动物学报 　50 (4): 686 - 690, 2004.A cta Zoologica S inica 　　　　　 　

　 Received Dec. 28 ,2003 ;accepted Jan. 31 ,2004　3 This research was funded by a grant from World Bank (No. MM2Ⅲ\ 18)33 Corresponding author. 　E2mail :nbfgr @sancharnet . in ;kulvin 100 @hotmail. com

333 NBFGR Cochin Unit ,CMFRI Campus , PB # 1603 , Ernakulam , Kochi :682018 , Kerala , India. E2mail :nbfgrcochin @vsnl. net3333 Present Address :BGRRP ,World Fish Center ,PO Box 500 GPO ,10670 ,Penang ,Malaysia. E2mail :a. ponniah @cgiar. org　 ν 2003 动物学报 Acta Zoologica Si nica

Microsatell ite DNA markers to assess population structure of redtailed barb Gonoproktopterus curmuca 3

Achamveettil GOPALA KRISHNAN333 , Kochikkaran Kunjumohammed MUSAM2M IL U333 , Peringady Mohammed ABDUL MUN EER333 , Kuldeep Kumar LAL33 ,Dhurendra KAPOOR , Alphis Geethanand PONN IAH3333 , Vindhya MOHINDRANational Bureau of Fish Genetic Resources (NBFGR2ICAR) , Canal Ring Road , P1O. Dilkhusha , Telibagh , Lucknow2226002 ( UP) ,India

用微卫星标记评估红尾　的种群结构 3

Achamveettil GOPALA KRISHNAN333 , Kochikkaran Kunjumohammed MUSAM2M IL U333 , Peringady Mohammed ABDUL MUN EER333 , Kuldeep Kumar LAL33 ,Dhurendra KAPOOR , Alphis Geethanand PONN IAH3333 , Vindhya MOHINDRANational Bureau of Fish Genetic Resources (NBFGR2ICAR) , Canal Ring Road , P1O. Dilkhusha , Telibagh , Lucknow2226002 ( UP) ,India

摘 　要 　本文检测了三种鲤科鱼的 16 对微卫星引物在红尾　中的适用性 , 其中 6 对引物可以成功扩增 , 且 5 个位点具有多态性。对采自两条不同河流的标本 , 通过检测这些多态微卫星位点的遗传变异情况 , 评估了它们在红尾　种群结构分析的适合性。结果显示这 5 个多态位点在上述两个样本中的平均表观杂合度分别是 01293 和01471。这两个样本显著的基因异质性表明我们所确定的微卫星标记可用于红尾　的种内遗传分化研究 [动物学报 50 (4) : 686 - 690 , 2004 ]。关键词 　红尾　 　微卫星 　遗传变异Key words 　Red tailed barb , Gonoproktopterus curm uca , Microsatellite , Genetic variation

　 　Red tailed barb Gonoproktopterus curm uca( Hamilton2Buchanan , 1807) is endemic to the riversoriginating exclusively from southern part of theWestern Ghats in Peninsular India ( Gopalakrishnanand Ponniah , 2000) . The Western Ghats are recog2nized as one of the twenty five biodiversity“hotspots”of the world (Myers et al. , 2000) . G1 curm uca hascommercial value as a food fish as well as for orna2mental t rade and also considered as a potential speciesfor aquaculture. The sharp decline in abundance ofG1 curm uca and its endangered status is of serious

concern ( Gopalakrishnan and Ponniah , 2000) . Forthe significance attached to the species , effective con2servation and propagation2assisted rehabilitationstrategies need to be planned. However , such an ap2proach needs data on the genetic variation and popula2tion structure of G1 curm uca across its natural dist ri2bution. To generate population genetics data , identi2

fication of polymorphic markers with consistentscorable alleles is a crucial step ( Fergusan et al. ,1995) . Until now , no information is available on anyclass of genetic markers in G1 curm uca .

