50 ( 4): 686 - 690, 2004.

A ct a Zoologica S i nica ¡¡¡¡¡¡¡¡¡¡ ¡¡

¡¡ ¡¡ Red tailed barb Gonop roktopterus cu rm uca ( Hamilton2Buchanan , 1807) is endemic to t he rivers originating exclusively f rom sout hern part of t he Western Ghat s in Peninsular India ( Gopalakrishnan and Ponniah , 2000) . The Western Ghat s are recog2 nized as one of t he twenty five biodiversity hot spot s¡– ¡ of t he world ( Myers et al. , 2000 ) . G1 cu rm uca has commercial value as a food fish as well as for orna2 mental t rade and also considered as a potential species for aquacult ure. The sharp decline in abundance of G1 cu rm uca and it s endangered stat us is of serious concern ( Gopalakrishnan and Ponniah , 2000 ) . For t he significance attached to t he species , effective con2 servation and propagation2assisted rehabilitation st rategies need to be planned. However , such an ap2 proach needs data on t he genetic variation and popula2 tion st ruct ure of G1 cu rm uca across it s nat ural dist ri2 bution. To generate population genetics data , identi2

¡¡ This research was funded by a grant from World Bank (No. MM2 \ 18) 3 33 Corresponding aut hor. ¡¡ E2mail :nbfgr @sancharnet . in ;kulvin 100 @hot mail. com 333 NBF GR Cochin Unit ,CMFRI Campus , PB # 1603 , Ernakulam , Kochi :682018 , Kerala , India. E2mail :nbfgrcochin @vsnl. net 3333 Present Address :B GRRP ,World Fish Center ,PO Box 500 GPO ,10670 ,Penang ,Malaysia. E2mail :a. ponniah @cgiar. org ¡¡ ν 2003 ¶fl˛§–¤ A cta Zoologica S i nica

Achamveettil GO PALA KRISHNAN333 , Kochikkaran Kunjumohammed MU SAM2 M IL U 333 , Peringady Mohammed ABDUL MUN EER333 , Kuldeep Kumar LAL 33 , Dhurendra KAPOOR , Alp his Geet hanand PONN IA H3333 , Vindhya MO HINDRA
National Bureau of Fish Genetic Resources ( NBF GR2ICAR) , Canal Ring Road , P1O. Dilkhusha , Telibagh , L ucknow2226002 ( U P) , India ˆ˛¢˛—˙–Œ…˙˘„”˛†¡¡˜¨”‰Æ„„ National Bureau of Fish Genetic Resources ( NBF GR2ICAR) , Canal Ring Road , P1O. Dilkhusha , Telibagh , L ucknow2226002 ( U P) , India “ ¡¡ “ ¡¡ ˛˜…†`¸¨¿˘ª˜ – 16 ¶˛¢˛—˙˛”˛†¡¡—˜˚˚ˆ— , ˘— 6 ¶˛¿‡„ƒ' ˛»ª—¶‹— ¡£¶†`‰ı†»˝‹”`˜–Œ– , ˝¤„…†—'¶‹˛¢˛—˙˛»ª˜¯·«–˙Ø¿ ”˛†¡¡¨”‰Æ„„•˛˜˚˚”ˇ— ¡£‰Æ„ߡ˚ 5 ‚¶‹˛»ªˇ˚`‰‚ø–—˜˘‰ø–„”ˇ¶¨•–˚˙ 01471 ¡£`‰‚ø–ˇł˜»ø˚—–ˆ˛ˆ˙¸ø¨•¶¤˜˛¢˛—˙–Œ…˙¿ˆ”˛†¡¡˜˜¯·«•»fl—¿ –¤ 50 ( 4) : 686 - 690 , 2004 ] ¡£ „…·˚ ¡¡ ˛†¡¡ ¡¡˛¢˛—˙ ¡¡ ” ¯·«– Key words ¡¡ Red tailed barb , Gonoproktopterus curm uca , Microsatellite , Genetic variation

Achamveettil GO PALA KRISHNAN333 , Kochikkaran Kunjumohammed MU SAM2 M IL U 333 , Peringady Mohammed ABDUL MUN EER333 , Kuldeep Kumar LAL 33 , Dhurendra KAPOOR , Alp his Geet hanand PONN IA H3333 , Vindhya MO HINDRA
¡¡ Received Dec. 28 ,2003 ;accepted Jan. 31 ,2004
, ˙ 5‚ , ˘„`¸¸ˆ˙ 01293 ”˝ [ ¶fl˛§

