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Razvoj postopka za analizo polimorfnih regij v genomu riža
ID Kavčič, Ema (Author), ID Dolinar, Marko (Mentor) More about this mentor... This link opens in a new window

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Abstract
Riž je pomemben vir hrane. Poznamo več tisoč različnih sort riža, med katerimi lahko razlikujemo na osnovi DNA. Zaradi značilnosti rastlinske celice izolacija DNA iz rastlinskih tkiv ni enostavna. Ugotovili smo, da s trenjem vzorca v tekočem dušiku in inkubacijo v pufru CTAB pri 60 °C lahko DNA izoliramo tako iz listov kot tudi iz semen riža. Na stopnjo fragmentacije DNA je imel največji vpliv vir izhodiščnega materiala. Izolirana DNA je bila dovolj kvalitetna za pomnoževanje s PCR, saj smo lahko pomnožili regijo ITS. Za razlikovanje med sortami riža uporabljajo več različnih označevalcev. Iz zbirke Gramene smo s seznama 50 najpogosteje uporabljenih mikrosatelitnih označevalcev izbrali 9 označevalcev in preverili, ali so uporabni za razlikovanje med izbranimi sortami (hajajuki, jukihikari, duborskian, titanio, uz rosk, ponta rubra, vialone nano, ribe, riž za kuhanje neznane sorte, rdeči, črni in čajni riž). Najprej smo poskusili s spreminjanjem temperature prileganja začetnih oligonukleotidov preprečiti nastanek nespecifičnih produktov pri reakciji PCR. Produkti PCR naj bi se med sortami razlikovali po dolžini, vendar je pričakovana razlika manjša kot 30 bp. Ločevanje z AGE ima premajhno ločljivost, da bi lahko opazili tako majhne razlike. Dokazali smo, da s PAGE lahko dosežemo ločljivost 3 bp, kljub temu pa nismo opazili razlik v dolžini amplikonov med sortami pri označevalcih RM536 in RM431. Očiščene produkte onačevalca RM431 smo poslali na sekvenciranje brez predhodnega kloniranja PCR-produktov. Določeno zaporedje večine vzorcev ni bilo dovolj kvalitetno za primerjavo med sortami. Vzorca sort hajajuki in ponta rubra pa se v zaporedju označevalca RM431 nista razlikovala od referenčnega zaporedja.

Language:Slovenian
Keywords:riž, mikrosatelit, izolacija DNA, variabilnost med sortami
Work type:Bachelor thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2023
PID:20.500.12556/RUL-149547 This link opens in a new window
COBISS.SI-ID:164448515 This link opens in a new window
Publication date in RUL:07.09.2023
Views:623
Downloads:38
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Secondary language

Language:English
Title:Development of a procedure for analysis of polymorphic regions in the genome of rice
Abstract:
Rice is an important food source. There are several thousand different cultivars which can differentiated among using DNA. Due to cahracteristics of a plant cell, DNA isolation from plant tissues is not trivial. We demonstrated that DNA can be isolated from both rice leaves and seeds using mortar with liquid nitrogen, followed by incubation in CTAB buffer at 60 °C. The fragmentation of gnomic DNA depended primarily on the source of plant material. Purity of isolated DNA was high enough to perform PCR. We have successfully amplified ITS regions from several samples. There are many molecular markers that can be used to differentiate among rice species. In the Gramene data base there is a selection of 50 most commonly used microsatellite markers. We chose 9 of them to check if they are appropriate to detect differences among selected rice varieties (Hayayuki, Yukihikari, Duborskian, Titanio, Uz Rosk, Ponta Rubra, Vialone Nano, Ribe, unknown variety of cooking rice, red, black and tea rice). We tried to minimize nonspecific PCR products by increasing the annealing temperature. The PCR products of different varieties are expected to have different lengths, the difference being less than 30 bp. Separation by AGE was not appropriate to detect such small differences. We proved that we can detect a difference of 3 bp by PAGE instead. Nevertheless, we could see no size difference among varieties using markers RM536 and RM431. The purified PCR product of the marker RM431 has been sequenced. The quality of analysed sequences was not good enough to perform a comparison between all rice samples. The sequences of Hayayuki and Ponta Rubra were identical to the reference sequence of RM431.

Keywords:rice, microsatellite, DNA isolation, inter-cultivar differences

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