MOLECULAR GENETIC CHARACTERIZATION OF

ENDEMIC RED - TAILED BARB,

Gonoproktopterus curmuca (Hamilton - Buchanan, 1807)

~ ~ b ~ ~ i n p c v l t i a e ~ $ t h

-@tf=+$

Doctor of Tfiihsop/ir

~ n ~ S ~ $ ~

c ? m t ? k ~ i t y 4 S E i e n c e a n d 9 ~

CocAin - 682 022,3&

J*

K.K. MUSAMMILU

National Bureau of Fish Genetic Resources Cochin Unit Central Marine Fisheries Research Institute

(Indian Council of Agricultural Research) Post Box No.1603, Cochin - 682 01 8, Kerala, India

April, 2008

THE ILI13RARY C E N ~ [ ~ \ ! , fi;,:;ll'l~ FI:li< 2:: : . i-!LSrXRCti INSTITUTE

\</d~.; 1 1 - :,..:I> 1

DECLARATION

I hereby declare that this Ph.D thesis entitled "MOLECULAR

GENETIC CHARACTERIZATION OF ENDEMIC RED - TAILED

BARB, Gonoproktoptems c ~ m t t c a (Hamilton - Buchanan, 1807)" is the

authentic and boiafide record of the research work done by me at

National Bureau of Fish Genetic Resources (NBFGR) Unit, Central

Marine Fisheries Research Institute (CMFRI), Cochin and it has not

previously formed the basis for the award of any degree, diploma,

associateship, fellowship or other sirmlar titles or recognition.

Emakulam,

18t" April, 2008.

K.K. MUSAMMILU

(Reg. No. 2582, Ph.D, CUSAT)

THE I_IRRP,RY m p J $ t j A R : ' E FiC 1.'

' '.S\RCH INSTITUTE

Ii(.)C;III - 6132 018

, '3, ,, ' ,' ; :,I ' :., .' :g #

.* m 37lTjciSlh Fk fm * !. . ? $ .. NATIONAL BUREAU OF FISH GENETIC RESOURCES

7 r.r

vrI:l, TI-r NBFGR Cochin Unit, CMFRl Campus, P.B. No. 1603, Cochin-682 018, Kerala, India

I C A I ~ Tel. & Fax: (0484) 2395570; E-mail: nbfgrcochin@eth.net

Dr. A. GOPALAKRISHNAN, Senior Scientist, Officer-in-Charge & Supervising Teacher

07th April, 2008.

CERTIFICATE

This is to certify that the thesis entitled, "MOLECULAR GENETIC

CHARACTERIZATION OF ENDEMIC RED - TAILED BARB,

Gonoproktopterus curmuca (Hamilton - Buchanan, 1807)" is an authentic

record of the original and bonafide research work carried out by

Sri. K.K. Musammilu (Reg. No.. 2582) at National Bureau of Fish Genetic

Resources (NBFGR) Unit, Central Marine Fisheries Research Institute

(CMFRI), Cochin under my supervision and guidance for the award of the

degree of Doctor of Philosophy in the Faculty of Marine Sciences, Cochin

University of Science and Technology, Cochin, Kerala. It is also certified

that no part of the work presented in this thesis has been submitted earlier

for the award of any other degree, diploma or any other similar title.

A. Gopalakrishnan

THE I lF'P,A?Y CENTi??I- ?d,',:sf' I l"l, C l l INSTITUTE

. . a :. , I - I .: L'J ; f i

(With hz&h esteem and comphcelzcy, I eqress nty gratitude and ilzdc6t.ncss to I?ZY mentor and supervisor, Doctor ~c l iamvee t t i l S;opnh&ishnalz, ~ h . 0 , JW, S C I Z ~ O I , Scientist a~zdOficer-in-Charge, Watio~zal@u.reaz~ ofFi.sh Gerzetic @sources (WBFGcii) Cochiiz Unit, C N Y R I for his constant he& e,~erzded i~z solvirzg various problems a116 clifficuhies that arose d i i ~ ~ g 11y research work and aho for g i ~ k g me tlic wziqzre guidznce Jor structz~ri~zg a proper work phrz, which unquestionnb6y assisted ill.

comphtilzg n1.y theslj. ~ I L tinic. I f e e l v e ~ y fortunate for liavirzg beer2 allowed nze to hariz mohcuhr techniques - even though I h a d v e ~ y Gitth kriowhdge of mo6ecular tccliniqzles - in your urzique h6. W h a t I appreciate most is how you treat aKofyour students with ~~espect, rzot as mirzdhss ilferiors, I n particuh< I realb app~eci~zte the h,rjcb o j trust you phcedziz me. I IZ short, I thinkyou were the pc fect advisorJor nze.