Microsatellite D NA markers to assess population structure of red tailed barb Gonoproktopter us curmuca 3
3

fication of polymorp hic markers wit h consistent scorable alleles is a crucial step ( Fergusan et al. , 1995) . U ntil now , no information is available on any class of genetic markers in G1 cu rm uca . Microsatellites are short tandem repeat motif s wit h high level of allelic polymorp hism and co2domi2 nant inheritance , usef ul for direct assessment of pat2 tern and dist ribution of genetic variability at int ra specific level ( O¡fl Connell and Wright , 1997 ) . The flanking sequences of microsatellites wit hin related taxa are highly conserved. The potential of t hese markers is enhanced when primers designed for one species amplify homologous loci in ot her species ( Scribner and Pearce , 2000 ) . Successf ul amplifica2 tion of homologous microsatellite loci has been demon2 st rated in some cyprinid fishes ( Zheng et al. , 1995 ; Mohindra et al. , 2001 ;Lal et al. , 2004) . The pre2 sent st udy examines cross2species amplification of


¡¡¡¡

Achamveettil GOPALA KRISHNAN et al. :Microsatellite markers in Gonoproktopterus curm uca

687

primers ,

p hic microsatellite loci and evaluate suitability of t he identified loci in population st ruct ure analysis of

1 ¡¡ Materials and met hods

111 ¡¡ Sampl ing sites and sample collection The G1 cu rm uca specimens were obtained t hrough commercial catches f rom two rivers , Cha2 ( Vazhachal , n = 15 ) , lakkudi Periyar (Bhut hat hanket uu , n = 14 ) . The riverine locations were chosen to cover geograp hically distant popula2 tions of G1 cu rm uca . The samples were obtained during J uly 1999 to August 2001 and total lengt h ranged f rom 200 mm to 600 mm ;collection was done at act ual fishing sites. Blood samples , collected t hrough caudal punct ure , were fixed in 95 % et hanol ( 1¡ˆ5) and stored at 4 ¡ till use. 112 ¡¡ PCR ampl if ication and electrophoresis Total genomic DNA was ext racted f rom blood samples following t he procedure of Ruzzante et al. ( 1996 ) . PCR amplifications were performed ( MJ Research t hermal cycler P TC2200 ) in a final volume of 25ƒ l , containing 25 - 50 ng of genomic DNA , 1 ¡` PCR buffer ( 10 mmol Tris2HCl , p H 910 ; 50 mmol KCl ; 0101 % gelatin) , 210 mmol MgCl2 , 012 mmol of each dN TP , 5 p mol of each primer and 115 unit s of Taq DNA polymerase. Amplification conditions were 94 ¡ for 5 min followed by 25 cycles at 94 ¡ for 30 s , Ta for 30 s and 72 ¡ for 1 min , wit h a final extension of 72 ¡ for 4 min. After amplification , 8 ƒ l of PCR product s were elect rop horesed on non2denat uring polyacry2 lamide ( 19¡ˆ1 acrylamide bisacrylamide) gels ( size 10 cm ¡` 1015 cm , Amersham Biosciences Lt d. ) . The gel concent ration was optimised according to allele size for better resolution. Elect rop horesis was done at 4 ¡ wit h 1 ¡` TB E buffer for 5 hours at 150 V . The gels were silver stained ( Silver Staining Kit , Amer2 sham Biosciences , U SA ) to visualize microsatellite loci and allelic patterns ;and known DNA size marker ( Ms pI cut pB R 322 DNA) was run in every gel. The size of t he amplified product s was determined wit h ID Elite ( Amersham Biosciences) software. The alleles wit h dinucleotide repeat s could be resolved and were designated according to PCR product sizes. Genotype of each individual at each locus was assigned manual2 ly. 113 ¡¡ Screening of primers and genetic diversity analysis Microsatellite primers f rom Cy p ri n us carpio , B arbodes gonionot us and Catl a catl a were tested for amplification of homologous loci ( Table 1 ) . The t hree species are termed as resource species in t he

G1 cu rm uca .

