ofmicrosatellites for parent-age and kinship analyses in animalsrdquo Methods in Enzymologyvol 395 pp 222ndash238 2005


[38] M M Hansen E Kenchington and E E Nielsen ldquoAssigningindividual fish to populations using microsatellite DNA mark-ersrdquo Fish and Fisheries vol 2 no 2 pp 93ndash112 2001

[39] M Sanetra F Henning S Fukamachi and A Meyer ldquoAmicrosatellite-based genetic linkage map of the cichlid fishAstatotilapia burtoni (Teleostei) a comparison of genomicarchitectures among rapidly speciating cichlidsrdquo Genetics vol182 no 1 pp 387ndash397 2009

[40] X Lu H Xu Z Li H Shang R P Adams and KMao ldquoGeneticdiversity and conservation implications of four Cupressusspecies in China as revealed by microsatellite markersrdquo Bio-chemical Genetics vol 52 no 3-4 pp 181ndash202 2014

[41] G Nikbakht A Esmailnejad and N Barjesteh ldquoLEI0258microsatellite variability in Khorasan Marandi and Arianchickensrdquo Biochemical Genetics vol 51 pp 341ndash349 2013

[42] P Choudhary S M Khanna P K Jain C Bharadwaj J KumarP C Lakhera et al ldquoMolecular characterization of primary genepool of chickpea based on ISSR markersrdquo Biochemical Geneticsvol 51 pp 306ndash322 2013

[43] A Arias-Perez J Fernandez-TajesM B Gaspar and JMendezldquoIsolation of microsatellite markers and analysis of geneticdiversity among east atlantic populations of the sword razorshell Ensis siliqua a tool for populationmanagementrdquo Biochem-ical Genetics vol 50 pp 397ndash415 2012

[44] R Zhou Y Li J-Q Li and N-F Liu ldquoSeasonal changes inthe genetic diversity of two rodent populations Midday Gerbil(Meriones meridianus) and NorthernThree-Toed Jerboa (Dipussagitta) Detected by ISSRrdquo Biochemical Genetics vol 50 pp350ndash371 2012

[45] C R M Fernandes C F Martins K M Ferreira andM A Del Lama ldquoGene variation population differentiationand sociogenetic structure of nests of Partamona seridoensis(Hymenoptera ApidaeMeliponini)rdquo Biochemical Genetics vol50 pp 325ndash335 2012

[46] P Upadhyay C N Neeraja C Kole and V K Singh ldquoPopu-lation structure and genetic diversity in popular rice varietiesof India as evidenced from SSR analysisrdquo Biochemical Geneticsvol 50 pp 770ndash783 2012

[47] R K Joshi S Mohanty B Kar and S Nayak ldquoAssessment ofgenetic diversity in Zingiberaceae through nucleotide bindingsite-based motif-directed profilingrdquo Biochemical Genetics vol50 pp 642ndash656 2012

[48] Q Xu and R Liu ldquoDevelopment and characterization ofmicrosatellite markers for genetic analysis of the swimmingcrab Portunus trituberculatusrdquo Biochemical Genetics vol 49no 3-4 pp 202ndash212 2011

[49] P Supungul P Sootanan S Klinbunga W Kamonrat PJarayabhand and A Tassanakajon ldquoMicrosatellite polymor-phism and the population structure of the black tiger shrimp(Penaeus monodon) in Thailandrdquo Marine Biotechnology vol 2no 4 pp 339ndash347 2000

[50] P M A Muneer R Sivanandan A Gopalakrishnan V SBasheer K K Musammilu and A G Ponniah ldquoDevelopmentand characterization of RADP and microsatellite markers forgenetic variation analysis in the critically endangered yellowcatfish Horabagrus nigricollaris (Teleostei Horabagridae)rdquo Bio-chemical Genetics vol 49 no 1-2 pp 83ndash95 2011

[51] A Gopalakrishnan K K Musammilu V S Basheer et al et alldquoLow genetic differentiation in the populations of the malabarcarp labeo dussumieri as revealed by allozymes microsatellitesand RAPDrdquo Asian Fisheries Science vol 22 no 2 pp 359ndash3912009

