Isozyme Analysis on the Populations of Ruditapes philippinarum

Journal of Ocean University of China ( Oceanic and Coastal Sca Research )ISSN .1672-5182 January 30 2006 ,Vol.5 No.1 pp.58-62http :// awnww. ouc. edu. cn/xbyvb/E-mail :xbyrvb@ mail. OUC. edu. cnIsozyme Analysis on the Populations ofRuditapes philippinarumREN Yiping* , GAO Tianxiang , and YANG TianyanCollege of Fisheries , Ocean University of China , Qingdao 266003 , P. R. China( Received January 24 2005 ; accepted November 4 2005 )Abstract Horizontal starch gel electrophoresis was used to investigate the genetic structure of six populations of Ruditapesphilippinarum in the coast of China. Seven enzymes revealed eleven putative loci , and seven of them were polymorphic ,PGM* ,MDH-1* , MDH-3* ,LAP-1* ,LAP2* , MPI-1 * and MPI-2*. The proportions of polymorphic loci of R. philip-pinarum populations varied from 0.454 5 to 0.6364. The values of observed and expected heterozygosities were from 0.039 4to 0.1545 and from 01111 to 0.238 5 , respectively. Nei's genetic distance varied from 0.0033 to 0.0253 with an average of0.0135. It is suggested that the genetic diversity of R. philippinarum was high.Key words Ruditapes philippinarum ; isozyme ; genetic variationwere collected from six different locations in 2004( Fig.1 ) , 30 individuals each location. All the individ-1 Introductionuals were collected in the intertidal zone and broughtManila clam ，Ruditapes philippinarum , whichto the Key Laboratory of Mariculture , Ministry of E-belongs to the family Veneridae , distributes mainly a- ducation ,Ocean University of China either alive or inlong the coasts of the Philippines , Japan and Chinafrozen. Upon the individuals' arrival ，the muscles( Zhuang ,2001 ). R. philippinarum is an intertidal were immediately taken out and kept at- 80C.bivalve, which prefers to inhabit in the inner bay117"125°129°E( Zhuang ,2001 ;Xie ,2003 ). It has high commercial41"Nvalue and is also an important aquaculture species inintertidal zones. In recent years , because of over- fish-Dalianing and environmental pollutions ，the yield of R.philippinarum has decreased ( Liu et al.，1999 ).37°Laizhou. There were many studies on C. gigas ，haemastomaQingdaoand Mytilus edulis ( Fujio, 1982 ; Garton, 1984 ;Koehn , 1984 ) and other organisms( Zheng and Wu，Yellow Sea1994 ; Stephen and Richard ，1997 ; Buso et al. ,331998 ). In order to investigate the causes for the highmortality and outbreak of diseases , it is important tcexamine the genetic structure of R. philippinarumNingbo F East China Sea29and compare the level of population heredity. Themuscles of R. philippinarum of six different popula-tions collected from the coasts ( Ningbo ，Putian ,Tianjin , Qingdao , Laizhou and Dalian )of China were25°t ( Putiangranalyzed using the method of horizontal starch gelelectrophoresis in this paper.br中国煤化工2 Materials and MethodsMHCNMHG2.1 SamplingFig.1 Locations of six populations.A total of 180 individuals of R. philippinarum2.2 Isozyme Analysis* Correspondingutbor. Tel :0086 532- 82032960All individuals to be analyzed for isozyme were hcE- mail xeHyi区扼. edu. cnmogenized with approximately equal volume of dis-REN Y.P. et al. : Isozyme Analysis on the Populations of Ruditapes philippinarum59tilled water. The homogenate was centrifuged at 12000 the heterozygosity( H ) which involved the observedrmin-' for 12 min at 4C. The supernatant was ab-heterozygosity( Ho ) and the expected heterozygositysorbed by filter paper and used for electrophoresis.