1 .epre.r.r my gra tc j t f i~cr~~ tn iDrdW$Kd-fiEoi G%FE&CW of CWFqI, Cocfiifj, Yernfir$r firu@ng mom $IF nt j Pfi,a ~ c g ~ ~ f r n t i ~ ~ z ~ ~ n h r Cochiu. U~~fuers i~ if LTc i t~~c~ mid Ttcfin~bgy (LW%SJT), Cocfiin, 17+r&h mid for tfie prornpiws.r i ~ r fd$r~,q irK r ~ c c c s ~ ~ ~ ~ rsclinmfor tfiis srrmntfi f~fr~timring of tfic rcrcarcfi jmcqri-nrrtue and TdLT La&g C h z c t d ~ . fl,f,D%~Gx Luc(?tow, %+far rpradtfi) for yennittiq arc to tokc i h f i ru#r( form3 PhQ! thesir. T441hgrei7rpL0.~xir~, 1 f f i a n k ~ o f : (99 Wofin~zJoseplI W#dfly( Ncnfirr, J~n'C~if i~mTScient~ts' q~cruitmertt ~ o a r d ( f~w) a n d Former ainctor ?f CMFRI, Dr Pon.rziah a ~ z d D r D Kapoor, Former Directors of WBFG$ cL)r 38 Singh (Pri~zcz$al~nvestz&ator, WJTP Nissiorz Node-18) forp?,ovid?ng allfacz&t,ics and time5 lie& to complete this workas scheduled: I aho thanif, the coizcerr~ednz~tliorities of CZIJAT forgiving me the registration for Ph.D. I am indebted to D r R P a u h j Scientist-in-Charge, Post-Graduate Programme in Waricuhure (PGW), C N F R I and {DT PC floma.t, Pri~~cipalJcGrrh:~t (ie~tctic,~), CMmI$r tfirir c ~ - o p ~ u t ~ o ~ ~ , mfimfih czd:~dte, nadri?rrc(y fit@ du.pa'n8 my Ph.rD pgrmrne a?rdfn~. critjtiz6 pind tf rou~f i the

lr$t thh o~lp~rt7mit-j to eVra.7 BY n(ifihufion t o @of: (Or) TP jafi€tichy Gnnaent of ZmbgY, Nafiaroja's ~ a f i g ~ , Errla&~hrn find {Dr qlS rNdrh~t?q

. ,~wor Scirnhkt, MPR'FC~RCDCB~~ ~ n d $ tlirir tinre@ gmiidoam, ndwie ondruppun throughout my entire work

W i t h great respect andregar&, I acif,nowEdge D r m L a l ; D r 'Ilindhya 5l4ohWdi.a and D r Peyush Punia, SeniorScie~ztists, JVBFGR Lucknow, for the he& reildered by them and novel ideas shared 6y them on various practical aspects of the topic, without which this research work woul;t~zot have been so nice$ accomplished: D r L a l was of great help in equippzrzg the Unit with mohrn equi;z?ments, carrying out the statistical analjsis of dath and iizte.rpretation of ~'esults. D r 'Iliizdhya worked u,ztir.ing6y to dentzjq poljmorphic microsatelfite markers ilz tliis species, designed primers; and taught various steps of cloni~zg and D N A sequencing t o cor$rm the occurrelice of repeats in ampficons. I remain o6Liged to iD7, R Neelht&anteswar for his prompt

respoitse to my never ending request for Giteratz~re that hebed rrze in varioz~s stages of the Ph.D work I ah0 eqress my siizcere gratitude to Dr CPShaji, CSI~,~ooGOfliccu, ?$?&'I, Peechi, Trichur andDr TVSajeev, Scientist, I(,FRI, NilamnGu.r for their he& i?i.

colbction triys to upstreams of Chah/C.&dy and Chafiya?, @;vers ill Nih111.Gur aiid Chah/C./C.udy Forest Ra~zges of G r a b State.