[52] A Gopalakrishnan P M Abdul Muneer K K MusammiluK K Lal D Kapoor and V Mohindra ldquoPrimers fromthe orders Osteoglossiform and Siluriform detect polymor-phic microsatellite loci in sun-catfish Horabagrus brachysoma(Teleostei Bagridae)rdquo Journal of Applied Ichthyology vol 22 no5 pp 456ndash458 2006

[53] T-J Xu X-Q Quan Y-N Sun K-C Zhao and R-X WangldquoA first set of polymorphic microsatellite loci from the marbledrockfish sebastiscus marmoratusrdquo Biochemical Genetics vol48 no 7-8 pp 680ndash683 2010

[54] A Mandal K K Lal V Mohindra et al ldquoEvaluation ofgenetic variation in the clown knifefish Chitala chitala usingallozymes RAPD and microsatellitesrdquo Biochemical Geneticsvol 47 no 3-4 pp 216ndash234 2009

[55] T Chauhan K K Lal V Mohindra et al ldquoEvaluating geneticdifferentiation in wild populations of the Indian major carpCirrhinus mrigala (Hamilton-Buchanan 1882) evidence fromallozyme and microsatellite markersrdquo Aquaculture vol 269 no1ndash4 pp 135ndash149 2007

[56] AD Rogers SMorley E Fitzcharles K Jarvis andMBelchierldquoGenetic structure of Patagonian toothfish (Dissostichus elegi-noides) populations on the Patagonian shelf and Atlantic andwestern Indian Ocean Sectors of the Southern Oceanrdquo MarineBiology vol 149 no 4 pp 915ndash924 2006

[57] A Reilly and R D Ward ldquoMicrosatellite loci to determinepopulation structure of the patagonian toothfish Dissostichuseleginoidesrdquo Molecular Ecology vol 8 no 10 pp 1753ndash17541999

[58] S A Appleyard R D Ward and P M Grewe ldquoGenetic stockstructure of bigeye tuna in the Indian Ocean using mitochon-drial DNA and microsatellitesrdquo Journal of Fish Biology vol 60no 3 pp 767ndash770 2002

[59] P Bentzen C T Taggart D E Ruzzante and D CookldquoMicrosatellite polymorphism and the population structureof Atlantic cod (Gadus morhua) in the northwest AtlanticrdquoCanadian Journal of Fisheries and Aquatic Sciences vol 53 no12 pp 2706ndash2721 1996

[60] P F Larsen E E Nielsen K Meier P A Olsvik M M Hansenand V Loeschcke ldquoDifferences in salinity tolerance and geneexpression between two populations of Atlantic cod (Gadusmorhua) in response to salinity stressrdquo Biochemical Geneticsvol 50 pp 454ndash466 2011

[61] D P Drinan S T Kalinowski N V Vu B B Shepard C CMuhlfeld and M R Campbell ldquoGenetic variation in westslopecutthroat trout Oncorhynchus clarkii lewisi implications forconservationrdquo Conservation Genetics vol 12 no 6 pp 1513ndash1523 2011

[62] C A Davies EMGosling AWas D Brophy andN TysklindldquoMicrosatellite analysis of albacore tuna (Thunnus alalunga)population genetic structure in the North-East Atlantic oceanand Mediterranean Seardquo Marine Biology vol 158 no 12 pp2727ndash2740 2011

[63] M I Taylor L Ruber and E Verheyen ldquoMicrosatellites revealhigh levels of population substructuring in the species-poorEretmodine cichlid lineage from Lake Tanganyikardquo Proceedingsof the Royal Society B Biological Sciences vol 268 no 1469 pp803ndash808 2001

[64] A F J Jamsari T Min-Pau and M N Siti-Azizah ldquoIsolationand multiplex genotyping of polymorphic microsatellite DNAmarkers in the snakehead murrel Channa striatardquoGenetics andMolecular Biology vol 34 no 2 pp 345ndash347 2011


