Genetic differences between Capoeta capoeta capoeta, Capoeta capoeta umbla, and Capoeta tinca were determined using PCR-RFLP of mtDNA cytochrome-b (Cyt-b) by amplifying approxi- mately 400-500 bp of this region from each of the three subspecies. The restriction enzymes SpeI and HinfI did not indicate genetic differences but AluI and HpaII did. Thus, PCR-RFLP of the mtDNA was used to distinguish between closely related subspecies without having to ana- lyze the entire DNA sequence of specimens. Use of this technique demonstrated that the Cyt-b regions of the three subspecies have different base sequences. The similarity between C. c. capoeta and C. c. umbla is 71.4% while C. tinca is more distant (50% for C. c. umbla and 33.3% for C. c. capoeta). The Cyt-b similarity is consistent with morphological and taxonomical similar- ities. PCR-RFLP can serve as a tool for genetically identifying subspecies of fish in nature and in aquaculture

Aksakal, Ecrument

Erdogan, Orhan

eVols at University of Hawaii at Manoa

The Open Access Israeli Journal of Aquaculture – BamidgehAs from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published  exclusively  as  an  on-line  Open  Access  (OA) quarterly  accessible  by  all AquacultureHub  (http://www.aquaculturehub.org)  members  and  registered  individuals and institutions.  Please visit our website (http://siamb.org.il) for free registration form, further information and instructions.  This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible.Editor-in-ChiefDan Mires Editorial BoardSheenan Harpaz Agricultural Research OrganizationBeit Dagan, IsraelZvi Yaron Dept. of ZoologyTel Aviv UniversityTel Aviv, IsraelAngelo Colorni National Center for Mariculture, IOLREilat, IsraelRina Chakrabarti Aqua Research LabDept. of ZoologyUniversity of DelhiIngrid Lupatsch Swansea UniversitySingleton Park, Swansea, UKJaap van Rijn The Hebrew University Faculty of AgricultureIsraelSpencer Malecha Dept. of Human Nutrition, Food and Animal SciencesUniversity of HawaiiDaniel Golani The Hebrew University of JerusalemJerusalem, IsraelEmilio Tibaldi Udine UniversityUdine, ItalyCopy Editor Ellen Rosenberg Published under auspices ofThe Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), University of Hawaii at Manoa Library andUniversity of Hawaii Aquaculture Program in association withAquacultureHub http://www.aquaculturehub.org              ISSN 0792 - 156X Israeli Journal of Aquaculture - BAMIGDEH.PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH -Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAELPhone: + 972 52 3965809http://siamb.org.il The Israeli Journal of Aquaculture – Bamidgeh  59(4), 2007, 206-211.206Use of PCR-RFLP Analysis of mtDNA Cytochrome-bGene to Determine Genetic Differences in Capoeta spp.Ercument Aksakal* and Orhan ErdoganDepartment of Aquaculture, Agriculture Faculty, Ataturk University, 25240 Erzurum, Turkey (Received 1.4.07, Accepted 3.5.07)Key words: Capoeta spp., Cytochrome-b, MtDNA, PCR, RFLPAbstract Genetic differences between Capoeta capoeta capoeta, Capoeta capoeta umbla, and Capoetatinca were determined using PCR-RFLP of mtDNA cytochrome-b (Cyt-b) by amplifying approxi-mately 400-500 bp of this region from each of the three subspecies. The restriction enzymesSpeI and HinfI did not indicate genetic differences but AluI and HpaII did. Thus, PCR-RFLP ofthe mtDNA was used to distinguish between closely related subspecies without having to ana-lyze the entire DNA sequence of specimens. Use of this technique demonstrated that the Cyt-bregions of the three subspecies have different base sequences. The similarity between C. c.capoeta and C. c. umbla is 71.4% while C. tinca is more distant (50% for C. c. umbla and 33.3%for C. c. capoeta). The Cyt-b similarity is consistent with morphological and taxonomical similar-ities. PCR-RFLP can serve as a tool for genetically identifying subspecies of fish in nature andin aquaculture.Introduction Capoeta are widely distributed throughoutsouthern China, northern India, Turkmenistan,Lake Aral, the Middle East, and Anatolia,inhabiting gravel and stony areas of fast flow-ing rivers. There are five species and six sub-species of this genus