N y profuse thank are h e to Dr BaGu Philip, 8rofess01; School of Warii1.e Scieizces, Cochin 'Lliziversity of Science and CTech~zolbgy for sparzrzg hi^ valuaGh time as the E~temza6Expcrt of my DoctoralQsearch Committee. I a60 take this opportunity to thank Prof: I(,I'JmmaLu, Fo~?m~er .%cad; Department of Zoology, Naharaja 's Colhge, Erna/Zuhm and Or 2/ Grrerzce %'jeGello, ,Se~zior Lecturer, St. JlGert's ColGege, Erna@hnz for their vah~abb advices, eizcouragenzent, @izdguidance aizd sutjgcstiomu &ring 7 1 9 6h.D programnte.

I fee[ happy to e q n s s nzy thank to N r $ tP ~ a u l t o n (Senior I'echiticalJssistai~.t, CNFRI, Cocliiil) and N r Wanda&nar %go (Gchnical-?lssistant, CWFRI) for, their tinze6y he& di~ring the period of Ph.D work I express my thank to my colkagues especial4 to Dr PjVA Mz~neer, Dr N Wagarajan, Dr Christopher ROY, Or Jrrup MnndaGandDrNarishaizkarfor their encouragement aizdsupport during this dbctoral work

I sincere6y ack$owl;!dge the Senior @search Fellbwsh@ and the e~celhnt facifities for research from the Wational~gricuhural Technolbgy Project (W-?~T~- ICJR (34ission

ode Sub Project -18) because of which no3nancial/iardsh@s were encounteredwliil;! rsuing the ~ t i . D .

Np q e & l tha~& are abo h e to ~ p : ~h~~drase@aran (FGW) alrd WE Joy (g3W)fnr t f i t ix tiv~c$ he& d~ri72g uarious ~xcrls-riom. Jfl tfiase irl PC$?M, LiGrq

d PAFD of M ~ I mrd ~ n t c t i c cfinrnct&ation @imk>,r end Libmy nnd ~clrrnenta~ion Elivisi~n nf$ff4~GR ~yckfiaw, w h hm8 s m ur other way 6 e @ d duting my t c ~ u 7 ~ fire S ~ T T L ' C ~ C ~ ~ J tfialrhd, For ffie aromCs~qpart tx@en&d to ~ c , t f ie

ientihs cf . M F ~ ~ n d CNFnqr and the m t z d ~ r i n y m r t circh if my f ~ e n d s , :luding my Gatchmates, hbmates, seniors and juniors ?om ~h .c i ) and N.Sc are

,FectionateGy remembered:

I WOUH &e to thank my father, mother, grandmother, s is te~ Grother-in-hw, niece, nephew and other rehtives who have encouraged arzd supported me in my work Tina[@, I dedcate this Ph.D thesis to belbvedpcreizts andteachers, who mouldid me to take up this nice piece of wor/iand to continue nty journey iiz the f i e u of life sciences!

'Final@, I wouH6e remiss if1 didnot thanktlie ~ o d f o r aLTthe Qbssiizgs in 119 hfc! t

Kochikkaran I<unjumohammed MUSAMMILU Emakulam, the Friday 18t" April, 2008.

fHP 1 IfJPAW\Y CENTTU;! f,',~ ' ' ' ' t I:7Cfl /N§#fflb

I.., C ' I ! ' , ' o / q

Declaration Certificate Acknowledgenients Contents List of tables List of figures Abbreviations

v X .,.

Xl l l

XV

INTRODUCTION .................... ..,.n,q.*.......... .......... , ....... . O l - 12 1.1 Scope of the study 1.2 Objective of the study 1.3 Technical progsainine of the study 1.4 Description of the species

1.4.1 Taxonomic status 09 1.4.2 Confusion over the scientific name of the species 10 1.4.3 Distinguishing characters 10

I 1.4.4 Colour 11 1.4.5 Common names 11 1.4.6 Habitat and distribution 11

Chapter 2

REVIEW OF LITERATURE .....................~.,.,~~m,.~.~.,.,...~.,. ~.e.~.I.I.I 13 - 41

2.1 Type 1 Molecular markers L 5

2.1.1 Allozylne markers 18

2.2 Type 2 Molecular markers 2 3

2.2.1 Random anlplified polyil~orphic DNA (RAPDs) 24 2.2.2 Microsatellites 28

2.3 Genetic markers in cypriilids 3 6

Chapter 3

MATERIALS AND METHODS . .... .. ....... .. .. ...... . .. ..... ...... . . . . , . , 42 - 74

3.1 Fish Specimen collection 42

3.2 Collection of tissue s a ~ ~ i p l e s

3.2.1 Collectioil of blood saillples for DNA marker studies 3.2.2 Collection of liver and nluscle for allozyme ailalysis

1 3.3 Allozylne analysis

3.3.1 Sanlplepreparatioil 3.3.2 Selection of allozymes 3.3.3 Electropl~oresis 3.3.4 Staining and imagillg 3.3.5 Scoring of alleles 3.3.6 Analysis of data

