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VYTAUTO DIDŽIOJO UNIVERSITETAS Irena VYTAUTO DIDŽIOJO UNIVERSITETAS GAMTOS MOKSLŲ FAKULTETAS BIOLOGIJOS KATEDRA Irena JAKUBANIS DNR MIKROSATELITINIO POLIMORFIZMO PANAUDOJIMAS LIETUVOS TAURIŲJŲ ELNIŲ (CERVUS ELAPHUS) GENETINĖS ĮVAIROVĖS TYRIMUOSE Magistro baigiamasis darbas Molekulinės biologijos ir biotechnologijos programa, 62101B180 Biologijos studijų kryptis Vadovas: prof. A.Paulauskas __________ ________ (Parašas) (Data) Apginta: prof. habil. dr. G. Kamuntavičius _________ _______ (Parašas) (Data) KAUNAS, 2008 Darbas atliktas : 2007 – 2008 m. Vytauto Didžiojo universitete, Biologijos katedroje, Kaunas, Lietuva ir Telemark University College, Department of Environmental and Health Studies, Bø i Telemark, Norvegija Recenzentas: dr. Daiva Ambrasienė, Vytauto Didžiojo Universitetas Darbas ginamas: viešame magistrų darbų gynimo komisijos posėdyje 2008 metų birželio 5 d. Vytauto didžiojo universitete, Biologijos katedroje, 101 auditorijoje 9.00 valandą. Adresas: Vileikos 8, 3035 Kaunas, Lietuva. Protokolo Nr. Darbo vykdytojas: I. Jakubanis Mokslinis vadovas: prof.A.Paulauskas Biologijos katedra, vedėjas: prof. A.Paulauskas Padėka Norėčiau nuoširdžiai padėkoti savo darbo vadovui prof. Algimantui Paulauskui už visokeriopą pagalbą, medžiagos surinkimą, moralinę paramą ir vertingas pastabas atliekant šį darbą. Dėkoju prof. Olav‘ui Rosef‘ui už suteiktą galimybę atlikti tyrimus Telemark universitete, bei moralinį palaikymą, Monai Sabo už tyrimo metodinės pusės paaiškinimą. Už tyrime naudotos medžiagos surinkimą esu dėkinga medžiotojams: doc.dr. Egidijui Bubelskui ir Robertui Išganaičiui. Taip pat nuoširdus ačiū dr. Vykintui Baubliui už vertingus patarimus. Dėkoju savo tėvams ir sesėms už paskatinimą ir tvirtą palaikymą, bei Julijai už moralinį ir psichologinį palaikymą. 3 Turinys Santrauka...............................................................................................................................................5 Abstract..................................................................................................................................................6 Įvadas.....................................................................................................................................................7 1. Literatūros apžvalga........................................................................................................................9 1.1.Tauriojo elnio (Cervus elaphus) bendra charakteristika...........................................................9 1.2. DNR žymenų polimorfizmas bei taikomi metodai populiacijų genetikoje............................14 1.2.1. Polimerazės grandininė reakcija (PGR)........................................................................14 1.2.2. Mikrosatelitinės sekos...................................................................................................17 1.3. Genetiniai tauriųjų elnių tyrimai............................................................................................19 1.3.1. Mikrosatelitai ir jų panaudojimas tauriųjų elnių genetiniuose tyrimuose.....................20 1.3.2. „Konservatyvų“kanopinių mikrosatelitinių lokusų panaudojimas tauriųjų elnių genėtinuose tyrimuose...................................................................................................22 2. Medžiagos ir metodika..................................................................................................................25 2.1. Tyrimo medžiaga....................................................................................................................25 2.1.1. DNR iškyrimas, koncentracijos nustatymas................................................................25 2.2. Mikrosatelitų amplifikacija.....................................................................................................29 2.2.1. Elektroforezė poliakrilamido gelyje.............................................................................30 3. Rezultatai ir jų aptarimas............................................................................................................33 3.1. Naudotų mikrosatelitinių pradmenų amplifikacijos optimizavimas......................................33 3.2. Lokusų charakteristika............................................................................................................34 3.3. Lietuvos tauriųjų elnių genetinė įvairovė ..............................................................................36 3.4. Lietuvos taurųjų elnių populiacijų genetinė diferenciacija....................................................42 3.4.1. Taurųjų elnių DNR mikrosatelitinio ir izofermentinio genetinio kintamumo palygimas .....................................................................................................................................44 