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Acta Mologica Academiae Scientiarum Hungariciae 46 J); Py. 2β9-264. 2000
ES TABLIS HING MICROS ATELLITE ANALYSIS FOR LOCALLY ENDANGERED POPULATION SOU ROOT VOLE MICROTUS OECONOMUS)
of the amplis ted PCR producis rat sed the problem that tengili polymorphism may be the
result of variation os a compotand microsatellite locus. Key words: Microlus Deconomi S. microsatellite analysi S, sequence analysis. Hungary
The probabili ty of extinction os i solated populations Shows negati Ve correlation with population si Ze SOULE l98J). Natural demographic fluctuation and variation in en viro iamental conditions may strongly affect the genetic parametersos populations and cata lead to genetic erosion, heiace making the structure of populations more fragile GAINES & WHITTAM l 980. BIII SMA & LOESCREl99 ). For this reason, the screening of genetic variation has a great importance in effective conservation meas ures of the largeted populations CIOFI et a l. l 998). The root vole Microtus oeconomus PALLAS. lJJ6J) is the rarest volespecies in Hungary according to the Red Data Book RAMONCZAY l 989). The populations in the Carpathian Bas in are enlisted as a locat sub species of root voles MicrotuS oeconomi S mehebi IEHI K. l 928 l) and their scattered populations are considered as a glaciat relicis sJANOS SY l 986). The species has a Holarctic distribution with 23 distinguis hed subspecies sontine database litip Πwww.mnet. filpubiscimiolii Imam malia rodentia arvi colidae microtus) and is a common rodent in Alaska. Siberia, and Northeria Europe sTAST l 982). The variabili ty of the North
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Although the Hungari an subspecies is under strict protection, iis ecologicaldemands, the si Ze of populations, and iis genetic diversi ty are stili obscure be- cause pre Vious studies have only been concerned with iis distribution EHIK
are only mur areas in Hest-Hungary S ZigetkoZ, HanSag, Ferto-to, Kis-Balaton)where populations remain ed. However, sui table habitats are continuousty shrin king main ly as consequences of human disturbance. The best known ex ample of habitat deteri oration is the drying off welland s in the S ZigetkoZ area, whicli is a result of the diversion of the Danube ri Ver. For the above mentioned reasons, fui ther investigation of locat populations of root voles is essentiat. Our ongo ing research is abolit to estimate the average genetic variabili ty with in and belween populations using microsatellite markers. Microsatellite analysis is more likely to detect considerable genetic variationcompared to other methods PAT EAU et a l. l99 ). The feas ibi lily of two different sana pling methods for DNA extraction and the applicability of l0 heterolo-gous primer patrs for the PCR amplification os presumably polymorphic micro- satellite loci are described below.
mole collectio rRoot voles were collected using wooden live traps stom 5 localities of three different livingareas in Hungary during the years l99J and l998 Al together 86 root vole individuals and a fewSpecimen os 3 reference murine species see later) were sampled. Tissues of disserent origi n. stichas pieces of tali and uprooted hair. were collected in order to compare PCR yields. Tail cuttings REUS et al. l 998) proved to be relative ly harnaless, and it also hel ped to a void res ampling. Pieces of tail were froZen in liquid nitrogeia untii storage at -50'C. Tufis of hair were atr-dried and thensiored at Φ4'C. Samples were gi ven unique idenfication codes generaled frona the year os collection, species names abbreviation, sirst letter of the locali ty and the ordinal number of the specimen the numbering Skipped when an animal was recaptured). E. g. 98)MoL2 is the second Microtus oeconomus in l998 collected at Lipol.
Two different DNA extraction methous were applied using smali pieces sto in the tallo cithera standard phenolichtorosorin extraction MANIATIS et til. l 982) or a simplissed DNA isolation procedure LAIRII et til. l99 l). For tuns of hair we tested the standard protocol used sor humanhairs at the Hungari an Forensic Research Institute FUREDI perS. comm.). This method concentrates the phenolichtorosorin extracted DNA on a Micron l00 Amicon) membrane si iter. Ten heterologous DNA primer patrs were tested sor ampli sying polymorphic microsatellite loci using PCR. Five commercialty avat labie 'mouse primer patrs' s Murine Screening Set. 4l0
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MICROSATELLITE ANALYSIS FOR MICROTUS OECONOMUS RODENTIA
worked in a very narrow range of reaction conditions. The other three 'moti se
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Amplification of root vole samples by 'vole-primers' MSCRB-5. MSMM-2 MSMM-3 and MSMM-4 also yiel ded specific producis. These pri mers worked in a wider range of reaction conditions and most of them gave producis with hankVole Clethrisnon S glareoluS) samples as well. Primer pair MSMM-l did notgive a distinct PCR produci for the examined species. nor did they amplify bankvole refere iace samples PAPΡ Ω GUBANYI unpubi. data). Presently the number of alleles in root voles at these microsatellite loci has not been completely determined. although we have shown two loci MSCRB-5 and MSMM-2 to be polymorphic. In vestigations continue to estimate the extentos variabili ty of these loci.
The sequence analysis of PCR producis obtained by primer MSCRB- Fig. l)has revealed a possibie problem. The polymorphism os a locus might be the re
sult of the repeat variation of more than one repetitive region close to each Other.
In the Gen Bank Repori ΙSHIBASHI et al. l 995) eight repetitive regions os di mersand tetramers were described frona the identicat locus of the grey red-backed vole
N uncertain. - deletion insertiora.
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MICROSATELLITE ANALYSIS FOR MICROTUS OECONOMUS RODENTIA)
Gen Bank accession number D 3J836). We have mund si x presumptive microsatellite repeat motis close to each other in this region. These are the following with
lengths may ari se frona different combinations of repeat motis patierias. Obvious-ly. we had to determine whicli one of the above mentioned motis s is responsi hiefor the polymorphism of our PCR producis. Fortunate ly. the animal coded 98)MOS4 was a heteroZygote for this locus. By analysing the DNA seque iaceand separating the two alleles. it hecame apparent that the ' Ps)GT n times' was the repeat main ly responsi bie for tengili polymorphism l in the case of 98)Moson 8 and n l l on homo logotis chromosomes J. Therefore. locus M SCRB-5 is applicabie for population genetic studies, since allele tengili depends Only on the repeat number of one satellite. Ne Veriheles s. situations such as this indicate the importance of sequela cing at least a few PCR producis of each locus to avo id mi sinterpretation of allele tengili polymorphis m. The possibili ty of the presence of nullalleles should be considered as weli PEMBERTON et al. l99 β). In conclusion, we have successsul ly extracted and amplis ted root vole DNAs amples with the hel p of si x primer pati s . The analysis of tengili polymorphismfor each locus and the evaluation of the variabili ty at these loci are both in pro
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publis hed 2l si September. 2000
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ACTA ZOOLOGICA ACADEMIAE SCIENTIARUM HUNGARICAE
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