3.3.6.1 Allele ji.eqz~encies, polyrnorplzic loci ui~cl heterozygosity

3.3.6.2 Linkage cliseqz~ilibriz~in 3.3.6.3 Hardy- Weirzberg Equilibriunz 3.3.6.4 Estiinutes ofpopt~latiorz d#ererztiatiorz 3.3.6.5 Genetic si17zilarity uizd distuizce 3.3.6.6 Aiinl~eis ofMoleciilar- Varia~zce (AMOVA) 3.3.6.7 Denclrogranz 3.3.6.8 Bottleneck

3.4 Microsatellites analysis

3.4.1 Genonlic DNA isolatioil 3.4.2 DNA Quailtification 3.4.3 Designing of priiners for nlicrosatellite sequences 3.4.4 Develop~lleilt of microsatellite illarkers through cross-

species aillplificatioil 3.4.5 PCR alnplification 3.4.6 Polyaciylalllicle gel electrophoresis (PAGE) 3.4.7 Visualization of inicrosatellite proclucts 3.4.8 Calculatioil of the molecular weights of the bands 3.4.9 Final selectioil of illicrosatellite primer pairs 3.4.10 Confii~llation of nlicrosatellite by cloning and

sequencing 3.4.11 Elutioil of amplified products from agarose gel 3.4.12 Constiuction of recoillbinant plasmid 3.4.13 Competent cell preparation 3.4.14 Transforn~ation of reconlbiilant plasinid 3.4.15 Selection of recoinbinants 3.4.16 Confinllation of cloning

3.4.16.1 Screeniizg by PCR to check for. r~zicroscrtellite re1)euts

3.4.17 Sequencing of microsatellite loci 3.4.1 8 Population stiucture ailalysis

3.4.18.1 Scoringofalleles 3.4.18.2 Analysis of Duta

3.5 Randoill Ainplified Pol yillorphic DNA (RAPD) analysis

3.5.1 Screening of RAPD prilliers 3.5.2 PCR aillplification 3.5.3 Agarose electropl~oresis and visualization of bands 3.5.4 Analysis of Data

3.5.4.1 Scoring of l>u~i~ls 3.4.5.2 Allele,fi-eyuencie a17dpolj/1norphic loci 3.5.4.3 Average gene diversity (H) 3.5.4.4 Gerletic clfferentiatior~ (Glr) 3.5.4.5 Genetic ~ i ~ i z i l u ~ i t j ~ CIIICI cli.stn~ice 3.5.4.6 Dendrogrnn~

Chapter 4

RESULTS m................m.m.,.am ......... .... ..... . - 138 4.1 Allozyille analysis 75

4.1.1 Selectioil of allozynles 75 4.1.2 Polyn~oi-phic Eilzynies 76

4.1.2.1 Asl~arfute Anzir~o Pansfiruse (AAT 2.6.1.1) 76 4.1.2.2 Creatine Ki~mse (CK 2.7.3.2) 76 4.1.2.3 Esteruse (EST 3.1. I.-) 76 4.1.2.4 a-Glyceroplzosphate Delzydrogenase

(aGPDH-1.1.1.8) 76 4.1.2.5 Glucose-6-Phosphate Delzj~drogenuse

(G#DH 1.1.1.49) 77 4.1.2.6 Glzlcose Phosphate Isomerme (GPI-5.3.1.9) 77 4.1.2.7 Lactate Dehj~cEl-ogenase (LDN-1.1.1.27) 78 4.1.2.8 Malute Dehydrogenase (1MDH- I. I. 1.37) 7 8 4.1.2.9 Phosphogl~~conute clelzydroge~~ase (6PGDH-I. 1.1.44) 78 4.1.2.10 Phosphoglz~co~nutase (PGM-5.4.2.2) 78 4.1.2.1 1 Szperoxide Disr7zt~tase (SOD-1.15.1.1) 79 4.1.2.12 Xanthiize Dehj~clrogenme (XDH- I. 1.1.204) 79