G1 cu rm uca . The objective was to identify polymor2

developed

for

t hree

cyprinids

in

Of t he 16 heterologous primer pairs tested , six ( 23100 %) provided successf ul amplification of ho2 mologous loci in G1 cu rm uca ( Table 1) . It is evident ( Table 2 ) t hat t he optimum annealing temperat ure ( Ta ¡ ) observed in G1 cu rm uca differed f rom t hat reported in t he resource species for respective primer pair. Primer B gon 22 amplified but produced monomorp hic band in all t he individuals tested. In t he present st udy , five polymorp hic mi2 crosatellite loci ( M FW 1 , 11 , 19 , 26 , Ccat G1 21) , ex2 hibiting 2 to 5 alleles , could be successf ully identified for G1 cu rm uca . The parameters of genetic variation at each locus and over all loci differed between t he two sample set s ( Table 3 ) . The observed heterozy2 gosity values over all loci were 01293 ( Chalakkudi ) and 01471 ( Periyar) . Mean number of alleles per lo2 cus ranged f rom 4120 ( Chalakkudi ) to 4140 ( Peri2 yar) . The probability test provided t he evidence t hat t he observed allele f requencies significantly ( P < 0105 ) deviated f rom t hat expected under Hardy2 Weinberg equilibrium. Deviation was observed in bot h t he sample set s , at t hree to four loci ( Table 3 ) wit h significant deficiency of heterozogotes. Except at locus M FW 1 ( Chalakkudy samples ) , t he score test also confirmed significant heterozygote deficiency at ot her t hree loci. Significant heterogeneity ( P < 0105 ) in geno2 type proportions was observed at t hree out of five loci

st udy. This cross2species amplification experiment was done wit h eight specimens of G1 cu rm uca . The optimum annealing temperat ure to get scorable band pattern was determined t hrough experimental stan2 dardization for each primer pair. The primers yielding scorable amplified product were again evaluated wit h larger sample size ( 29 individuals , f rom 2 rivers) to evaluate t heir suitability in quantification of genetic divergence in G1 cu rm uca . The data was analyzed using software Genetix 4102 ( Belkhir et al. , 1997 ) to obtain allele f requencies , mean number of alleles per locus , heterozygosity values , expected ( He ) and observed ( Ho ) . Test s for conformity to Hardy2Wein2 berg expectations ( probability and score test ) were performed by t he Markov chain met hod wit h parame2 ters dememorization = 1 000 , batches = 100 and it2 eration = 100 ( Genepop ver. 313 , probability test ) . Genetic homogeneity of four sample set s was deter2 mined t hrough an exact test ( G based test ) t hat as2 sumes random samples of genotypes ( Genepop ver. 313 , Genotype differentiation test ) ( Raymond and Rousset , 1995b) . This test is performed on genotype tables and possible non2independence of alleles wit hin genotypes will not affect test validity ( Raymond and Rousset , 1995c ; Goudet et al. , 1996) .

2 ¡¡ Result s

688 ¡¡¡¡

¶fl ¡¡¡¡ ¡¡¡¡ ¡¡¡¡ ¤ ˛ § –

50

¡¡

Total tested

Table 1 ¡¡ Primers of Microsatellite loci tested for cross species amplif ication in G1 curmuca
Species No . of primer pairs tested 1 Locus Genbank accession No. AF045380 ¡¡¡¡¡“ ¡¡¡¡¡“ Reference
Catla catla Ccat G1 Cypri nus carpio

Naish and Skibinski , 1998

10

MFW 1 , 2 , 9 , 11 , 15 , 17 ,

19 ,20 , 24 , 26

Crooijmans et al. , 1997 Chenuil et al. ,1999

B arbodes gonionot us

5

Bgon 22 , 69 , 75 ,79 ,17

16

probability (p) of genotype homogeneity between samples is given. Significant probability values are marked , 33 P < 0105 , 3 Critical probability level adjusted for sequential bonferroni correction.

Table 2 ¡¡ Characteristics of amplif ied microsatellite loci in G1 curmuca
Resource species Species Locus Primer sequence (5¡œ 3¡ ) ¡
Cypri nus M FW 1

Repeat motif CA

Ta ( ¡ ) 55

GTCCA GACT GTTCA TCA GGA G

carpio

GA GGT GTACACT GA GTCACGC

M FW 11

GCA TTT GCCTT GA T GGTT GT G

CA

55

TCGTCT GGTTTA GA GT GCT GC GAA TCCTCCA TCA T GCAAAC

M FW 19

CA

55

CAAACTCCACA TT GT GCC

M FW 26

CCCT GA GA TA GAAACCACT G

CA

55

CACCA T GCTT GGA T GCAAAA G

Catla catla

Ccat G1 - 1

A GCA GGTT GA TCA TTTCTTCC T GCT GT GTTTCAAA T GTTCC

[ GA TA ] n ¡› ¡› [ CCA ] n CCT

61

B arbodes

B gon 22

TCTT GTT GA TCACACGGACG

-

gonionot us

ACA GA T GGGGAAA GA GA GCA

Table 3 ¡¡ Parameters of genetic variability for each microsatellite locus in G1 curmuca samples from t wo locations
Locus homogeneity River Ch Pr Size range (bp) 175 - 190 No. of alleles at each locus 4 Ho He HW(p)
M FW 1