[65] L E Perales-Flores A M Sifuentes-Rincon and F J Garcıade Leon ldquoMicrosatellite variability analysis in farmed catfish(Ictalurus punctatus) from Tamaulipas Mexicordquo Genetics andMolecular Biology vol 30 no 3 pp 570ndash574 2007

[66] J Ribolli C M R Melo and E Zaniboni-Filho ldquoGeneticcharacterization of the Neotropical catfish Pimelodus maculates(Pimelodidae Siluriformes) in the Upper Uruguay RiverrdquoGenetics and Molecular Biology vol 35 no 4 pp 761ndash769 2012

[67] M M De Abreu L H G Pereira V B Vila F Forestiand C Oliveira ldquoGenetic variability of two populations ofPseudoplatystoma reticulatum from the Upper Paraguay RiverBasinrdquo Genetics and Molecular Biology vol 32 no 4 pp 868ndash873 2009

[68] W Salzburger S Baric and C Sturmbauer ldquoSpeciation viaintrogressive hybridization in East African cichlidsrdquoMolecularEcology vol 11 no 3 pp 619ndash625 2002

[69] G M Leclerc H Kaiping G J Leclerc and E Bert ldquoCharac-terization of a highly repetitive sequence conserved among theNorth American Morone speciesrdquoMarine Biotechnology vol 1no 2 pp 122ndash130 1999

[70] B D Neff P Fu and M R Gross ldquoMicrosatellite evolutionin sunfish (Centrarchidae)rdquo Canadian Journal of Fisheries andAquatic Sciences vol 56 no 7 pp 1198ndash1205 1999

[71] K A Kellogg J A Markert J R Stauffer Jr and T D KocherldquoMicrosatellite variation demonstrates multiple paternity inlekking cichlid fishes from Lake Malawi Africardquo Proceedings ofthe Royal Society B Biological Sciences vol 260 no 1357 pp 79ndash84 1995

[72] W-J Lee and T D Kocher ldquoMicrosatellite DNA markersfor genetic mapping in Oreochromis niloticusrdquo Journal of FishBiology vol 49 no 1 pp 169ndash171 1996

[73] A L Brooker D Cook P Bentzen J M Wright and RW Doyle ldquoThe organization of microsatellites differs betweenmammals and cold-water teleost fishesrdquo Canadian Journal ofFisheries and Aquatic Sciences vol 51 pp 1959ndash1966 1994

[74] M P Small T D Beacham R EWithler and R J Nelson ldquoDis-criminating coho salmon (Oncorhynchus kisutch) populationswithin the Fraser River British Columbia using microsatelliteDNAmarkersrdquoMolecular Ecology vol 7 no 2 pp 141ndash155 1998

[75] T D Beacham and J B Dempson ldquoPopulation structureof Atlantic salmon from the Conne river Newfoundland asdetermined from microsatellite DNArdquo Journal of Fish Biologyvol 52 no 4 pp 665ndash676 1998

[76] S Arslan and F Bardakci ldquoGenetic structure of brown trout(Salmo trutta) populations from turkey based on microsatellitedatardquo Biochemical Genetics vol 48 no 11-12 pp 995ndash1014 2010

[77] K-A Naish and D O F Skibinski ldquoTetranucleotide microsatel-lite loci for Indian major carprdquo Journal of Fish Biology vol 53no 4 pp 886ndash889 1998

[78] P Das A Barat P K Meher P P Ray and D MajumdarldquoIsolation and characterization of polymorphic microsatellitesin Labeo rohita and their cross-species amplification in relatedspeciesrdquoMolecular EcologyNotes vol 5 no 2 pp 231ndash233 2005

[79] P M A Muneer A Gopalakrishnan R Shivanandan V SBasheer and A G Ponniah ldquoGenetic variation and phylo-genetic relationship between two species of yellow catfishHorabagrus brachysoma andH nigricollaris (Teleostei Horaba-gridae) based on RAPD and microsatellite markersrdquoMolecularBiology Reports vol 38 no 4 pp 2225ndash2232 2011

