Identifikacija različitih sorata lucerke primenom molekularnih markera

Abstract

Lucerka je, posle kukuruza, najvaţnija krmna vrsta u našoj zemlji, zahvaljujući ne samo povoljnom hemijskom sastavu i visokom sadrţaju proteina, već i visokim prinosima i veoma dobrim biološkim osobinama. Plava lucerka, Medicago sativa L. (2n = 32) je prirodni tetraploid. Cilj ovog rada je da se ispitivanjem deset razliĉitih sorata lucerke odredi varijabilnost i grupisanje srodnih genotipova lucerke primenom RAPD molekularnih markera. Za ekstrakciju DNK iz biljnog materijala korišćeni su prvi zeleni listići klijanaca lucerke (kotiledoni). Za analizu podataka dobijenih molekularnom karakterizacijom korišćeni su statistiĉki programi za populaciono-genetiĉke analize. Primećeno je da se u RAPD analizi genoma biljaka mogu pratiti repetativne sekvence koje ĉine znaĉajan deo genetiĉkog materijala. Lanĉana reakcija polimeraze bazirana na repetativnim sekvencama (RAPD-PCR) korišćena je za utvrđivanje genetiĉke raznovrsnosti između razliĉitih sorata lucerke. OPA 01, OPA 02, OPA 13 i OPB 10 prajmeri su korišćeni da bi se dobili razliĉiti DNK profili. Na osnovu RAPD DNK profila moţe se videti razdvajanje DNK fragmenata razliĉite veliĉine od 100 bp do 3 kb. Razlike između fragmenata su ocenjivane vizuelno na osnovu njihovog položaja na gelu. Na gelu na kome se nalaze razliĉite sorte lucerke sa prajmerom OPB 10 mogu se uoĉiti razlike između sorata lucerke. Jasno se vidi da se sorta lucerke Zajeĉarska 83 razlikuje od svih ostalih sorata lucerke. Sorte Osjeĉka 99, Osjeĉka 88 i Osjeĉka 66 pokazuju visok nivo međusobne sliĉnosti, kao i sorte Kruševaĉka 22 i Kruševaĉka 28, što je i oĉekivano sa obzirom da su navedene sorte koje su sliĉne selekcionisane u istim selekcionim kućama. Ovo istraţivanje je potvrdilo da prouĉavana kolekcija sorti lucerke poseduje varijabilnost neophodnu za uspešan selekcioni proces.

After maize, alfalfa is the most important forage species in our country due to favorable chemical composition and high content of protein. It is also characterized with high yields and very good biological properties. Blue alfalfa, Medicago sativa L. (2n = 32) is a natural tetraploid. The aim of this work was to determine variability and grouping related alfalfa genotypes using RAPD molecular markers, by testing ten different alfalfa cultivars. The first green leaves of alfalfa seedlings (cotyledons) were used for DNA extraction from the plant material. For the analysis of data obtained by the molecular characterization the statistical programs for population genetic analysis were used. It is observed that in the RAPD analysis of the genome can be monitored repetitive sequences that make up a significant part of the genetic material. The polymerase chain reaction based on repetitive sequences (RAPD-PCR) was used to determine the genetic diversity among the different cultivars of alfalfa. OPA 01, OPA 02, OPA 13, and OPB 10 primers were used to obtain the various profiles of DNA. Based on RAPD DNA profiles, the separation of DNA fragments of different size from 100 bp to the 3 kb were noticed. The differences between the fragments were evaluated visually on the basis of their position on the gel. On the gel containing different varieties of alfalfa with primer OPB 10, differences between alfalfa varieties were noticed. It is clear that the cultivar Zajeĉarska 83 differs from all other cultivars of alfalfa. Cultivars Osjeĉka 99, Osjeĉka 88, and Osjeĉka 66 showed a high level of similarity, as well as cultivars Kruševaĉka 22 and Kruševaĉka 28. This is expected, taking into account that these two groups of similar cultivars were selected in the same companies. This study confirmed that studied collection of alfalfa cultivars has variability necessary for successful selection process.