175 - 195

5 5 5 3 6 4 3 5 4

M FW 11

Ch Pr

180 - 201

180 - 201

M FW 19

Ch Pr

215 - 220

201 - 225

M FW 26

Ch Pr

147 - 160

147 - 157

Mean over all loci

(Ch = Chalakudy , Pr = Periyar) . The observed ( Ho) and Hardy2 Weinberg expected ( He) heterozygosity values wit h associated probability (p) ;

Ccat G1 21

Ch Pr

185 - 201

190 - 201 -

Ch Pr

-

4140

4120

01714 01267 01143 01200 01786 01267 01286 01400 01429

01333

01293

01471

01745 01718 01704 01487 01791 01649 01582 01660 01599

01571

Successful primer pair amplified in G1 curm uca No. ( %) 1 (100) 4 (20) 1 (20) Ta ( ¡ ) 59 58 51 57 51 55 < 010001 3 010101 33 010199 33 010005 3 010020 3 010319 33 010204 33 010003 3 010029 3 011391 6 (23100)
G1 curm uca

No. of alleles 5

5

6

4

5

1

Genotype (p) 011185

01684

01617

< 010001 3

010005 3 010008 3

010370 33

015254


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689

The st udy demonst rates successf ul cross2priming of microsatellite loci in red tailed barb , G1 cu rm uca and identified five polymorp hic loci. The result is consistent wit h t he earlier report s , suggesting t he possibility of using primers interspecifically among cyprinids ( Zheng et al. , 1995 ) . Mohindra et al. ( 2001) demonst rated amplification of homologous mi2 crosatellite locus in L abeo rohit a using primer devel2 oped for ot her cyprinid Catl a catl a. Successf ul iden2 tification of polymorp hic microsatellite markers for Ci rrhi n us m ri gal a was achieved t hrough use of primers of ot her cyprinid fishes (Lal et al. , 2004) . The presence of null alleles could be one of t he possible factors responsible for t he observed heterozy2 gote deficiency. Null alleles are not represented in PCR amplification due to mutation at primer binding site and cont ribute towards homozygote excess ( Paet kau and St robeck , 1995) . Raymond and Rous2 set ( 1995a) suggested t he score test was more power2 f ul t han t he probability test when t he alternate hy2 pot hesis of interest is heterozygote deficiency. Inter2 estingly , except at locus M FW 1 ( Chalakkudy sam2 ples) , t he result s of t he score test were consistent wit h t he probability test . This may not be a conclu2 sive interpretation for t he absence of null alleles , however it suggest s t he likelihood of deficiency of het2 erozygotes at least at t hree loci in bot h populations. If t rue , a serious concern is t hat t he assumptions under2 lying t he Hardy2Weinberg equilibrium relevant to nat2 ural population of G1 cu rm uca are violated ( Ferguson et al. , 1995) . One of t he reasons could be reduction in effective breeding population size in G1 cu rm uca possibly due to overexploitation , rest ricted migrations and habitat alterations , etc. Genetic heterogeneity was tested on t he tables based on t he genotype rat her t han allele f requencies in view of t he observed nonconformity to Hardy2Wein2 berg expectations ( Raymond and Rousset , 1995c ; Goudet et al. , 1996) . The various estimates provided st rong evidence t hat t he two sample set s were not drawn f rom t he same random mating gene pool. Analysis of larger sample sizes f rom more geograp hical locations will provide fine scale assessment of popula2 tion st ruct ure of G1 cu rm uca and also more insight into t he observed homozygote excess. In t he given

( Table 3) . After t he sequential bonferroni correction ( P < 010071) was made to t he probability levels , still two loci ( M FW 19 and 26) exhibited significant het2 erogeneity. Combined proba2bility over all t he loci was less t han 010001 ( Table 3) . Gst for small sample size ( Nei and Chesser , 1983) of 01049 provided f ur2 t her evidence of population subst ruct uring i n

3 ¡¡ Discussion

G1 cu rm uca .