3.5. Lietuvos tauriųjų elnių populiacijų palygimas su kitomis Europos populiacijomis.................46 Išvados.................................................................................................................................................53 Magistrinio darbo tema publikuoti I. Jakubanis darbai ......................................................................54 Literatūra.............................................................................................................................................55 Priedai..................................................................................................................................................59 4 Santrauka Šiame darbe mikrosatelitinių DNR sekų metodu buvo ištirta 24 taurieji elniai iš 4 rajonų, 8 skirtingų Lietuvos miškų. Pagrindinis tyrimo objektas buvo nustatyti tauriųjų elnių DNR mikrosatelitų kintamumą Lietuvoje. Elnių audinių bei kepenų pavyzdžiai surinkti 2003 - 2005 metų medžioklės sezonuose iš medžiotojų nušautų individų. Ištyrus 7 mikrosatelitinius lokusus (RT1, RT23, NVHRT16, NVHRT21, NVHRT48, NVHRT73, BM888) nustatyta, kad lokusai BM888, NVHRT48 bei NVHRT21 nepilnai tinka Lietuvos taurųjų elnių populiacinei analizei, kadangi po amplifikacijos ne pas visus tirtus individus buvo matoma pradmenų amplifikacija. Panevėžio rajone aptiktas vienas retas alelis (0,042 dažnumas) RT1 lokuso alelyje esančiam 224 bp srityje. Pastebėta, kad Lietuvoje gyvenančių taurųjų elnių teorinis ir faktinis heterozigotiškumas artimas į kitų šalių taurųjų elnių populiacijų heterozigotiškumus. Jurbarko rajono populiacija pasižymi didžiausiu faktiniu heterozigotiškumu, kuris siekė 0,75. Didžiausias efektyvus alelių skaičius (ne=3,45) pastebėtas Panevėžio rajono elnių populiacijoje. Tiek pagal DNR tiek pagal izofermentine analizę labiausia genetiškai artimos populiacijos yra Minčios ir Ažvinčių, labiausiai nutolusios pagal mūsų tirtus polimorfinius lokusus yra Mantvilių ir Sipailiškių miškų tauriųjų elnių populiacijos. Lietuvos taurųjų elnių populiacija yra vienoje šakoje su Vengrijos bei Škotijos taurųjų elnių populiacijomis, tačiau nutolusi nuo Švedijos taurųjų elnių populiacijos. 5 Abstract Author of term paper: Irena Jakubanis Full tittle of term paper: The application of microsatellite polymorphism for the genetical variability research of Lithuanian Red Deer (Cervus elaphus). Term paper advisor: prof. A. Paulauskas Presented at: Vytautas Magnus University, Faculty of Nature Sciences, Kaunas, 2008, June Number of pages: 58 Number of tables: 16 Number of pictures: 25 Number of appendixes: 4 We investigated tissue and liver of 24 red deers (Cervus elaphus). Samples for analysis were collected from 4 localities, 8 different forests of Lithuania during 2004-2005 hunting seasons. We used 7 microsatellite primers (RT1, RT23, BM888, NVHRT16, NVHRT21, NVHRT48, NVHRT73) derived from reindeer (Rangifer tarandus) to find out which of them is the best suitable for genetical investigations of red deer. BM888, NVHRT48 and NVHRT21 did not appeared suitable for whole population analysis because not in all samples was detected amplification. The analysis of alleles showed the rarest alleles is RT1 locus 224 bp alleles (0,042). Lithuanian red deers observed and expected heterozygosities are within the range of values previously found for reed deers. It was estimated that the biggest observed heterozygosity were Jurbarko localities forest (Ho= 0,75). The biggest effective number of alleles (ne=3,45) was observed in Panevėžio locality red deer population. According to our DNA and antedate done isoenzyme investigation the most genetically similar were Minčios and Ažvinčių red deer subpopulations; the most different were Sipailiškių and Mantvilių ones. Lithuanian red deer are in one cluster with Hungarian and Scottish red deers populations, they are in separate cluster with Swedish red deer population. 6 Įvadas Filogenetinių ryšių, tokių kaip genetinės medžiagos ir jos elementų formavimasis, kitimas bei mutacinis kintamumas evoliucijos eigoje, nustatymas tarp įvairių organizmų iki šiol daugiausiai rėmėsi morfometrinių duomenų, baltymų ir izofermentinių sistemų tyrimais bei įvertinimu. Dabartiniu metu, filogenetinių ryšių įvertinimui, yra naudojami DNR tyrimu pagrįsti metodai, kurie, dėl tikslumo ir platesnio pritaikymo spektro, suteikia detalesnę ir patikimesnę informacija apie organizmus, molekulinės ekologijos, sistematikos, populiacinės ir evoliucinės genetikos tyrimų srityse. Pagrindinai DNR tyrimo pranašumai, lyginant juos su anksčiau naudotais metodais yra tokie, kad šiuo atveju yra tiriama tiesioginė medžiaga, o ne jos produktai, o patys DNR tyrimo metodai pasižymi dideliu specifiškumu ir gauti rezultatai yra labai tikslūs. Molekulinei metodai kuo
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