4.1.3 Mono~norphic enzymes 79

4.1.3.1 Glyceraldehj)de-3-Phosphate dehydrogenase (GAPDH-1.2.1.12) 7 9

4.1.3.2 Malic Enzyrne (ME-1.1.1.40) 79

4.1.4 Genetic variability 82 4.1.5 Number and perceiltage of polymorphic loci 8 2 4.1.6 Observed and effective ~lunlber of alleles 8 2 4.1.7 Frequencies of alleles 8 3 4.1.8 Stock-specific illaskers (private alleles) 84 4.1.9 Observed and expected heterozygosities 85 4.1.10 Hardy-Weinberg expectations 85

4.1.1 1 Linkage disequilibriun~ 4.1.12 Genetic differentiation 4.1.13 Genetic relationship between populations 4.1.14 AMOVA 4.1.15 Dendrogram 4.1.16 Bottleneck analysis

4.2 Microsatellite analysis

4.2.1 Isolation of DNA 4.2.2 Quantification and puiity of DNA 4.2.3 Selection of prilners 4.2.4 Confirmatioil of microsatellites 4.2.5 Confinnation of cloiling

4.2.5.1 Tlzroz~gh PCR

4.2.6 Microsatellite loci collfinned after sequencing 4.2.7 Type and relative frequency of inicrosatellites 4.2.8 Variations in microsatellite band pattern 4.2.9 Genetic variability 4.2.10 Number and percentage of polyinorphic loci 4.2.1 1 Observed and effective number of alleles 4.2.12 Frequency of alleles 4.2.13 Agreement with Hardy-Weinberg expectations 4.2.14 Frequency of ilull alleles 4.2.15 Observed (Hobs) and expected (H,,,) heterozygosities 4.2.16 Private alleles (Stock-specific markers) 4.2.17 Linkage disequilibrium 4.2.1 8 Genetic differentiation 4.2.19 AMOVA 4.2.20 Genetic distance and similarity 4.2.2 1 Dendrogram 4.2.22 Bottleneck analysis

4.3 RAPD Analysis

4 . l 1 Selection of primers 4.3.2 Reproducibility of RAPD pattern 4.3.3 Genetic variability

I 4.3.3.1 Nunzber oJ'ar~zplzJiedfr.ag~1zei2ts 4.3.3.2 Linkage ~Eiseqziilibriza~z 4.3.3.3 Genetic differelztiation 4.3.3.4 Stock-speciJic inarke~~s (Piivate alleles) 4.3.3.5 Genetic dktance and similarity index 4.3.3.6 Dend~*ograin

4.4 Coinparative assessment of resuIts of three markers

4.4.1 Number of loci and alleles 4.4.2 Percentage of polymosphic loci 4.4.3 Observed and expected heterozygosities 4.4.4 Genetic differentiation

4.4,5 Private alleles 4.4.6 Genetic distance and sin~ilarity 4.4,7 The UPGMA based dendrogam

Chapter 5

DISCUSSION ..........,.,......,.,.4.,.......... .............. 139 - 177

5.1 Allozynles 140

5.1.1 Polyrnoiplic allozylne markers 140 5.1.2 Amount of genetic variability and Hardy-Weinberg Equilibriuln 14 1 5.1.3 Linkage disequilibriuin 145 5.1.4 Private alleles 145 5.1.5 Population genetic sti-ucture 146 5.1.6 Genetic distance values 148 5.1.7 Bottlei~eck Analysis 148

1 5.2 Microsatellites 15 1

5.2.1 Type and relative frequency of microsatellite arrays observed 155 5.2.2 Genetic variability and Hardy-Weinberg Equilibrium 155 5.2.3 Null alleles 158 5.2.4 Linkage disequilibiiurn 160 5.2.5 Stock-specific markers 160 5.2.6 Genetic differentiation 161 5.2.7 Genetic relationships ainoilg populations 163 5.2.8 Bottleneck analysis 163

5 Random amplified polyi~~oi-phic DNA (RAPDs) 165

5.3.1 Reproducibility of RAPD ~narkers 166 5.3.2 Genetic variability in RAPD analysis 167 5.3.3 The size and number of the RAPD ainplicons 169 5.3.4 Linkage disequilibriun~ 169 5.3.5 Population specific RAPD ~narkers 169 5.3.6 Genetic differentiation 170 5.3.7 Genetic relationship between populations 170