(He). A locus was considered to be polymorphic ifEnzyme electrophoresis was carried out using stan-the most common allele was equal to or less than 0.95dard horizontal starch gels following the method of the at one or more localities.. The genetic deviation indexJapan Fisheries Resource Conservation Association defined as d =( Ho/He)- 1 was calculated for each( 1989 )and Pasteur et al. ( 1988 ). Electrophoresis w- polymorphic locus , where Ho was determined by di-as performed in 11.8% horizontal starch gels using TC rect count and He was estimated from average values-7.0 buffer system. Seven isozymes tested included across all samples( Nei , 1987 ). This estimation of theaspartate aminotransferase( AAT ,E.C. 2.6. 1.1 )，deviation from Hardy-W einberg equilibrium was usedleucine aminopeptidase ( LAP，E. C.3.4. 11. 1 )，to estimate the null. alele frequencies.malate dehydrogenase ( MDH, E. C.1.1.1.37 ),In order to estimate the degree of genetic divergencemalic enzyme( ME, E. C.1. 1.1. 40 ), mannose-6-among the samples , Nei's genetic distance formulaphosphate isomerase( MPI ,E.C. 5.3. 1.8 ), phos-was used. The dendrograms from the matrix of genet-phoglucomutase( PGM ,E.C. 5.4.2.2 ) , and super-ic distances were constructed using the UPGMAoxide dismutase( SOD ,E.C. 1.15.1.1 ). The names，method( Nei ,1972 ).E. C. numbers and abbreviations of the enzymes werethe same as those described by Shaklee et al.( 1990 ).3 ResultsGenotypes were computed for each individual inpopulations and used to estimate genetic parameters.Those isozymes obtained consistently and clearly inThe allele frequencies were also calculated for eachII individuals , were considered to be useful geneticpopulation. The extent of genetic variability in each markers for R. philippinarum population analysispopulation was quantified by determining the propor- (Fig.2). The seven enzyme systems revealed elevention of polymorphic loci per population( P ) ,the aver-putative loci ，of which ，seven were polymorphicage number of efficient allele( Ae ) , the genetic identi-( LAP-1* ,LAP-2* ,MDH-1* , MDH-3* ,MPI-1* ,ty( I ), the genetic distance( D ) which were calculat-MPI-2 * PGM* ),while theothers( AAT* ,MDH-2* ,ed for pairwise comparisons of the six populations toprovide an estimate of their genetic relationships , and ME* , SOD * ) were monomorphic in all populations.MDH-1#:MPI-I#MDH-2*MDH-3#*MPI 2*MDH-!"*奶0b/b*b/cMDH-3* *avb畅/MP-2*talcobbec"ddebice+|:一". :LAP-1中PGM*:一-二LAP-2*+a/b *b/b *a/a *clc *b/c*ale b/e */x *de *ele °dJ *elgLAP-2* *a/b *a/a *$b/b *clc *d/d *b/c+中国煤化工MHCNMHGME*SOD*AAT**ala*a/aFig.2 Electrophoretic patterns of eleven isozymes.60Journal of Ocean University of ChinaVol.5 ,No. 12006Genetic variabilities were estimated based on the erage observed and expected heterozygosities rangedproportion of polymorphic loci and mean heterozy-from 0.0394 to0.1545 and from 0.111 1 to 0.2385 ,gosity( Table 1 ). The average proportion of polymor-respectively. The average number of efficient allelephic loci( P ) varied from 0.4545 to 0.6364. The av- was from 1.3109 to 1 .4779.Table 1 Allele frequency , proportion of polymorphic loci( P* ) , average observed andexpected heterovygosities( Ho and He ) , and the average number of efficient allele( Ae)LocusAlleleNingboPutianTianjinQingdaoLaizhouDalianAAT”a1.00001. 000LAP-1*0. 13330.0333.0. 25000. 26670.16670.0833b0.78330.93330.63330.56670.71670. 76670. 0330.1670. 16670.11670. 1500LAP-2 °0. 06670.01670.05000.783 30.60000.61670.68330. 05000.25000.21670.26670.. 3500d0. 01670. 00000.00000.0000.033MDH-1。0.10000.0000 .0.90001. 0000MDH-2“MDH-3 *0. 