Belkhir K , Borsa P , Goudet J , Chikhi L , Bonhomme F , 19971 GEN ETIX , logiciel sous Windows pour la ge¡ ne¡ tique des popula2 tions , http/ / www1univ2montp21fr/ ¡« genetix/ genetix1 ht m1 Brookfield J F Y , 19961 A simple new met hod for estimating null allele frequency from heterozygote deficiency. Mol. Ecol. 5 : 452 - 4551 Chenuil A , Galtier N , Berrebi P , 19991 A test of t he hypot hesis of an autopolyploid vs allopolyploid origin of a tetraploid lineage. Applica2 tions to t he genus B arbus (Cyprinidae) . Heredity 82 : 373 - 380 1 Croojimans RPMA , Bierbooms VAF , Komer J , Vander Poel JJ , Groe2 nen MAM , 19971 Microsatellite markers in common carp Cypri2 nus carpio . Animal Genetics 28 : 129 - 134 1 Ferguson A , Taggart JB , Prodohl PA , McMeel O , Thompson C , Stone C , Mc Ginnity P , Hynes RA , 19951 The application of molecular markers to t he study and conservation of fish populations wit h special reference to S al mo. J . Fish Biol. 47 ( Suppl. A) : 103 - 1261 Gopalakrishnan A , Ponniah A G , 2000 1 Cultivable , food , sport and or2 namental fish species endemic to Peninsular India wit h special refer2 ence to t he Western Ghats. In : Ponniah A G , Gopalakrishnan A ed. Endemic Fish Diversity of t he Western Ghats. NBF GR2NA TP Publication , No 11. L ucknow , India : National Bureau of Fish Ge2 netic Resources , 13 - 321 Goudet J , Raymond M , de Meeus T , Rousset F , 19961 Testing differ2 entiation in diploid populations. Genetics 144 : 1 933 - 1 940 1 Lal Kuldeep K , Chauhan T , Mandal A , Rajeev K. Singh , Lavie Khulbe , Ponniah A G , Vindhya Mohindra , 20041 Identification of microsatellite DNA markers for population structure analysis in In2 dian major carp , Ci rrhi nus m rigala ( Hamilton2Buchanan , 1882) . J . Appl. Icht hyol. 20 : 1 - 5 ( In press) . Myers N , Mittermiler RA , Mittermiler CG , Da2Fonseca GAB , Kent J , 20001 Biodiversity hotspots for conservation priorities. Nature 403 : 853 - 8581 Mohindra V , Mishra A , Palanichamy M , Ponniah A G , 20011 Cross2 species amplification of Catla catla microsatellite locus in L abeo ro2 hita . Indian J . Fish. 48 : 103 - 1081 Naish , KA , Skinbiski DOF , 1998 Tetranucleotide microsatellite loci for Indian major carps. J . fish. Biol. 53 : 886 - 8891 Nei M , Chesser R K , 19831 Estimation of fixation indices and gene di2 versities. Ann. Hum. Genet . 47 : 253 - 2591 O¡fl Connell M , Wright J M , 1997 1 Microsatellite DNA in fish. Rev. Fish Biol. 7 : 331 - 363 Paet kau D , Strobeck C , 19951 The molecular basis and evolutionary history of a microsatellite null allele in bears. Mol. Ecol. 4 : 519 5201 Raymond M , Rousset F , 1995a. Testing heterozygote excess and defi2 ciency. Genetics 140 : 1 413 - 1 419 1 Raymond M , Rousset F , 1995b. GEN EPOP (Ver. 112) : a population

sit uation , cautious use of t he identified loci is suggest2 ed. The met hod of estimating null allele f requencies (Brookfield , 1996 ) may help in deriving appropriate conclusions. In conclusion , t he present st udy identified five polymorp hic microsatellite loci t hat exhibit promise to determine genetic divergence in nat ural populations of G1 cu rm uca . This will also provide monitoring mechanism against t he possible genetic bottlenecks ; t he populations may be facing and help to plan st rate2 gy for rehabilitation of declining nat ural resources. Acknowledgements ¡¡ Thanks are accorded to PIU , NA TP for financial support and Dr1 S1 P1 Singh , Principal Investigator , NA TP sub project ( MM 18) . References

690 ¡¡¡¡

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50

¡¡

genetics software for exact test and ecumenicism. Journal of Hered2 ity 86 : 248 - 2491 HYPERL IN K http/ / www1cefe1cnrs2mop 1fr/ Raymond M , Rousset F , 1995c. An exact test for population differenti2 ation. Evolution 48 : 1 280 - 1 283 Ruzzante DE , Taggart CT , Cook D , Goddard S , 19961 Genetic differ2 entiation between inshore and offshore Atlantic cod Gadus morhua off Newfoundland microsatellite DNA variation and antifreeze level. Can. J . Fish. Aquat . Sci. 53 : 634 - 6451

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