54 Coinparative analysis of results wid1 three markers in G. czrrl11zicu. 172

Chapter 6

SUMMARY ............................. ............................ 178 - 183

Chapter 7

CONCLUSION ~...~..........m....................m.I......4...pm.m~~.~.~.m.~.~.m.~~.~p...... 184 - 1138

7.1 Conselvation and inanage~nent of natural populations of Gorzopr-okctopter~~s czlrinzlcu based on the present findings 184

7.2 Approaches for in-sitz~ conservation 186 7.3 Action plan suggested for 'propagation-assisted, stock-

specific restocking' of red-tailed barb with the help of 'supportive breeding prograinine' 186

7.4. Current status of ex-sitz~ conselvation of Gonoproktoptert~s C Z / ~ " ~ ~ ~ Z L C ~ I B X

. REFERENCES ................................................................................ 189 223

APPENDICES

1. Publications 2. NCBI accessions submitted

LIST OF TABLES -- --- --- - .. Page No.

Table 01 Sanlple size of Gonoprokoptert~s czlrrnzlca and sampling period at three riverine locations. 42

Table 02 Naine of enzymes wit11 their enzylne colnnlission (E.C.) nulnber used in allozyne analysis in Gorzoproktopterz~s cz~rrnz~ca. 46

Table 03 Microsatellite primers of related species tested for cross- species anlpli fication in Gorzoprokctopter.z~s czirmuca . 60

Table 04 The sequence, concentration and the annealing tei~lperature of selected inicrosatellite prinlers. 64

Table 05 Selected prilners with concentration and inolecular weight used in RAPD analysis in Gonoprokctopterws czirnzztca (the priiners asterisked are selected for population analysis). 7 1

Table 06 Distribution of diineric G6PDH genotypes in inale and female G. cz~rrnz~ca from different river systems. 77

Table 07 The naines of enzyine loci, nuinber of loci and obseived alleles for allozyme analysis in Gonopr-oktopterzis curmzlca. The enzymes inark '11s' did not yield any scorable activity. 80

'l'able Or( The distribution of allozyine genotypes and their Rf values in G. currnuca from three iiveiine systems. 8 I

Table 09 Obseived (na) and Effective (ne) number of allozyille alleles in three riverille populations of G. cz~rr~zzica. 83

~ilble 10 Allozylne alleles and allele fi-equencies in G. cz~rr~zuca from three riveiine populations and ainong populations. X 4

1 able 11 Private allele in allozyine and its frequency. 85

ble 12 Sumnary of genetic variation and heterozygosity statistics of fourteen allozyme loci in Gorzopr~okctopter-us czlrrnzlca. 56

Table 13 Fisher's exact test of allozyi~~e allele hon~ogeneity for all the population pairs of Gonoproktopterus cz~r.rnzicu. R8

Table 14 F-statistics (FsT) for overall populations of Gonoprolitopter~,is curnzz~ca using allozyines. 3 13.

Table 15 Pair-wise Fisher's FsT (8) (above diagonal) and their sibmificance levels (below diagonal) between livesine populations of Gor~opl.oktoptert~s nir71~zica using allozyne markers. !XI

Table I6 Nei's (1978) genetic identity (above diagonal) and genetic distance (below diagonal) using allozynie inarkers in G. czlrmuca; geographical distances (in IOn) are given in bracket. I

Table 17 Analysis of Molecular Variance (AMOVA) based on allozyne markers in t h e e populations of G. cz~r*mz~ca

Analysis of genetic bottleneck in G, currnuca with allozyne inarkers under infinite allele inutation inodel (IAM) and two- phased inutation inodel (TPM).

Table 18

Characteristics of polymorphic inicrosatellite loci in G. CzLI-lnzLca.

Table 19

Obseived (na) and Effective (ne) nuinber of lnicrosatellite alleles in three riverine populations of G. cur7~zz~ca.

Table 20

Microsatellite alleles and allele frequencies in G. czLrnzzLcu from three riverine populations and overall populations.

Table 2 1

Suminary statistics of null allele fi-equencies in G. czimzzlca. Table 22

Table 23 Sun~inary of genetic variation and heterozygosity statistics of eight inicrosatellite loci in Gorzoproktopter~~s cz~rnzz~ca.

Private alleles in microsatellite and their frequencies. Table 24

Table 25 Fisher's exact test of microsatellite allele homogeneity for all the population pairs of G. currntrca.