0000. 10000.9330.96670. 95001. 0000.0. 90000.0333EM *1.000MPI-1 *0. 03330.06670.233 30.18330. 23330. 31670. 56670. 61670.73330.1333MPI-2”0. 20000.15000. 66670. 58330. 75000.066 70.07670. 11670. 1330.2000PGM *0. 000. 85000.7330.66670.2833gSOD“1. 000.)”0.54550.45450.63640. 5455Ho0.07270.03940. 10300.11210.15450. 1394He0.111 10.14140.20960.238 50.22960.2305Ae1.391 71.31091.32401.45221.47791.4399Table 2 The genetic deviation indices of six different populations- 0.5392-0.5537- 0.5402- 0.2509-0.4775LAP-2*-0. 6336-0.4714- 0.6396- 0.9388- 0.7824- 0. 3970MDH-1'0.1111MDH-3*0.05730.0526MPI- 1*-0.9411- 0.5337中国煤化工o5- 0.6764PMI-2 "0.9438-0.9193HCNMHG39- 0.5708- 0.0105- 0.05880.048 00.4101- 0.0958Because the expectation values of the genotype fre-1990 ). The genetic deviation indices at the PGM * ofquency were all less than5 ,the x~ test for the Hardy- populations of Putian ，Qingdao and Laizhou ，theWeinberg瓦市数据um could not be performed( Liu ,MDH1 * of populations of Qingdao , and the MDH3'REN Y.P. et al. : Isozyme Analysis on the Populations of Ruditapes philippinarum6:of populations of Ningbo , Qingdao and Dalian were all less than zero demonstrated slight absence of heterozy-more than zero , which showed the allele frequenciesat gotes( Table 2 ). Nei's genetic distances( Nei's D )a-these loci deviated from expected Hardy- Weinberg e- mong all samples used on the 11 loci were given inquilibrium and the heterozygotes were surplus at theseTable 3. The Nei's D of R. philippinarumn popula-loci. On the contrary , the rest genetic deviation index tions varied from 0.0033 to 0.025 3.Table3 Nei's genetic distance D and genetic identity Iamong R. philippinarum populations based on eleven lociGenetic identityGenetic distanceNingboPutianTianjinQingdaoLaizhouDalian0. 9939.0. 98480. 98640. 98840. 98830. 00620.97550. 97500. 98430. 97810.01540.02490. 99670.99170. 98900.01370.02530.00340.98950. 98820.01170.01580.00890.010590. 9904Dalian .0.01180.0220.01110.011870.0097portion of polymorphy loci of marine invertebrate isabout 0.587 ( Selander , 1976 ). The results indicated4 Discussionthat the genetic variation of R. philippinarum was aSince Market and Mullor developed the concept oflttle higher. The average observed and expectedisozyme' during 1957- 1959，with the development heuer-ozygosities over all loci were from 0.0394 toof the methods of isozyme analysis ,it has become an 0.1545 and from0.111 1 to 0.2385 , respectively. Com-important technique for studying biological phenomena pared with other marine clams , such as Farrer scallopand been widely applied. An understanding of the ex-( Chlamys farreri ) with the mean of observed and ex-tent and distribution of genetic variability within and pected heterozygosities of 0.1295 and 0.1695 ( Liamong R. philippinarum populations is essential foret al. ,2001 ) ,and three congeneric clams A. borealis ,devising strategies. It is also valuable in offering evi-A. elliptica , A. striata belong to the genus Astartdence of the evolutionary forces shaping natural popu-with the Ho and He values of 0.2577 ,0.1775 ,0.2200 ,lations. Furthermore ，it contributes to the under-and 0.6058 ,0.5678 ,0.6565 respectively( Busoet al. ,standing of the genetic variability available and its po-1998),the Ho and He values of R. philippinarumtential use in breeding programs.are at a middle level.' I he values of average heterozy-Much information has been obtained on aquatic or-gosities( Table 1 ) showed that the average heterozy-ganisms through isozyme analysis. The percentage of gosity of Qingdao population