in the inland waters ofTurkey (Geldiay and Balik, 1996). Fish species can be identified by analyz-ing proteins by isoelectric focusing (Pineiro etal., 2001) or immunoreaction (Carrera et al.,1997; Ochiai and Watabe, 2003). However, inmost cases, these methods of analysis areinapplicable to thermally processed productsas a result of protein denaturation. In suchcases, species can be identified using DNAnucleotide sequencing (Quinteiro et al., 1998),RFLP (Borgo et al., 1996; Quinteiro et al.,1998), or single strand conformation polymor-phism (SSCP; Marklund et al., 1995; Rehbeinet al., 1995). Analysis of nucleic acids, suchas mitochondrial and nuclear DNAs, hasadvantages over protein-based analyses, asthe former method is not dependent on tis-sues and the age of the individual. Further,mitochondrial DNA (mtDNA) has severaladvantages over nuclear DNA in the diagno-*  Corresponding author. Tel: +90-442-2311064, fax: +90-442-2360958, e-mail: eaksakal@atauni.edu.tr207sis of fish products. For example, the formerhas several times higher copy number and,therefore, is abundant in various tissues(Brand et al., 1994; Zhang and Hewitt, 2003).As mtDNA is haploid and a maternally inherit-ed sequence, ambiguities as a result of het-erozygous genotypes are theoretically avoid-ed (Lockley and Bardsley, 2000).DNA analysis for the identification ofclosely related fish species has already beenreported (Rehbein et al., 1995; Borgo et al.,1996; Birstein and Desalle, 1998; Sezaki etal., 2001). In this study we established analternative method for identifying Capoetaspp. by PCR-RFLP application and concludedthat PCR-RFLP can serve as a tool to geneti-cally identify subspecies of fish both in natureand in aquaculture (Quinteiro et al., 1998;Céspedes et al., 2000; Cocolin et al., 2000;Hisar et al., 2006).Materials and MethodsSample collection and DNA extraction. A castnet (1-2 km) was used to collect five adults(fork length>19 cm) from each population:Capoeta capoeta capoeta (Fig. 1) from theAras River, Capoeta capoeta umbla (Fig. 2 )from the Karasu River, and Capoeta tinca(Fig. 3) from the Coruh River, all in easternAnatolia, Turkey. The subspecies were identi-PCR-RFLP analysis of Capoeta spp.Fig. 1. Capoeta capoeta capoeta from the Aras River (39°59’13’N 41°42’55”E).Fig. 2. Capoeta capoeta umbla from the Karasu River (39°54’58”N 40°49’38”E).fied by methods proposed by Geldiay andBalik (1996). Muscle tissues were preserved immedi-ately in 95% ethanol and stored at ambienttemperature in the field and -20ºC in the labo-ratory. Sampled tissues were incubatedovernight at 55ºC in 500 µL of DNA extractionsolution (400 mM NaCl, 10 mM Tris, 2 mMEDTA, 1% SDS). Total genomic DNA was iso-lated according to Asahida et al. (1996) withminor modifications.Polymerase chain reaction (PCR) amplifi-cation. Approximately 400-500 bp of mito-chondrial Cyt-b gene were amplified with Cyt1(5’-cca tcc aac atc tca gca tga tga aa-3’) andCyt2 (5’-ccc ctc aga atg ata ttt gtc ctc a-3’)primers (Barlett and Davidson, 1991; Cocolinet al., 2000). Each 100-µl reaction contained50-100 ng DNA, 10 mM Tris HCl (pH 8.3), 50mM KCl, 1-5 mM MgCl2, 2-5 units Taq DNApolymerase, 150 mM of each dNTP, and 0-3mM of each primer, and was amplified with acycling profile of 1 min at 94ºC, 1 min at 53ºC,and 2 min at 72ºC for 35 cycles. Restriction fragment length polymorphism(RFLP) analysis. The PCR products weredigested with a restriction enzyme (AluI, HinfI,SpeI, or HpaII) and the fragments wereresolved on 1.5% agarose gels using aTris/Borate/EDTA (TBE) buffer. The size ofthe restriction fragments was estimated bycomparison to a 10,000 bp ladder. Statistical analysis. Differences amongCapoeta subspecies in genetic and proximalmatrix relationships were tested using the sta-tistical package SPSS for Windows (1999),version 10.0 (Cluster analysis, Jaccard’smodel, Nearest Neighboor).ResultsResults of the PCR-RFLP analysis are shownin Fig. 4. The estimated molecular lengths ofthe fragments in each restriction morph aresummarized in Table 1. A dendrogram of theresults is presented in Fig. 5. Discussion          Capoeta species are widespread in lakes,rivers, and other freshwater bodies in Turkey.While there