[80] P D V N Sudheer S G Mastan H Rahman C Ravi PrakashS Singh andM P Reddy ldquoCross species amplification ability of

novel microsatellites isolated from Jatropha curcas and geneticrelationship with sister taxa cross species amplification andgenetic relationship of Jatropha using novel microsatellitesrdquoMolecular Biology Reports vol 38 no 2 pp 1383ndash1388 2011

[81] R YasodhaM Ghosh R Sumathi andK Gurumurthi ldquoCross-species amplification of eucalyptus SSR markers in Casuari-naceaerdquoActa Botanica Croatica vol 64 no 1 pp 115ndash120 2005

[82] K-S Kim M-S Min J-H An and H Lee ldquoCross-speciesamplification of Bovidae microsatellites and low diversity of theendangered Korean goralrdquo Journal of Heredity vol 95 no 6 pp521ndash525 2004

[83] C Schlotterer B Amos and D Tautz ldquoConservation of poly-morphic simple sequence loci in cetacean speciesrdquo Nature vol353 no 6348 pp 63ndash65 1991

[84] S S Moore L L Sargeant T J King J S Mattick MGeorges and D J S Hetzel ldquoThe conservation of dinucleotidemicrosatellites among mammalian genomes allows the useof heterologous PCR primer pairs in closely related speciesrdquoGenomics vol 10 no 3 pp 654ndash660 1991

[85] S Gupta K Kumari MMuthamilarasan A Subramanian andMPrasad ldquoDevelopment andutilization of novel SSRs in foxtailmillet (Setaria italica (L) P Beauv)rdquo Plant Breeding vol 132 no4 pp 367ndash374 2013

[86] P Galbusera S Van and E Matthysen ldquoCross-species ampli-fication of microsatellite primers in passerine birdsrdquo Conserva-tion Genetics vol 1 no 2 pp 163ndash168 2000

[87] A Gupta K K Lal P Punia R K Singh VMohindra R S Sahet al ldquoCharacterization of polymorphic microsatellite markersand genetic diversity in wild bronze featherback Notopterusnotopterus (Pallas 1769)rdquoMolecular Biology Reports vol 40 pp6625ndash6631 2013

[88] K Praebel W Jon-Ivar A Per-Arne et al ldquoA diagnostic tool forefficient analysis of the population structure hybridization andconservation status of European whitefish (Coregonus lavaretus(L)) and vendace (C albula (L))rdquo Advances in Limnology vol64 pp 247ndash255 2013

[89] V Dubut M Sinama J-F Martin et al ldquoCross-species ampli-fication of 41 microsatellites in European cyprinids a tool forevolutionary population genetics and hybridization studiesrdquoBMC Research Notes vol 3 article 135 2010

[90] B May C C Krueger and H L Kincaid ldquoGenetic variation atmicrosatellite loci in sturgeon primer sequence homology inAcipenser and Scaphirhynchusrdquo Canadian Journal of Fisheriesand Aquatic Sciences vol 54 no 7 pp 1542ndash1547 1997

[91] M Takagi ldquoPCR primers for microsatellite loci in tuna speciesof the genusThunnus and its application for population geneticstudyrdquo Fisheries Science vol 65 no 4 pp 571ndash576 1999

[92] D B Morris K R Richard and J M Wright ldquoMicrosatellitesfrom rainbow trout (Oncorhynchus mykiss) and their use forgenetic study of salmonidsrdquo Canadian Journal of Fisheries andAquatic Sciences vol 53 no 1 pp 120ndash126 1996

[93] N Kanda and F W Allendorf ldquoGenetic population structureof Bull trout from the Flathead river basin as shown bymicrosatellite and mitochondrial DNA markerrdquo Aquaculturevol 130 pp 92ndash106 2001

[94] B Angers and L Bernatchez ldquoUsefulness of heterologousmicrosatellites obtained from brook charr Salvelinus fontinalisMitchill in other Salvelinus speciesrdquo Molecular Ecology vol 5no 2 pp 317ndash319 1996

