Upotreba molekularnih markera i in vitro metoda u detekciji otpornosti suncokreta prema tribenuron-metilu

Abstract

U ovom radu su razvijeni in vitro test i molekularni markeri za detekciju tolerantnosti suncokreta prema tribenuron-metilu (gen Ahasl1-2). Pošto proces unošenja gena tolerantnosti povratnim ukrštanjem traje dugo, razvoj in vitro testova i molekularnih markera koji bi omogućili detekciju tolerantnih genotipova značajno bi skratio period konverzije linija i hibrida suncokreta u otpornu formu. Od posebnog je značaja razvoj testova i markera koji omogućavaju razlikovanje homozigotno i heterozigotno tolerantnih genotipova. Za razvijanje in vitro testa korišćeno je devet različitih genotipa suncokreta: četiri homozigotno tolerantna, četiri heretozigotno tolerantna i jedan osetljiv. Testiranje je vršeno različitim koncentracijama herbicida, u rasponu od 2,0 μM - 4,0 μM na dve pH, pH 7 i 8. Praćen je uticaj pH podloge i koncentracije herbicida na razvoj nadzemnog dela biljke i korena u cilju identifikacije optimalne kombinacije za detekciju tolerantnih genotipova suncokreta. Tokom testiranja tolerantnosti, razvoj osetljivog genotipa je zaustavljen tri dana nakon postavljanja klijanaca suncokreta u MS podlogu sa prethodno dodatim herbicidom. Sve testirane kombinacije su zaustavile razvoj osetljivog genotipa, dok se za razlikovanje homozigotno tolerantnih od heterozigotnih genotipova najbolje pokazala 3,0 μM na pH 7, a sveža masa korena kao najpovoljnija morfološka osobina za razlikovanje genotipova. Test je omogućio razlikovanje tolerantnih od osetljivog genotipa za 5 dana i homozigotno tolerantnih i heterozigota za 12 dana. U okviru molekularne analize tolerantnosti suncokreta prema tribenuron-metilu analizirane su tolerantna i osetljiva roditeljska linija, F1 i F2 potomstvo. Analizirana je mogućnost upotrebe SSR markera za detekciju tolerantnosti. SSR markeri se nisu pokazali kao pouzdani indikatori tolerantnosti zbog svoje udaljenosti od gena Ahasl1-2. Stoga su razvijeni CAPS markeri za detekciju tolerantnosti prema tribenuron-metilu. Ispitivane su različite kombinacije prajmera koji su dizajnirani na osnovu sekvence gena Ahasl1-2 i pronađena su tri restrikciona enzima čija se mesta digestije razlikuju između sekvence mutiranog gena Ahasl1-2 i divljeg tipa ahasl1. Neke kombinacije prajmera i enzima su omogućile razvoj dominantnih CAPS, a neke kodominantnih markera. Razvijeno je šest kodomnantnih CAPS markera koji će se koristiti u marker-asistiranoj selekciji.

tribenuronmethyl (Ahas1-2 gene) tolerance in sunflower is presented in this study. Since, introduction of tolecance in sunflower by conventional breeding takes years, quick tests and molecular markers that can detect tolerant genotypes will shorten the time for developing tolerant sunflower lines and hybrids. Special emphasis of this work was to develop methods for discrimination between homozygous and heterozygous tolerant genotypes, since those methods are the most useful for facilitating backcrossing. Nine sunflower genotypes: four homozygous tolerant, four heterozygous and one susceptible sulfonylurea genotype were used for development of an in vitro test. MS media supplemented with different concentrations of herbicide (in range between 2.0 μM - 4.0 μM) and with pH either 7 or 8 were used for resistance testing. The effect of medium pH and herbicide concentration on above-ground and root mass of sunflower seedlings was observed in order to identify the optimal tribenuron-methyl concentration and pH combination, as well as morphological parameter most useful for resistance testing. All tested herbicide concentrations were found to be suitable for discrimination of tribenuronmethyl susceptible genotype, since the growth of tested susceptible genotype was halted after 3 days of culture on all tribenuron-methyl supplemented media. The best pH and herbicide concentration combination for discrimination between homozygous and heterozygous tolerant sunflower genotypes was 3.0 μM at pH 7, while root mass was found to be the best parameter for discrimination between homozygous and heterozygous tolerant genotypes. The test enabled discrimination between tolerant and susceptible genotypes in 5 days, as well as discrimination between homozygous and heterozygous tolerant genotypes in 12 days. Molecular analysis of tribenuron-methyl tolerance included resistant and susceptible parental lines, F1 and F2 progeny. Analysis of use of SSR markers for tolerance gene detection showed that SSRs were not suitable because of the distance between markers and Ahasl1-2 gene. Therefore, CAPS markers were developed. Specific primers for Ahasl1-2 gene were designed and three restriction enzymes that have different patterns of restriction between resistant Ahasl1-2 gene and wild type, ahasl, were identified. Some of the combinations of primers and enzymes showed dominant nature, however six markers proved to be co-dominant. Those markers will be used in marker assisted selection and for shortening long period for backcrossing.