Microsatellites are short tandem repeat motifswith high level of allelic polymorphism and co2domi2nant inheritance , useful for direct assessment of pat2tern and dist ribution of genetic variability at intraspecific level (O’Connell and Wright , 1997) . Theflanking sequences of microsatellites within relatedtaxa are highly conserved. The potential of thesemarkers is enhanced when primers designed for onespecies amplify homologous loci in other species(Scribner and Pearce , 2000) . Successful amplifica2tion of homologous microsatellite loci has been demon2st rated in some cyprinid fishes ( Zheng et al. , 1995 ;Mohindra et al. , 2001 ;Lal et al. , 2004) . The pre2sent study examines cross2species amplification of

primers , developed for three cyprinids inG1 curm uca . The objective was to identify polymor2

phic microsatellite loci and evaluate suitability of theidentified loci in population structure analysis ofG1 curm uca.

1 　Materials and methods111 　Sampling sites and sample collection

The G1 curm uca specimens were obtainedthrough commercial catches from two rivers , Cha2lakkudi ( Vazhachal , n = 15 ) , Periyar(Bhuthathanketuu , n = 14) . The riverine locationswere chosen to cover geographically distant popula2tions of G1 curm uca. The samples were obtainedduring J uly 1999 to August 2001 and total lengthranged from 200 mm to 600 mm ;collection was doneat actual fishing sites. Blood samples , collectedthrough caudal puncture , were fixed in 95 % ethanol(1∶5) and stored at 4 ℃ till use.112 　PCR amplif ication and electrophoresis

Total genomic DNA was extracted from bloodsamples following the procedure of Ruzzante et al.(1996 ) . PCR amplifications were performed ( MJResearch thermal cycler PTC2200) in a final volumeof 25μl , containing 25 - 50 ng of genomic DNA , 1 ×PCR buffer (10 mmol Tris2HCl , p H 910 ; 50 mmolKCl ; 0101 % gelatin) , 210 mmol MgCl2 , 012 mmolof each dN TP , 5 pmol of each primer and 115 unitsof Taq DNA polymerase.

Amplification conditions were 94 ℃ for 5 minfollowed by 25 cycles at 94 ℃ for 30 s , Ta for 30 sand 72 ℃ for 1 min , with a final extension of 72 ℃for 4 min. After amplification , 8μl of PCR productswere electrophoresed on non2denaturing polyacry2lamide (19∶1 acrylamide bisacrylamide) gels (size 10cm ×1015 cm , Amersham Biosciences Ltd. ) . Thegel concentration was optimised according to allelesize for better resolution. Electrophoresis was done at4 ℃with 1 × TBE buffer for 5 hours at 150 V. Thegels were silver stained ( Silver Staining Kit , Amer2sham Biosciences , USA) to visualize microsatelliteloci and allelic patterns ;and known DNA size marker( MspI cut pB R322 DNA) was run in every gel. Thesize of the amplified products was determined with IDElite (Amersham Biosciences) software. The alleleswith dinucleotide repeats could be resolved and weredesignated according to PCR product sizes. Genotypeof each individual at each locus was assigned manual2ly.113 　Screening of primers and genetic diversityanalysis

Microsatellite primers from Cyprinus carpio ,B arbodes gonionot us and Catla catla were tested foramplification of homologous loci ( Table 1 ) . Thethree species are termed as resource species in the

study. This cross2species amplification experimentwas done with eight specimens of G1 curm uca . Theoptimum annealing temperature to get scorable bandpattern was determined through experimental stan2dardization for each primer pair. The primers yieldingscorable amplified product were again evaluated withlarger sample size (29 individuals , f rom 2 rivers) toevaluate their suitability in quantification of geneticdivergence in G1 curm uca . The data was analyzedusing software Genetix 4102 (Belkhir et al. , 1997)to obtain allele f requencies , mean number of allelesper locus , heterozygosity values , expected ( He) andobserved ( Ho) . Tests for conformity to Hardy2Wein2berg expectations (probability and score test ) wereperformed by the Markov chain method with parame2ters dememorization = 1 000 , batches = 100 and it2eration = 100 ( Genepop ver. 313 , probability test) .Genetic homogeneity of four sample sets was deter2mined through an exact test ( G based test ) that as2sumes random samples of genotypes ( Genepop ver.313 , Genotype differentiation test ) ( Raymond andRousset , 1995b) . This test is performed on genotypetables and possible non2independence of alleles withingenotypes will not affect test validity ( Raymond andRousset , 1995c ; Goudet et al. , 1996) .

