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Jeļena Oreha Daugavpils Universitāte DZTI Ekoloģijas departaments Jeļena Oreha REPŠA (COREGONUS ALBULA (L.)) POPULĀCIJU ĢENĒTISKĀ STRUKTŪRA UN MORFOLOĢISKĀ MAINĪBA LATVIJAS EZEROS Promocijas darbs bioloģijas doktora zinātniskā grāda (Dr. biol.) iegūšanai (ģenētikas apakšnozarē) Darba zinātniskais vadītājs: Dr. biol., asoc.prof. Nataļja Škute Daugavpils 2016 Promocijas darbs izstrādāts: Daugavpils Universitātes DZTI Ekoloģijas departamentā laika periodā no 2003. līdz 2016. gadam. Ieguldījums tavā nākotnē! Promocijas darbu atbalstīja: Šis darbs ir izstrādāts ar Eiropas Sociālā fonda atbalstu projektos „Atbalsts doktorantūras studijām un pēcdoktorantūras pētījumiem dabas zinātnēs un informācijas tehnoloģijās” (Vienošanās Nr. ESF2004/0003/VPD1/ESF/PIAA/04/NP/3.2.3.1./0003/0065) un Starpdisciplinārās zinātniskās grupas izveidošana Latvijas lašveidīgo zivju ezeru ilgtspējības nodrošināšanai” (Vienošanās Nr. 2009/0214/1DP/1.1.1.2.0/09/APIA/VIAA/089 Darba raksturs: promocijas darbs bioloģijas nozares ģenētikas apakšnozarē. Promocijas darba zinātniskais vadītājs: Dr. biol., asoc.prof. Nataļja Škute (Daugavpils Universitāte, Latvija) Recenzenti: Dr. biol., asoc.prof. Inese Kokina (Daugavpils Universitāte, Latvija) Dr. habil. biol., prof. Īzaks Rašals (Latvijas Universitāte, Latvija) PhD, prof. Jana Radzijevskaja (Vītauta Dižā Universitāte, Lietuva) Promocijas padomes priekšsēdētājs: Dr. biol., profesors Arvīds Barševskis Promocijas darba aizstāvēšana notiks: Daugavpils Universitātes Bioloģijas Promocijas padomes atklātajā sēdē 2016. gada 07. decembrī, plkst. 13.00, Parādes ielā 1A, 209. auditorijā, Daugavpils Universitātē, Daugavpilī. Ar promocijas darbu un tā kopsavilkumu var iepazīties Daugavpils Universitātes bibliotēkā, Parādes 1, lasītavā, Daugavpilī un http://du.lv/zinatne-un-petnieciba/promocija/aizstavesanai- iesniegtie-promocijas-darbi/ Atsauksmes sūtīt: Promocijas padomes sekretārei, Parādes iela 1A-207, Daugavpils, LV- 5401; mob. +37126002593; e-pasts: [email protected]. Padomes sekretāre: Dr. biol., Jana Paidere, Daugavpils Universitātes pētniece. 2 SATURS IEVADS ................................................................................................................................ 5 1. Literatūras apskats ............................................................................................................. 9 1.1. Repsis kā sīgu dzimtas pārstāvis un tā bioloģija ............................................................ 9 1.1.1. Repša vieta dzīvo organismu sistemātikā .................................................................... 9 1.1.2. Repša izplatība ........................................................................................................... 10 1.1.3. Repša morfoloģija ..................................................................................................... 10 1.1.3.1. Plastisko pazīmju mainība sīgu zivīm .................................................................... 12 1.1.3.2. Meristisko pazīmju mainība sīgu zivīm.................................................................. 15 1.1.4. Repša bioloģijas vispārīgs raksturojums ................................................................... 17 1.1.5. Repša aizsardzība un tautsaimnieciskā nozīme ......................................................... 20 1.2. Ģenētiskais monitorings ............................................................................................... 23 1.3. Ģenētiskā daudzveidība ................................................................................................ 28 1.3.1. Ģenētiskās daudzveidības vispārējie parametri ......................................................... 29 1.3.2. Ģenētiskās daudzveidības marķieri ........................................................................... 30 1.3.3. Izofermentu polimorfisms ......................................................................................... 32 1.3.4. Kodola genoma anonīmo secību polimorfisms ......................................................... 37 1.3.5. Kodola genoma tandēmo atkārtojumu polimorfisms. ............................................... 42 1.3.5.1. Tandēmi atkārtojumi kodola genomā. .................................................................... 42 1.3.5.2. Mikrosatelītu secību mainība ................................................................................. 45 1.3.5.3. Mikrosatelītu lokusu mainība zivīm ....................................................................... 46 1.3.6. Molekulāro marķieru un morfometrisko parametru kombinēšana ............................ 53 2. Materiāli un metodes ....................................................................................................... 