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(Impatiens Noli-Tangere L.) Lietuvos Populiacijų Genetinė Įvairovė, Įvertinta Pagal Appd Žymenis

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(Impatiens Noli-Tangere L.) Lietuvos Populiacijų Genetinė Įvairovė, Įvertinta Pagal Appd Žymenis VYTAUTO DIDŽIOJO UNIVERSITETAS GAMTOS MOKSLŲ FAKULTETAS BIOLOGIJOS KATEDRA Julija Petkevičiūtė PAPRASTOSIOS SPRIGĖS (IMPATIENS NOLI-TANGERE L.) LIETUVOS POPULIACIJŲ GENETINĖ ĮVAIROVĖ, ĮVERTINTA PAGAL APPD ŽYMENIS Magistro baigiamasis darbas Molekulinės biologijos studijų programa, valstybinis kodas 621C1001 Molekulinės biologijos, biofizikos ir biochemijos studijų kryptis Vadovė: prof. habil. dr. E. Kupčinskienė _________ __________ (Parašas) (Data) Apginta: prof. habil. dr. A. Paulauskas _________ __________ (Parašas) (Data) Kaunas, 2013 PADĖKA Esu dėkinga už nuoširdžią pagalbą ir visokeriopą palaikymą: prof. habil. dr. E. Kupčinskienei L. Zybartaitei R. Vilčinskui 2 TURINYS SANTRUMPOS 3 SANTRAUKA 4 SUMMARY 5 ĮVADAS 6 1. LITERATŪROS APŽVALGA 7 1.1 Svetimkraščių rūšių invazija 8 1.1.1 Pasaulio klimato kaitos poveikis natūralių ir svetimkraščių augalų plitimui 8 1.1.2 Invazinių rūšių poveikis aplinkai 9 1.1.2.1 Lietuvos invaziniai augalai ir jų keliama grėsmė vietinei florai 10 1.1.2.2 Invazinių rūšių kontrolės priemonės 11 1.2 Sprigių genties biologija 12 1.2.1 Sprigių genties morfologija, ekologija ir geografinis paplitimas 13 1.2.2. Lietuvos natūralios ir invazinės sprigių genties rūšys 19 1.3. Metodai, naudojami tiriant natūralius ir invazinius augalus 22 1.3.1 Molekuliniai metodai, naudojami tiriant augalus 22 1.3.2 Sprigių genties molekuliniai tyrimai 25 2. TYRIMŲ OBJEKTAS, VIETOS IR METODAI 28 2.1 Augalinės medžiagos rinkimas 28 2.2. Genominės DNR išskyrimas 30 2.3. Išskirtos genominės DNR koncentracijos ir švarumo nustatymas 32 2.4. APPD metodo pritaikymas paprastajai sprigei 32 2.5. APPD PGR produktų elektroforezė 34 2.6. Duomenų statistinė analizė 35 3. REZULTATAI 36 3.1. Paprastosios sprigės genetinės įvairovės pagal APPD žymenis 36 IŠVADOS 43 LITERATŪROS SĄRAŠAS 44 PRIEDAI 52 3 SANTRUMPOS AFLP – amplifikuotų fragmentų ilgio polimorfizmas (angl. Amplified Fragment Length Polymorphism) SNP – vieno nukleotido polimorfizmas (angl. Single nucleotide polymorphism) APPD – atsitiktinai pagausinta polimorfinė DNR (angl. Random Amplified Polymorphic DNA, RAPD) SSR – mikrosatelitai, kartotinių sekų atkarpos (ang. simple sequence repeats) PGR – polimerazinė grandininė reakcija (angl. Polymerase Chain Reaction, PCR) SNP – vieno nukleotido polimorfizmo tyrimas (angl. Single nucleotide polymorphism) bp – bazių pora (angl. base pair) DNR – deoksiribonukleorūgštis (angl. Deoxyribonucleic acid, DNA) dNTP – deoksiribonukleotidai cDNR – chromosominė DNR QTL – kiekybinių lokusų požymiai (angl. Quantitative trait loci) EST – išreikštų sekų žymenys (angl. Expressed sequence tag) AMOVA – molekulinės genetinės įvairovės analizė (angl. Analysis of Molecular Variance) UPGMA – porų grupavimo pagal aritmetinius vidurkius metodas su vienodais svoriais (angl. Unweighted Pair-Group Method of arithmetic Averages) PKA – principinių koordinačių analizė (angl. Principal Coordinates Analysis, PCA) GDxy – genetinis atstumas tarp x ir y genotipų (angl. Genetic Distance) Gst – genetinės