2 　ResultsOf the 16 heterologous primer pairs tested , six

(23100 %) provided successful amplification of ho2mologous loci in G1 curm uca ( Table 1) . It is evident( Table 2) that the optimum annealing temperature( Ta ℃) observed in G1 curm uca differed from thatreported in the resource species for respective primerpair. Primer B gon22 amplified but producedmonomorphic band in all the individuals tested.

In the present study , five polymorphic mi2crosatellite loci ( M FW 1 ,11 ,19 ,26 , Ccat G121) , ex2hibiting 2 to 5 alleles , could be successfully identifiedfor G1 curm uca . The parameters of genetic variationat each locus and over all loci differed between thetwo sample sets ( Table 3) . The observed heterozy2gosity values over all loci were 01293 ( Chalakkudi)and 01471 ( Periyar) . Mean number of alleles per lo2cus ranged from 4120 ( Chalakkudi) to 4140 ( Peri2yar) . The probability test provided the evidence thatthe observed allele f requencies significantly ( P <0105 ) deviated from that expected under Hardy2Weinberg equilibrium. Deviation was observed inboth the sample sets , at three to four loci ( Table 3)with significant deficiency of heterozogotes. Exceptat locus M FW 1 ( Chalakkudy samples) , the scoretest also confirmed significant heterozygote deficiencyat other three loci.

Significant heterogeneity ( P < 0105) in geno2type proportions was observed at three out of five loci

7864 期 Achamveettil GOPALA KRISHNAN et al. :Microsatellite markers in Gonoproktopterus curm uca　　

Table 1 　Primers of Microsatellite loci tested for cross species amplif ication in G1 curmuca

SpeciesNo. of primer

pairs testedLocus

Genbank accessionNo.

ReferenceSuccessful primer pair amplified

in G1 curm uca No. ( %)

Catla catla 1 Ccat G1 AF045380 Naish and Skibinski , 1998 1 (100)

Cypri nus carpio 10MFW 1 , 2 , 9 , 11 , 15 , 17 ,

19 ,20 , 24 , 26　　— Crooijmans et al. , 1997 4 (20)

Barbodes gonionot us 5 Bgon 22 , 69 , 75 ,79 ,17 　　— Chenuil et al. ,1999 1 (20)

Total tested 16 6 (23100)

Table 2 　Characteristics of amplif ied microsatellite loci in G1 curmuca

Resource species G1 curm uca

Species Locus Primer sequence (5′→ 3′) Repeat motif Ta ( ℃) Ta ( ℃) No. of alleles

Cypri nus M FW1 GTCCAGACTGTTCATCAGGAG CA 55 59 5

carpio GAGGTGTACACTGAGTCACGC

M FW11 GCATTTGCCTTGATGGTTGTG CA 55 58 5

TCGTCTGGTTTAGAGTGCTGC

M FW19 GAATCCTCCATCATGCAAAC CA 55 51 6

CAAACTCCACATTGTGCC

M FW26 CCCTGAGATAGAAACCACTG CA 55 57 4

CACCATGCTTGGATGCAAAAG

Catla catla Ccat G1 - 1 AGCAGGTTGATCATTTCTTCC [ GATA] n ⋯ 61 51 5

TGCTGTGTTTCAAATGTTCC ⋯[ CCA] n

Barbodes B gon 22 TCTTGTTGATCACACGGACG CCT - 55 1

gonionot us ACAGATGGGGAAAGAGAGCA

Table 3 　Parameters of genetic variability for each microsatellite locus in G1 curmuca samples from two locations

Locus homogeneity River Size range (bp) No. of alleles at each locus Ho He HW(p) Genotype (p)