was higher than the rest ,polymorphic loci and the mean heterozygosity are two which indicated that the genetic diversity of Qingdaoimportant parameters for the analysis of genetic diver-population was more abundant comparatively , and thesity. The percentage of polymorphy of R. philip- genetic background was more complex.pinarum was from 0.4545 to 0.6364. GenerallyNei's genetic distance calculated from allele frequen-speaking，the percentage of polymorphy of teleost iscies of isozyme genes is also a useful measurement in0.215( Fujio and Kato, 1979 ), and the average pro- estimating the degree of genetic divergence. Nei( 1987 )中国煤化工一FYHCNMHG0.000 0.0020.004 0.006 0.008 0.010 0.012 0.0140.0160.018ig.3 Dendrogram constructed using UPGMA cluster analysis of Nei's genetic distance show-ing the relationship among the six populations of R. philippinarum.62Journal of Ocean University of ChinaVol.5 ,No. 12006summarized that the genetic distance was distributed57 : 520-543.around 0.01 between local races , around 0.1 between Japan Fisheries Resource Conservation Association ; 1989.subspecies and around 1.0 between species for variousPopulation Differentiation of Marine Organism by Isozymeorganisms. The results showed that Nei's D of six R.Analysis. Japan Fisheries Resource Conservation Associa-tion , Tokyo , 28 -209.philippinarum populations ranged from 0.0033 toKoehn, R. K., 1984. Gentic heterozygosity and growth0.0253 , which indicated that the individuals from sixrate in mytilus edulis. Mar. Biol. ,82 :1-7.different locations were the same species despite exist-Liu,R. Y., Y. B. Liang ,and X. C. Zhang , 1999. Pre-ing morphological differences( Table 3 ). Moreover ,liminary analysis and comparison with isozymes elec-the dendrogram of six populations showed that thetrophoresis of Ruditapes philippinaru in the north of Chi-Nei' s genetic distance was more or less related to geo-na. Mar. Environ. Sci. ,18 :24 -28.graphical distance ( Fig.3 ). The individuals from theLiu ,Z. D.，1990. The statistic of genetic data. In : TheGenetics. High Education Press , Beijing ,34- -42.locations near each other had the closer relationships.Ma,S. S. ,S. L. Zhou,J. F. Chen,F. Y. Xin, Y. Cui ,The individuals from Ningbo and Putian were neigh-et al. ,1997. On the death of the short-necked clam( Ru-boring and they were first assembled in the tree dia-ditapes philippinarum Adams et Reeve ) in the intertidegram.However，partly because of the extensivezone and ecologic environment effect. Mar. Fish. Res. ,breeding of R. philippinarum , there existed a lttle18:1-8. .confusion in the northern populations( Wu and Lii ，Nei , M.，1972. Genetic distance between populations.1993 ;Ma et al. ,1997 ). Generally speaking , we stillAmer. Nat. , 106 :283 - -292.could see the obvious differences between the northernNei , M. , 1987. Molecular Evolutionary Genetics. Columbiapopulations and the southern ones from the dendro-University Press ,New York ,128- 134.Pasteur ,N. ,G. Pasteur ,and F. Bonhomme ,1988. Practi-The results of isozyme analysis showed that therecal Isozyme Genetics. Elis Horwood Limited , Chichester ,509 pp.were genetic differences existing between the northernSelander ,R. K. , 1976. Molecular Evolution. Sinaver Asso-populations and the southern ones. Of course , the ge-ciates , Sunderland ,21 -45.netic relationship of R. philippinarum could not have Shaklee,J. B..F. W. Allendorf ,D. C. Morizot , and G.been revealed completely and further studies are stillS. Whitt , 1990. 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