diferenciacijos koeficientas (Nei, 1973) H – genetinė įvairovė (Nei, 1973) I – Šenono (Shannon’s) informacinis indeksas Na – stebimas alelių skaičius lokuse Ne – efektyvių alelių skaičius lokuse Nm – genų srautas tarp populiacijų (angl. Gene flow) P – polimorfinių lokusų procentas SDS – natrio dodecilsulfatas (angl. Sodium dodecyl sulphate) SN – standartinis nuokrypis V – vidurkis 4 SANTRAUKA Autorius: Julija Petkevičiūtė Pavadinimas: Paprastosios sprigės (Impatiens noli-tangere L.) Lietuvos populiacijų genetinė įvairovė, įvertinta pagal APPD žymenis Vadovas: prof. habil. dr. Eugenija Kupčinskienė Pristatyta: Vytauto Didžiojo Universitetas, Gamtos mokslų fakultetas, Kaunas, 2013, birželis. Puslapių skaičius: 54 (įskaitant priedus) Lentelių skaičius: 9 Paveikslų skaičius: 10 Priedų skaičius: 3 Literatūros nuorodų skaičius: 95 Balsaminaceae šeimai priklauso daugiau nei 1000 sprigių rūšių, penkios iš jų yra randamos Europoje. Lietuvoje auga 4 rūšys, iš jų 1 auginama balzamininė sprigė (Impatiens balsamina L.), 2 sulaukėjusios smulkiažiedė sprigė (Impatiens parviflora DC.), bitinė sprigė (Impatiens glandulifera L.) ir 1 natūraliai auganti - paprastoji sprigė (Impatiens noli-tangere L.). Lyginant skirtingų sprigių rūšių prisitaikymo ir kintamumo duomenis, galima gauti daug vertingos informacijos. Lietuvoje molekuliniai tyrimai buvo atliekami tik invazinių rūšių, todėl šio darbo tikslas buvo įvertinti paprastosios sprigės genetinį kintamumą pagal Lietuvos geografiškai skirtingas populiacijas, naudojant atsitiktinai pagausintos polimorfinės DNR (APPD) metodą. Augalai buvo surinkti iš 15 Lietuvoje geografiškai skirtingų vietovių. Išbandžius kitų sprigių rūšių tyrimuose naudotus APPD oligonukleotidinius pradmenis iš jų paprastajai sprigei tiko aštuoni oligonukleotidiniai pradmenys: OPA-20, OPD-20, 222, 250, 269, 340, 474, 516. Tarp paprastosios sprigės populiacijų genetiniai duomenys svyravo tokiuose intervaluose: 3–15 % polimorfinių fragmentų skaičius, 0.005–0.049 Nei genetinė įvairovė, 0.009–0.074 Shannon’o indeksas. UPGMA dendograma pagal individus parodo, kad skirtingų populiacijų individai nesimaišė, sudarė atskiras šakas. Dendogramoje individai suskirstyti į 7 klasterius Genetiniai atstumai tarp populiacijų svyravo 0.185–0.467. Genetinių atstumų dendrograma pagal 220 individus, išsiskirstė į 7 populiacijų grupes. Pagal APPD žymenis UPGMA individų ir populiacijų dendrogramose genetinių ir geografinių atstumų ryšys nebuvo nustatytas Lietuvos paprastosios sprigės populiacijoms, tą patį galima teigti ir apie principinių koordinačių analizę. 5 SUMMARY Author: Julija Petkevičiūtė Title: RAPD analyses of genetis diversity among Lithuania population of Impatiens noli- tangere L. Supervisor: prof. hab. dr. Eugenija Kupčinskienė Presentation: Vytautas Magnus University, Faculty of Nature Sciences, June, 2013, Kaunas. Number of pages: 54 (including appendices) Number of tables: 9 Number of figures: 10 Number of Appendices: 3 Number of references: 95 Balsaminaceae family comprises more than 1000 species, among them five species are present in Europe. There are 4 species in Lithuania, one of them is cultivated – Impatiens balsamina L., two species are known as aliens – Impatiens parviflora DC., Impatiens glandulifera L. and only one is growing naturally – Impatiens noli-tangere