56 2.1. Pētījumu objekts ........................................................................................................... 56 2.2. Materiāla vākšanas vietas ............................................................................................. 56 2.3. Materiāla vākšanas apstākļi un laiks ............................................................................ 57 2.4. Materiāla morfometrisko parametru mērījumi ............................................................. 58 2.5. Repša īpatņu vecuma un dzimuma noteikšana ............................................................. 58 2.6. Morfometrisko datu statistiskā apstrāde un analīze ...................................................... 59 2.7. Paraugu sagatavošana izofermentu analīzei ................................................................. 60 2.8. Izofermentu elektroforēze poliakrīlamīda gēlā............................................................. 61 2.9. Izofermentu vizualizēšana, analīze un statistiskā apstrāde ........................................... 61 2.10. Kodola DNS izdalīšana .............................................................................................. 63 2.11. DNS kvantitātes un kvalitātes noteikšana .................................................................. 63 2.12. RAPD - PĶR amplifikācija ........................................................................................ 64 2.13. Analītiskā elektroforēze agarozes gēlā ....................................................................... 65 2.14. RAPD-PĶR fragmentu vizualizēšana, analīze un statistiskā apstrāde ....................... 65 3 2.15. Mikrosatelītu praimeru testēšana ................................................................................ 66 2.15.1. Polimerāzes ķēdes reakcijas apstākļi ....................................................................... 66 2.15.2. Analītiskā elektroforēze poliakrīlamīda gēlā ........................................................... 66 2.15.3. Mikrosatellītu fragmentu vizualizēšana .................................................................. 67 2.16. Mikrosatelitu lokusu ģenētiskā analīze ...................................................................... 67 2.16.1. Polimerāzes ķēdes reakcijas apstākļi ....................................................................... 67 2.16.2. Amplificēto fragmentu izmēru noteikšana un statistiskā analīze ............................ 68 3. Rezultāti ........................................................................................................................... 71 3.1. Pētāmo populāciju repša īpatņu vecuma struktūra ....................................................... 71 3.2. Pētāmo populāciju repša īpatņu morfometriskā analīze ............................................... 71 3.2.1. Repša īpatņu ķermeņa masa un garums ..................................................................... 71 3.2.2. Repša īpatņu kondīcija un augšanas ātrums .............................................................. 72 3.2.3. Repša īpatņu plastiskie parametri .............................................................................. 74 3.2.4. Repša īpatņu meristiskie parametri ........................................................................... 77 3.3. Izofermentu sistēmu polimorfisms ............................................................................... 79 3.3.1. Populāciju alēļu pārpilnība ........................................................................................ 84 3.3.2. Pētāmo repša populāciju heterozigotātes līmenis ...................................................... 85 3.3.3. Alēļu sastopamība pētāmās repša populācijās ........................................................... 86 3.3.4. Pētāmo repša populāciju ģenētiskā mainība un diferenciācija .................................. 87 3.4. Anonīmu secību jeb RAPD polimorfisms .................................................................... 89 3.5. Mikrosatelītu polimorfisms .......................................................................................... 93 3.5.1. Mikrosatelītu praimeru testēšana ............................................................................... 93 3.5.2. Mikrosatelītu polimorfisms un alēļu pārpilnība analizētos lokusos .......................... 93 3.5.4. Populāciju heterozigotātes līmenis ............................................................................ 97 3.5.5. Populāciju ģenētiskā struktūra un diferenciācija ....................................................... 99 4. Diskusija ........................................................................................................................ 102 4.1. Pētāmo repša populāciju bioloģiskie un morfometriskie parametri ........................... 102 4.2. Pētāmo repša populāciju meristiskie
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