Table 26 F-statistics (FST) and Rho-statistics (RsT) for overall populations in G. a~r17zuca.

Table 27 Pair-wise Fisher's FST (0) (above diagonal) and RST (below diagonal) between riveiine samples of Gor~op7-olrto~~tcr-~~s currnuca using rnicrosatellite markers.

Table 28

Table 29

Analysis of Molecular Variance (AMOVA) based on lnicrosatellite inarkers in three populations of G. ct~rnzuca.

Nei's (1978) genetic identity (above diagonal) and genetic distance (below diagonal) using lnicrosatellite inarkers in G. cunnuca; geographical distances (in I h l ) are given in bracket.

Table 30 Analysis of genetic bottleileck in G. curnzz~ca with microsatellite inarkers under infinite allele mutation inodel (IAM) and two-phased nlutation inodel (TPM).

Table 3 1 Number of RAPD fiagnents and their size range for each Operon primer.

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

The total number of RAPD fi-aglients; nunlber & % of polymorphic bands and average gene diversity for each and overall populations of Gonopr.olctopter.~~s cz~r17za~ca.

Co-efficient of genetic differentiation (Gsr) for overall populations.

Stock- specific RAPD markers with size for each population.

Nei's genetic identity (above diagonal) and genetic distance (below diagonal) using RAPD markers in G. cuimuca; geographical distances (in Ian) are given in bracket.

Coinparative assesslllellt of results of three markers.

The coinparison of genetic distance, genetic differentiation estiiliates between pair-wise populations using three maslcers.

-.*------- - LIST OF FIGURES . -- - - - - - . - . -. - - - - .

Figure 0 1 Gonoprolctopter-us czlrnzuca (Adult)

Figure 02 Gonopi-olctopterus czirunuca (Juvenile)

Page No.

Figure 03 Map showing the distribution of the sarnpling sites of Gorzoprolctoptert~s czlr-nzuca 43

Figure 04 Aspartate amino transferase (AAQ pattern in G. currnuca 93

Figure 05 Creatine kinase (CK) pattern in G, czirmzica 93

Figure 06 Esterase (EST) pattern in G. curmuca 94

Figure 07 Glyceraldehyde-3-Pl~osphate Dehydrogenase (GAPI) in G. ctirr~tzica 94

Figure 08 a-Glycerophosphate (Glycerol-3-phosphate) dehydroge~zase (aG3PDU) pattern in G. curmuca

Figure 09 Glucose-6-phosphate dehydrogenase (Gd'DH) pattern in G. curnzzica

Figure 10 Glucose phosphate isolnerase (GPO pattern in G, czlrinuca

Figure 11 Lactate dehydrogeanse (LDH) pattern in G. czlrinuca

Figure 12 Malate dehydrogenase (MDI-I) pattern in G. czirinuca

Figure 13 Malic enzyme (ME) pattern in G. czrru~zzica

Figure 14 6-Phosphogluconate dehydrogenase (6PGDH) pattei-n in G. curmuca

Figure 15 Phosphogluco~nutase (PGM) pattern in G. czirinuca

Figure 16 Superoxide dislnuate (SOD) pattern in G, curnzzlca

Figure 17 Xanthine dehydrogenase (XDH) patten1 in G. curmzlca

Figure 17a Qualitative "mode-shift" indicator test to discrilninate , bottlenecked populations of Gonoprolctoptei-us curnzuca fi-om

three rivers based on allozyne allele frequency distribution.

Figure 18 Microsatellite pattern of locus CcatGl-1 in G. czlrmuca

Figure 19 Microsatellite pattern of locus MFWOI in G. ctir-unuca

Figure 20 Microsatellite pattern of locus MFWll in G. curnzuca

Figure 2 1 Microsatellite pattern of locus MFW19 in G. czlrnzzica

Figure 22 Microsatellite pattern of locus MFW26 in G. czirnzzlca

Figure 23 Microsatellite patteln of locus MFW72 in G. czirnzz~ca

Figure 24 Microsatellite pattern of locus Ppro48 in G. czlrnzzlcu

Figure 25 Microsatellite pattern of locus Ppro126 in G. curinzica

Figure25a Qualitative "mode-shift" indicator test to discriminate bottlenecked populations of Gorzopr.oktoptertis currnzlca fiom three livers based on inicrosatellite alleIe frequency distribution.