L. Valuable information might be obtained comparing adaptation level and variability of alien species and native ones. In Lithuania, molecular studies have been carried out only for invasive species of Impatiens, so the objective of this study was to evaluate the genetic variability of populations of Impatiens noli-tangere differing in geography or habitats by the randomly amplified polymorphic DNA (RAPD). Plants were collected from Lithuania 15 different locations. For I. noli-tangere studies eight different oligonucleotide primers (OPA-20, OPD-20, 222, 250, 269, 340, 474, 516) were used. Among populations of I. noli- tangere genetic parameters ranged in the following intervals: 3–15 % of polymorphic DNA bands, 0.005–0.049 for Nei’s gene diversity, 0.009–0.074 for Shannon’s information index. Pairwise genetic distances between populations ranged in the interval 0.185–0.467. Genetic distance-based cluster analyses for 220 individuals indicated 7 major groups of populations, among which there was no clear geographical pattern. On the bases of RAPD markers prepared UPGMA dendrogram of individuals and populations did not show tight relation between population geography and geographical distances. The same was true when principal component analyses were done for genetical data. 6 ĮVADAS Antropogeninė veikla, kuri lemia pasaulinę klimato kaitą, melioraciją, taršą ir prekybą, įtakoja svetimkarščių augalų rūšių invazivumą, kurios daro poveikį vietinei augmenijai. Svarbiausi invazinių rūšių poveikiai vietinėms rūšims kyla dėl greito invazinių rūšių prisitaikymo prie pasikeitusių sąlygų ir dauginimosi. Biologinės invazijos yra svarbiausias faktorius veikiantis ekosistemų degradacijos priežastis ir biologinę įvairovę, todėl svarbu tirti ir įvertinti dabartinę padėtį Lietuvoje, tam, kad būtų galima imtis atitinkamų veiksmų biologinei įvairovei palaikyti. Invazinių augalų morfologijos, fiziologijos, ypač fenologijos ir genetikos pažinojimas, įskaitant žinias apie paprastąją sprigę yra būtinos vietinių rūšių išsaugojimui. Lietuvoje atliekami vietinių rūšių tyrimai, tačiau molekuliniais metodais dažniau tyrinėjami invaziniai augalai, o vietinių rūšių tyrimai atliekami neadvekačiai. Norint, pateikti kuo tikslesnius rezultatus reikia ištirti vietines rūšis ir gautus rezultatus lyginti su svetimkraštėmis rūšimis. Neigiamai invazijai Lietuvoje mažinti yra genetiniai molekuliniai tyrimai, kuriuos reikia naudoti atliekant vietinės paprastosios sprigės tyrimą. Siekiant rezultatų, remiantis užsienyje ir Lietuvoje darytais augalų morfologiniais, fiziologiniais, filogenetiniais, molekuliniais tyrimais, skirtais augalų populiacinės genetikos ir sistematikos analizei, atliksiu vietinės paprastosios sprigės tyrimą atsitiktinai pagaustintos polimorfinės DNR (APPD) metodu. Norint išsaugoti Lietuvos biologinę įvairovę reikia išsamiai pažinti vietines rūšis, todėl svarbu ištirti jų molekulinę įvairovę, kad būtų galima palyginti su svetimkraščių sprigių giminingų rūšių genetine įvairove. Todėl darbo tikslu buvo ištirti Lietuvos paprastosios sprigės 15 populiacijų genetinę įvairovę pagal APPD žymenis. Tikslui įgyvendinti iškeliami tokie uždaviniai: pritaikyti Impatiens genties kitų rūšių tyrimams naudojamą APPD metodą, parinkti pradmenų
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