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Phylogeny of the Genus Artemia Based on RFLP Analysis of Mtdna in IRAN

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Phylogeny of the Genus Artemia Based on RFLP Analysis of Mtdna in IRAN Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159) Phylogeny of the genus Artemia based on RFLP analysis of mtDNA in IRAN MAHBOBEH HAJIROSTAMLOO 1,SOHRAB REZVANIGILKOLAEI 2,SEIDMOHAMMAD REZA FATEMI 3,MAJEEDSADEGHIZADEH 4,SHAHROKHKAZEMPOUROSALOO 5 1DepartmentofBiology,Islamic AzadUniversity,MarandBranch,Marand54165161,IRAN 2IranianFisheriesResearchOrganization,Tehran141556116,IRAN 3DepartmentofMarinBiology,IslamicAzadUniversity,Science andResearchBranch,Tehran 14515775,IRAN 4DepartmentofGenetics,Facultyof BasicSciences,TarbiatModaresUniversity,Tehran14115175, IRAN 5DepartmentofPlantBiology,Facultyof BasicSciences,TarbiatModares University,Tehran14115 175,IRAN Email: [email protected] Abstract: - Phylogenetic relationships among the Iranianpopulations of Artemia belonging tothree species of Artemia parthenogenetica, A. franciscana and A. urmiana were inferredby PCR-RFLP analysis of a 1566bp segment of the mitochondrial 16S rRNA gene.A total of 270 specimens from 9Artemia populations inhabiting inthe inlandIranianlakes (Urmia,Shoor,Inche-Burun,Maharloo,Bakhtegan,Namak,Hoze-Soltan,Mighan and Nough) was surveyed using 10 restriction enzymes. Twelve different haplotypes were detected in A. parthenogenetica populations,including four haplotypes inShoor andInche-Burun,one inBakhtegan,one in Namak andHoze-Soltan,three inMighanandthree in Maharloo.Four different haplotypes of A. franciscana were foundinNoughpopulationandnine different haplotypes of A. urmiana were foundinUrmia population. Phylogenetic analysis showedthese nine populations were clusteredintothree clades: 1- Shoor,Inche-Burun, Hoze-Soltan,Namak,BakhteganandMaharloo2 Nough andMighanand3 Urmia.Inthis study referring to literature andbasing onthe oldness of A. urmiana,it selected as the outgroupe.Obtainedresults alsoshow the ability of usedtechnique for divisionof different populations of Artemia inIranandparthenogenetic Artemia`s derivationfrom bisexual populationsandhighgeneticallyresemblancewiththose of A. urmiana. Key-word: - Artemia,mtDNA,phylogeny,PCR-RFLP,rRNA,IRAN 1 Introduction [22].Inspite of its great interest andextensive use Iranis in southhalf of temporal regioninnorthern inaquaculture,little is knownabout the systematic hemisphere and most parts of country have warm ofthedifferent Artemiastrainsandspecies [18]. climate. Water of some river, lakes and pools are Molecular DNA tools have beenwidely applied brine because they pass or settling in lands with in different organisms during the post decade in highsalt content,most of this lakes andpools are studying genetic similarities and differences, habitats of Artemia, likes Urmia lake in West phylogenetic relationships andmolecular evolution. Azerbaijan, Shoor and Inche-Borun lakes in This is especially important in phyla where no Golestan, Hoze-Soltan and Namak lakes in Qom, fossil records are available and/or there are no clear Maharloo and Bakhteghan lakes in Fars, Nough morphological traits to be used as markers of the pool inKermanandMighanpool inMarkazi. different population [18]. Over the last decade, The brine shrimp Artemia (Family Artemidae, modern techniques and new molecular markers order Anostroca, class Branchiopoda, subphylum have beenincreasingly usedinArtemia research[1, Crustacean and phylum Arthropoda) is a widely 24, 22, 2, 3, 10, 11, 20, 12].Their application, in distributed throughout the world in inland salt combinationwithcomplementary informationfrom lakes, coastal lagoons, and salterns. The genus of other techniques,makes them particularly useful in Artemia consists of a set of bisexual species providing better descriptions andinterpretations of definedby the criteria of reproductive isolationand biological diversity[24,3] a large number of parthenogenetic forms canbe a Its high rate of evolution, maternal mode of greatvarietyof ploidiesgroupedunder the binomen inheritance, and lake of recombination has made A. partenogenetica, for taxonomic convenience mtDNA the most useful molecule in this kind of Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159) analysis [18].It is small,compactly organizedwith 2.2 DNA extraction and PCR few intergenic spaces and with few exceptions its DNA extractedusing the differential centrifugation gene content is strictly conserved in the animal and phenol-chloroform method with some kingdom [14]. It is well established that its modification[12].ExtractedDNAs electrophoresed mutation rate is quite high, 5-10 times higher on %1 agarose gel containing 0.5 µg/ml ethidium comparedwithsingle-copynuclearDNA,probably