[95] A Ambali ldquoThe relationship between domestication andgenetic diversity ofOreochromis species inMalawiOreochromis


shiranus shiranus (Boulner) and Oreochromis shiranus chilwae(Trewavas)rdquoDissertation Abstracts International Part B Scienceand Engineering vol 58 no 4 pp 1655ndash1661 1997

[96] E Bezault X Rognon K Gharbi J F Baroiller and BChevassus ldquoMicrosatellites cross-species amplification acrosssome African cichlidsrdquo International Journal of EvolutionaryBiology vol 2012 Article ID 870935 2012

[97] C Rico K M Ibrahim I Rico and G M Hewitt ldquoStockcomposition in North Atlantic populations of whiting usingmicrosatellite markersrdquo Journal of Fish Biology vol 51 no 3 pp462ndash475 1997

[98] J C Patton B J Gallaway R G Fechhelm and M A CroninldquoGenetic variation of microsatellite and mitochondrial DNAmarkers in broad whitefish (Coregonus nasus) in the Colvilleand Sagavanirktok rivers in northernAlaskardquoCanadian Journalof Fisheries and Aquatic Sciences vol 54 no 7 pp 1548ndash15561997

[99] W Zheng N E Stacey J Coffin andC Strobeck ldquoIsolation andcharacterization of microsatellite loci in the goldfish CarassiusauratusrdquoMolecular Ecology vol 4 no 6 pp 791ndash792 1995

[100] G H Yue F Chen and L Orban ldquoRapid isolation andcharacterization of microsatellites from the genome of Asianarowana (Scleropages formosus Osteoglossidae Pisces)rdquoMolec-ular Ecology vol 9 no 7 pp 1007ndash1009 2000

[101] A Gopalakrishnan K K Musammilu P M Abdul Muneer KK Lal D Kapoor and A G Ponniah VMohindra ldquoMicrosatel-lite DNA markers to assess population structure of red tailedbarb Gonoproktopterus curmucardquo Current Zoology vol 50 no4 pp 686ndash690 2004

[102] K K Musammilu P M Abdul Muneer A Gopalakrishnan etal ldquoIdentification and characterization of microsatellite mark-ers for population genetic structure in endemic red tailed barbGonoprokterus curmucardquoMolecular Biology Reports 2014

[103] X H Zheng C Y Lu Y Y Zhao et al ldquoA set of polymor-phic trinucleotide and tetranucleotide microsatellite markersfor silver crucian carp (Carassius auratus gibelio) and cross-amplification in crucian carprdquo Biochemical Genetics vol 48 no7-8 pp 624ndash635 2010

[104] C M Herbinger R W Doyle E R Pitman et al ldquoDNAfingerprint based analysis of paternal and maternal effects onoffspring growth and survival in communally reared rainbowtroutrdquo Aquaculture vol 137 no 1ndash4 pp 245ndash256 1995

[105] P J Smith P G Benson and S M McVeagh ldquoA comparisonof three genetic methods used for stock discrimination oforange roughy Hoplostethus atlanticus allozymes mitochon-drial DNA and random amplified polymorphic DNArdquo FisheryBulletin vol 95 no 4 pp 800ndash811 1997

[106] R Frankham ldquoGenetics and conservation biologyrdquo ComptesRendus Biologies vol 326 no 1 pp S22ndashS29 2003

[107] CAMP ldquoReport of the workshop lsquoConservation Assessmentand Management Plan (CAMP) for freshwater fishes of India1997rdquo Organized By Zoo Outreach Organization (ZOO) andNational Bureau of FishGenetic Resources (NBFGR) LucknowHeld atNBFGR in September 1997 ZooOutreachOrganizationCoimbatore India 1998

