动物学报 　50 ( 4): 686 - 690, 2004.

A ct a Zoologica S i nica 　　　　　 　

Microsatellite D NA markers to assess population structure of red

tailed barb Gonoproktopter us curmuca 3

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

用微卫星标记评估红尾　的种群结构 3

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

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 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
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 ’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

　 Received Dec. 28 ,2003 ;accepted Jan. 31 ,2004

　3 This research was funded by a grant from World Bank (No. MM2 Ⅲ\ 18)
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 动物学报 A cta Zoologica S i nica
 4期 Achamveettil GOPALA KRISHNAN et al. :Microsatellite markers in Gonoproktopterus curm uca
primers , developed for t hree cyprinids in
G1 cu rm uca . The objective was to identify polymor2
p hic microsatellite loci and evaluate suitability of t he
identified loci in population st ruct ure analysis of
G1 cu rm uca .
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
lakkudi ( Vazhachal , n = 15 ) , 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∶5) 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 25μl , 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 μl of PCR product s
were elect rop horesed on non2denat uring polyacry2
lamide ( 19∶ 1 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
687
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
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
 688 动 　　物 　　学 　　报 50 卷 　
　　
Table 1 　Primers of Microsatellite loci tested for cross species amplif ication in G1 curmuca
No . of primer Genbank accession Successful primer pair amplified
Species Locus Reference
pairs tested No. in G1 curm uca No. ( %)

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

MFW 1 , 2 , 9 , 11 , 15 , 17 ,
Cypri nus carpio 10 　　— Crooijmans et al. , 1997 4 (20)
19 ,20 , 24 , 26
B arbodes 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 FW 1 GTCCA GACT GTTCA TCA GGA G CA 55 59 5

carpio GA GGT GTACACT GA GTCACGC

M FW 11 GCA TTT GCCTT GA T GGTT GT G CA 55 58 5

TCGTCT GGTTTA GA GT GCT GC

M FW 19 GAA TCCTCCA TCA T GCAAAC CA 55 51 6

CAAACTCCACA TT GT GCC

M FW 26 CCCT GA GA TA GAAACCACT G CA 55 57 4

CACCA T GCTT GGA T GCAAAA G

Catla catla Ccat G1 - 1 A GCA GGTT GA TCA TTTCTTCC [ GA TA ] n … 61 51 5

T GCT GT GTTTCAAA T GTTCC …[ CCA ] n

B arbodes B gon 22 TCTT GTT GA TCACACGGACG CCT - 55 1

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

M FW 1 Ch 175 - 190 4 01333 01571 010029 3 011185

Pr 175 - 195 5 01714 01745 011391

M FW 11 Ch 180 - 201 5 01267 01718 010003 3 015254

Pr 180 - 201 5 01143 01704 < 010001 3

M FW 19 Ch 215 - 220 3 01200 01487 010101 33 010005 3

Pr 201 - 225 6 01786 01791 010199 33

M FW 26 Ch 147 - 160 4 01267 01649 010005 3 010008 3

Pr 147 - 157 3 01286 01582 010020 3

Ccat G1 21 Ch 185 - 201 5 01400 01660 010319 33 010370 33

Pr 190 - 201 4 01429 01599 010204 33

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 wit h 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.
 4期 Achamveettil GOPALA KRISHNAN et al. :Microsatellite markers in Gonoproktopterus curm uca
( 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
G1 cu rm uca .
3 　Discussion
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
689
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
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’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
 690 动 　　物 　　学 　　报 50 卷 　
　　
genetics software for exact test and ecumenicism. Journal of Hered2 Scribner KT , Pearce J M , 20001 Microsatellites : evolutionary and
ity 86 : 248 - 2491 HYPERL IN K http/ / www1cefe1cnrs2mop 1fr/ met hodological background and empirical applications at individual ,
Raymond M , Rousset F , 1995c. An exact test for population differenti2 population , and phylogenetic levels. In : Baker ed. Molecular
ation. Evolution 48 : 1 280 - 1 283 Met hods in Ecology. London , England : Blackwell Science Limit2
Ruzzante DE , Taggart CT , Cook D , Goddard S , 19961 Genetic differ2 ed , 235 - 2711
entiation between inshore and offshore Atlantic cod Gadus morhua Zheng W , Stacey N E , Coffin J , Strobeck C , 19951 Isolation and char2
off Newfoundland microsatellite DNA variation and antifreeze level. acterization of microsatellite loci in goldfish Carassi us aurat us .
Can. J . Fish. Aquat . Sci. 53 : 634 - 6451 Mol. Ecol. 4 : 791 - 792 1