bromide inTBE buffer (90 mM Tris,90mM boric be cause there are noDNA- repair mechanisms in acid,2mM EDTA,PH=8) andvisualizedwithgel the mitochondria [10]. Very preliminary attempts documentationsystemfortheirquality. based on mtDNA restriction fragment length One mtDNA segments amplified with PCR using polymorphism (RFLP) analysis,were ventilatedin thefollowing primers: the early 1990s for determining evolutionary Forward:5′ -CCGGTCTGAACTCAGATC–3′ divergence inArtemia [6,7].Tworibosomal RNAs Reverse: 5′ -CTAGGATTAGATACCCTA–3′ (12 S and 16 S) are fragments of mt DNA that These primers designed based on a part of the separated from each other by tRNA- Val. In this Artemia franciscana 16sr RNAs sequence [25]. research effort we use 16srRNA for designing EachPCR reactioncomposedof 1µl of extracted primers, because previous studies [11, 20, 12] DNA,5µl of 10X PCR buffer (500mM kcl,200 showedthis gene is a highly favorable fragment for mM Tris- Hcl),0.5µl of d NTP mix(10mM each genetically similarities and differences, to estimate of dATP,dTTP,dCTP and dGTP , PH=7.5),2µl of the genetic relationship among different Artemia eachof 2primers tofinal concentrationof 20pM, populationof Iranand modeling their phylogenetic 0.5 µl of Taq polymerase (5 unit per µl) , 2 µl pattern byPCR-RFLPanalysisofmtDNA. Mgcl (50 ml M) and deionized water added to a 2 final volume of 50µl.The amplificationconditions consisted of initial denaturation at 94oc for 4 min 2 Materials and Methods and30sec,30cycles performedat 94oc for 1min and 15 sec, 54oc for 50 sec, 72oc for 1 min as 2.1 Artemia population denaturation,annealing andextensionrespectively. Adults Artemia individuals were collected from Finally 72oc for 4minas final extensionperformed. night different population: Urmia, Shoor, Inche- Electrophoresis of 10 µl of the amplified product burun, Namak, Hoze-Soltan, Maharloo and performed for their quality on a %1 agarose gel. Bakhteganlakes andMighanandNoughflaques in The size of amplified fragments estimated with a Iran(Fig.1).Samples were fixedinethanol (%95) DNA size marker (Lambda DNA Marker,3) ona andtransferredtoCaspianSea Ecological Research %6 polyacrylamid gel in TBE buffer and bands Institute for molecular study. Molecular visualizedwithsilverstaining[12]. information of A.franciscana as reference species calculatedonits complete mtDNAsequence[25]. 2.3 RFLP analysis PCR products (47 µl) separately digested by 10 restriction endonucleases: AluI, EcoRI, Eco47I, HaeIII, HindIII, HinfI, MobI, MspI, RsaI, TaqI ina final volume 20 µl according to the manufacture suggestedprotocol,todisplayDNA polymorphism. Then the digested samples run on %6 polyacrylamide gel and staining. The size of restrictionfragments estimatedincomparisonwith aDNAsizemarker(Ladder50bp). 2.4 Phylogenetic analysis For eachendonuclease,producedrestrictedpatterns were identifiedandall alignedby size.Individuals were codedbasedonexistence or nonexistence of each restricted fragment with specific number Fig1.Sampling sites of IranianArtemia population: (1or0) and assigned a multi-number code that 1) Urmia 2) Inche-borun 3) Shoor 4) Hoze-Soltan describedtheircompositemtDNAgenotypes. 5) Namak 6) Mighan 7) Nough 8) Maharloo 9) Phylogentic analysis was performed on multi- Bakhtegan number codes using maximum parsimony method Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, 2006 (pp154-159) (MP) as implemented in the version 4.0b16 of %6polyacrylamidgel,M=Marker PAUP [23]. The strict Consensus and Distance trees were obtained and there Bootstrap values, 3.2 Digestion with restriction endonuclease Length, Consistency Index (CI), Retention Index PCR products digested using 10 restriction (RI), Rescaled Consistency Index (RC) were enzymes. All enzymes restricted amplified DNA, calculated.Withattendtoconditionthat Maddison, produced fragments were clear and there totally etal. [16] were pointed about correct selection of sizes incomparisonwithDNA marker ladder 50bp Out-grouplikes: Out-groupmust be older thanIn were same with PCR Product. Nine of ten group and there must be no far distance between endonucleases (MspI, HinfI, HindIII, Eco47I, AluI, Out and In-groups and with attention to previous MobI, HaeIII, RsaI, TaqI) were showed inter and studies that indicated to oldness of Artemia interapopulation polymorphism that indicated to Urmiana in compare with other genus [7] it was different cut site in nucleotides sequence of selected as Out-group. Strict consensus tree 16SrRNA within and among Iranian Artemia adequately summarizes the available evidence exist population. Results of enzymatic digestive indifferent phylogenic tree offeredwithPAUP and indicatedtoexistence of 25haplotypes of Artemia distance tree that able to explain genetically
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