[108] B K Padhi and P K Mandal ldquoApplied fish Geneticsrdquo FishingChimes Visakhapatnam Andhra Pradesh India 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the phenotype during genetic screening is known as a geneticmarkerThemost common use of geneticmarkers in fisheriesbiology is to determine if samples from culture facilities ornatural populations are genetically differentiated from eachother They are also used to identify different species in theevent of taxonomic disputes and to detect genetic introgres-sion in a species The detection of genetic differentiationwould imply that the source groups comprise different stocks[5] and should be treated as separate management units orstocks [6] A common objective ofmolecular genetic analysesis to find diagnostic differences among presumed stocksin either nuclear allelic types or mtDNA haplotypes [7]Polymorphic DNAmarkers can provide fisheries researcherswith new insights into the behavior ecology and geneticstructure of fish populations levels of inbreeding disassor-tative mating success of alternative reproductive strategiesand life histories and the intensity of natural and sexualselection [8] Microsatellites are one of the best suitablegenetic markers for analyzing pedigree population structuregenome variation evolutionary process and fingerprintingpurposes

Genetic markers are basically two typesmdashprotein andDNA (molecular) In the beginning of 1960s the proteinssuch as haemoglobin and transferrin were involved in allstudies In protein markers allozyme markers are very pop-ular and most of the genetic variation studies have been con-ducted based on this marker [9ndash14] Molecular markers canbe categorized into two classes nuclear DNA and mitochon-drial DNA (mtDNA) markers based on their transmissionand evolutionary dynamics [15] Nuclear DNA markers suchas random amplified polymorphic DNA (RAPDs) amplifiedfragment length polymorphisms (AFLPs) variable number oftandem repeats loci (VNTRs minisatellites microsatellites)and single nucleotide polymorphisms (SNPs) are bipar-ently inherited Mitochondrial DNA markers are maternallyinherited exhibit high rates of mutation and are non-recombining such that they have one-quarter the geneticeffective population size (Ne) of nuclear markers [8] Usingrestriction enzymes mtDNA sequence can be cut at specificsites to generate restriction fragment length polymorphisms(RFLPs) or sequence analysis of different genes of mtDNAcan be used to detect phylogenetic relationships undertakepedigree analysis and assess population differentiation inmany species

Detection of polymorphisms at the nucleotide sequencelevel represents a new area for genetic studies especially astechnologies become available which allow routine appli-cation with relative ease and low cost From the 1990s anincreasing number of studies have been publishedmaking useof random parts of a genome With the advent of thermo-cyclers the amplification of small fragment of DNA throughpolymerase chain reaction (PCR) gained popularityThePCRtechnique was discovered in 1985 and the development ofDNA amplification using the PCR technique has opened thepossibility of examining genetic changes in fish populationsover the past 100 years or more using archive materials suchas scales [8] The advent of PCR coupled with automatedDNA sequencers made feasible major technological innova-tions such as minisatellite variant repeat mapping [16] and

assessment of the variations at microsatellite loci [17] ThePCR based techniques have the added attraction of requiringonly extremely small amounts of DNA that has led to wideusage of this technique in aquaculture and fisheries In thisreview we discuss the application of the most prevalentgenetic marker microsatellites in population genetic struc-ture and its usefulness in conservation of fish fauna

2 Microsatellites Markers

Recently attention has turned to another type of genetic vari-ation that of differences in the number of repeated copiesof a segment of DNA These sequences can be classifiedbased on decreasing sizes into satellites minisatellites andmicrosatellites [13] Satellites consist of units of severalthousand base pairs repeated thousands or millions of timesMinisatellites consist of DNA sequences of some 9ndash100 bp inlength that are repeated from 2 to several 100 times at a locusMinisatellites discovered in human insulin gene loci withrepeat unit lengths between 10 and 64 bp were also referredto as ldquovariable number of tandem repeatsrdquo (VNTRs) DNA[18] Microsatellites have a unique length of 1ndash6 bp repeatedup to about 100 times at each locus [19] They are also calledas ldquosimple sequence repeatrdquo (SSR) by Tautz [13] or ldquoshorttandem repeatrdquo (STR) DNA by Edwards et al [20] Jeffreyset al [21] and Weber [22] opined that length variations intandemly arrayed repetitiveDNA inmini- andmicrosatellitesare usually due to an increase or decrease in repeat unit copynumbers Differences in repeat numbers represent the basefor most DNA profiling techniques used today Later onlymicrosatellites became very common in population geneticsstudies