are distinguishing taxonomic fea-tures among the species and subspecies, it isdifficult and even impossible to distinguishthem in some small fish. Without a method ofmorphological determination, analytical meth-ods to distinguish between C. c. capoeta, C. c.umbla, and C. tinca are of great importance infish biology. In this work, we focused on partof the mitochondrial Cyt-b gene for identifyingCapoeta spp., using this DNA-based methodto identify the Capoeta subspecies withouthaving to analyze the entire DNA sequence.MtDNA can be used as a marker inspecies identification instead of nuclear DNA(nDNA). MtDNA has advantages in that it iseasy to isolate, has a simple genetic structure,exhibits a straight-forward mode of genetictransmission, and, in vertebrates, has a highmutation rate (Unseld et al., 1995; Hisar et al.,208 Aksakal and ErdoganFig. 3. Capoeta tinca from the Coruh River (40°33’52”N 41°35’35”E). 2092006). The primers used in this study, Cyt1and Cyt2, were used to identify tuna species(Barlett and Davidson, 1991) and to distin-guish among flatfish (Céspedes et al., 1998)and have become universal primers. PCR-RFLP analysis of the mtDNA was selected dueto its simplicity, speed, and low cost(Céspedes et al., 2000). This method is applic-able to the small PCR products that are avail-able from highly processed materials and can,therefore, easily be adapted to distinguishamong fish species in juveniles and larvae.PCR-RFLP analysis of Capoeta spp.Fig. 4. Analysis by restriction fragment length polymorphism (RFLP; 400-500 bp PCR-amplikon) ofCapoeta capoeta capoeta (lanes 1, 4, 7, 10), C. tinca (lanes 2, 5, 8, 11), and C. c. umbla (lanes 3, 6, 9, 12)using SpeI, HpaII, HinfI, and AluI for digestion and compared to M, a DNA marker (50-10,000 bp).Restriction enzymeSubspecies SpeI HpaII HinfI AluIC. capoeta capoeta 100-200 bp 100-200 bp - -300-400 bp 300-400 bpC. capoeta umbla 100-200 bp 200-300 bp - <100 bp300-400 bp 200-300 bp 400-500 bpC. tinca 100-200 bp 100-200 bp - <100 bp300-400 bp 300-400 bp 400-500 bpTable 1. Approximate size of fragment patterns obtained by PCR-RFLPanalysis of the mtDNA of Capoeta spp.The PCR-RFLP analysis revealed threemitochondrial genotypes. While the restrictionenzyme HinfI did not cut the Cyt-b region,AluI, SpeI, and HpaII did. There are manystudies involving RFLP techniques in otherfish species (Szalanski et al., 2000; McDowelland Graves, 2002, Docker and Heath 2003),but none in Capoeta. Nor is there any geneticinformation about C. c. capoeta, C. c. umbla,and C. tinca in Genbanks. Therefore thisstudy can become a reference for futureresearch on these species.The genetic relationships revealed by ourPCR-RFLP analysis are similar to those sug-gested by authors using morphological charac-ters (Geldiay and Balik, 1996) and to the taxo-nomical classification of the subspecies. Ourstudy shows that these species can be identi-fied safely and simply by using PCR-RFLP.AcknowledgementsThis work was all of the master thesis entitledThe Determination of Genetical Differencesby Using PCR-RFLP Application of mtDNACytochrome-b Region of Capoeta sp.Captured from Aras, Karasu and Coruh Basin,supported by the Ataturk University ScientificResearch Project BAP 2005/45.ReferencesAsahida T., Kobayashi T., Saitoh K. and I.Nakayama, 1996. Tissue preservation andtotal DNA extraction from fish stored at ambi-ent temperature using buffers containing highconcentration of urea. Fish. Sci., 62:727-730.Barlett S.E. and W.S. Davidson, 1991.Identification of Thunnus tuna species by thepolymerase chain reaction and directsequence analysis of their mitochondrialcytochrome b gene. Can J. Fish Aquatic Sci.,48(2):309-317.Birstein V.J. and R. Desalle, 1998.Molecular phylogeny of Acipenserinae. Mol.Phylogenet. Evol., 9:141-155.Borgo R.M., Souty-Grosset C., Bouchon D.and L. Gomot, 1996. 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Use of PCR-RFLP Analysis of mtDNA Cytochrome-b Gene to Determine Genetic Differences in Capoeta spp.

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Use of PCR-RFLP Analysis of mtDNA Cytochrome-b Gene to Determine Genetic Differences in Capoeta spp.

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