M FW1 Ch 175 - 190 4 01333 01571 010029 3 011185

Pr 175 - 195 5 01714 01745 011391

M FW11 Ch 180 - 201 5 01267 01718 010003 3 015254

Pr 180 - 201 5 01143 01704 < 010001 3

M FW19 Ch 215 - 220 3 01200 01487 01010133 010005 3

Pr 201 - 225 6 01786 01791 01019933

M FW26 Ch 147 - 160 4 01267 01649 010005 3 010008 3

Pr 147 - 157 3 01286 01582 010020 3

Ccat G121 Ch 185 - 201 5 01400 01660 01031933 01037033

Pr 190 - 201 4 01429 01599 01020433

Mean over all loci Ch - 4120 01293 01617 - < 010001 3

Pr - 4140 01471 01684 -

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

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.

886 动 　　物 　　学 　　报 50 卷 　　　

( Table 3) . After the sequential bonferroni correction( P < 010071) was made to the probability levels , stilltwo loci ( M FW 19 and 26) exhibited significant het2erogeneity. Combined proba2bility over all the lociwas less than 010001 ( Table 3) . Gst for small samplesize (Nei and Chesser , 1983) of 01049 provided fur2ther evidence of population substructuring i nG1 curm uca.

3 　DiscussionThe study demonstrates successful cross2priming

of microsatellite loci in red tailed barb , G1 curm ucaand identified five polymorphic loci. The result isconsistent with the earlier reports , suggesting thepossibility of using primers interspecifically amongcyprinids ( Zheng et al. , 1995 ) . Mohindra et al.(2001) demonstrated amplification of homologous mi2crosatellite locus in L abeo rohita using primer devel2oped for other cyprinid Catla catla. Successful iden2tification of polymorphic microsatellite markers forCi rrhinus m rigala was achieved through use ofprimers of other cyprinid fishes (Lal et al. , 2004) .

The presence of null alleles could be one of thepossible factors responsible for the observed heterozy2gote deficiency. Null alleles are not represented inPCR amplification due to mutation at primer bindingsite and contribute towards homozygote excess(Paetkau and Strobeck , 1995) . Raymond and Rous2set (1995a) suggested the score test was more power2ful than the probability test when the alternate hy2pothesis of interest is heterozygote deficiency. Inter2estingly , except at locus M FW 1 ( Chalakkudy sam2ples) , the results of the score test were consistentwith the probability test . This may not be a conclu2sive interpretation for the absence of null alleles ,however it suggests the likelihood of deficiency of het2erozygotes at least at three loci in both populations. Ift rue , a serious concern is that the assumptions under2lying the Hardy2Weinberg equilibrium relevant to nat2ural population of G1 curm uca are violated ( Fergusonet al. , 1995) . One of the reasons could be reductionin effective breeding population size in G1 curm ucapossibly due to overexploitation , rest ricted migrationsand habitat alterations , etc.

Genetic heterogeneity was tested on the tablesbased on the genotype rather than allele f requencies inview of the observed nonconformity to Hardy2Wein2berg expectations ( Raymond and Rousset , 1995c ;Goudet et al. , 1996) . The various estimates providedstrong evidence that the two sample sets were notdrawn from the same random mating gene pool.Analysis of larger sample sizes from more geographicallocations will provide fine scale assessment of popula2tion structure of G1 curm uca and also more insightinto the observed homozygote excess. In the given

situation , cautious use of the identified loci is suggest2ed. The method of estimating null allele f requencies(Brookfield , 1996) may help in deriving appropriateconclusions.

In conclusion , the present study identified fivepolymorphic microsatellite loci that exhibit promise todetermine genetic divergence in natural populations ofG1 curm uca. This will also provide monitoringmechanism against the possible genetic bottlenecks ;the populations may be facing and help to plan strate2gy for rehabilitation of declining natural resources.

Acknowledgements 　Thanks are accorded to PIU ,NA TP for financial support and Dr1S1P1Singh ,Principal Investigator , NA TP sub project ( MM -18) .

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