Microsatellites are short tandemly arrayed di- tri- ortetranucleotide repeat sequences with repeat size of 1ndash6 bprepeated several times flanked by regions of nonrepetitiveunique DNA sequences [13] Polymorphism at microsatelliteloci was first demonstrated by Tautz [13] and Weber andMay [17] Alleles at microsatellite loci can be amplified bythe polymerase chain reaction [23] from small samples ofgenomic DNA and the alleles separated and accurately sizedon a polyacrylamide gel as one or two bands and they areused for quantifying genetic variations within and betweenpopulations of species [24]The very high levels of variabilityassociated with microsatellites the speed of processing andthe potential to isolate large number of loci provide a markersystem capable of detecting differences among closely relatedpopulations Microsatellites that have been largely utilizedfor population studies are single locus ones in which boththe alleles in a heterozygote show codominant expression[25] Individual alleles at a locus differ in the number oftandem repeats and as such can be accurately differentiatedon the basis of electrophoresis (usually PAGE) according totheir size Different alleles at a locus are characterized bydifferent number of repeat units They give the same kind ofinformation as allozymes distinguishable loci with codomi-nant alleles but they are generally neutral and more variablethan allozymes [26] Like allozymesmicrosatellites alleles areinherited in a Mendelian fashion [27] Moreover the allelescan be scored consistently and compared unambiguously


TT G

Sample collection



DNA fragment

Vector DNA

+

RecombinantDNA





3998400

3998400

5998400

5998400

CG

G

T

CG

T

A

A GC

C

GC

TA

A

A

CG

CG C C

C

C

G G

3998400

5998400

3998400

5998400

AT

A

A

A

A A

T

T

T




T G

3998400

3998400

5998400

5998400

CG

T

CG

T

A

A GC

C

GC

TA

A

A

CG

AT




Taqpolymerase

PrimerNucleotides

DNA

ATCG



Elute DNA from gel

Topoisomerase

Topoisomerase

Tyr-274P

P

O

O

OH

AAPCR product

Tyr-274

HO




CCCTTGGGA

AGGGTTCCC







Functionalgenomics

Forensic science

Genome mapping



of humandiseases


studies

Pedigreeand gender

identification


















M 1 2 3 4 5 6 7 8 9 10 11 12M M































7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TT G

Sample collection



DNA fragment

Vector DNA

+

RecombinantDNA





3998400

3998400

5998400

5998400

CG

G

T

CG

T

A

A GC

C

GC

TA

A

A

CG

CG C C

C

C

G G

3998400

5998400

3998400

5998400

AT

A

A

A

A A

T

T

T




T G

3998400

3998400

5998400

5998400

CG

T

CG

T

A

A GC

C

GC

TA

A

A

CG

AT




Taqpolymerase

PrimerNucleotides

DNA

ATCG



Elute DNA from gel

Topoisomerase

Topoisomerase

Tyr-274P

P

O

O

OH

AAPCR product

Tyr-274

HO




CCCTTGGGA

AGGGTTCCC







Functionalgenomics

Forensic science

Genome mapping



of humandiseases


studies

Pedigreeand gender

identification


















M 1 2 3 4 5 6 7 8 9 10 11 12M M































7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Functionalgenomics

Forensic science

Genome mapping



of humandiseases


studies

Pedigreeand gender

identification


















M 1 2 3 4 5 6 7 8 9 10 11 12M M































7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











M 1 2 3 4 5 6 7 8 9 10 11 12M M































7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5 6 7 8 9 10 11 12M M































7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























7 Conclusion




Acknowledgments


References


























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article Application of Microsatellite Markers in ...downloads.hindawi.com/archive/2014/691759.pdf · Application of Microsatellite Markers in Conservation Genetics and Fisheries