Figure 25b The nucleotide sequence of each inicrosatellite locus in G. czir~rnz~ca. Repeat sequences are given in red colour & prilner sequences are in blue colour.

Figure25c The nucleotide sequence of each nlicrosatellite locus in G. czlrmzlca. Repeat sequences are given in red colour & prilner sequences are in blue colour.

Figure 26 RAPD pattern of G. cur-rnzlca with primer OPA - 15

Figure 27 RAPD pattem of G. czlrinzlca with primer OPA - 16

Figufe 28 RAPD pattem of G. cuiflnarca with priiner OPAA-07

Figure 29 RAPD pattern of G, curnluca with primer OPAA-08

Figure 30 RAPD pattern of G. czirnzuca with primer OPAC-05

Figure 3 1 RAPD patteln of G. czlrmzlca with priiiier OPAC-06

Figure 32 RAPD pattern of G, cur-muca with priiner OPAH-03

Figure 33 RAPD pattelm of G. czlrrnzlca with prilner OPAH-17

Figure 34 RAPD pattelm of G. czarntlca with prilner OPAH- 19

Figure 35 UPGMA dendrogra~ns of three riverine populations of G. currnzlca based on Nei's (1978) pair-wise genetic distance using allozyine, inicrosatellite and RAPD markers

Pg

cd AAT

AFLP

AK AMQVA

APS

bp BPB CAGE

CAMP

CK CMFRI

CUSAT

Dn dNTPs

EDTA

GPI n

Fsr

FUM

G6PDH

GAPDH ;

aG2PDB :

Hobs

1 fc.r,,

I-nw IAM IUCN

KFRl

LDH

MDH

LIST OF ABBREVIA TlONS -- - . -- -- L J - ---A- - .

Microgsains

Microlitre

Aspaitate Anlino Trailsferase

Amplified Fragnleilt Length Polymoi~hism

Adenylate Kinase

Analysis of Molecular Variance

Aillnloiliuill persulphate

Base pairs

Bron~o Phenol Blue

Cellulose Agarose Gel Electrophoresis

Conselvation Assessnlent Managenlent I'lan

Creatine ICinase

Central Marine Fisheries Research Institute

Cochin University of Science and Technology

Dalton

Deoxyn~~cleoside tri-phosphates

Ethylene Diamine Tetra Acetic acid

Esterase

Co-efficient of inbreeding

Co-efficient of genetic differentiation

Glucose phosphate iso~llerase

Average gene diversity or heterozygosity

Obseived heterozygosity

Expected heterozygosity

Hardy-Weinberg Equilibrium

Infinite allele mutation model

I~lternatioilal Uilion for Conseivatioil of Nature and Natural Resources

Kerala Forest Research Institute

Lactate dehydrogenase

Malate dehydrogenase

"Aalic enzyme

MM MFRs

arltDNA

MW

NAD

NADP

N ATP

NBFGR

NCBI

''g Nm ODH

PAGE

PCR

6PGDH

PGM

PK

RAPD

RARS ,

RFLP

1yn1

SDS

s w SOD

SSRs STRS

TEMED

7,,, TPM

UPGMA

VNTRs

XDH

Mission Mode

Microsatellite Flanking Regioils

Mitochondria1 DNA

Molecular weight

Nicotinainide adenine dinucleotide

Nicotiilainide adenine di~lucleotide phosphate

National Agricultural Technology Project

National Bureau of Fish Genetic Resources

National Centre for Bioteclulology Infosnlatioll

Nuclear DNA

Observed nuinber of alleles

Effective ~luinber of alleles

Effective populatioil size

Nanogranls

Rate of gene flow

Octonol dehydrogenase

Poly Ac~ylamide Gel Electrophoresis

Polymerase Chain Reaction

Phosphogluconate dehydrogenase

Phospl~oglucon~utase

Pyruvate Kinase

Random Amplified Polymorphic DNA

Regional Agricultural Research Station

Restriction Fragn~elzt Length Polylnotphism

Revolutions per minute

Sodium Dodecyl Sulpl~ate

Stepwise mutation model

Superoxide disnlutase

Siinple Sequence Repeats

Short Tandem Repeats

N.N.N'.N'. Tetra Methyl Ethylene Dialnine

Annealing Teinperature

Melting Temperature

Two Phased Mutation inodel

Ullweighted Pair-Group Method with Arithinetic Mean

Variable Number of Tandem Repeats

Xanthine dehydrogenase Full T

ext N

ot A

vaila

ble