Avaliação de marcas epigenéticas com a análise da expressão gênica de rDNA 45S em Urochloa ruziziensis, Urochloa brizantha e seu híbrido

Abstract

The genus Urochloa P. Beauv. (including Brachiaria (Grin.) Griseb.) has a prominent role in the Brazilian agricultural scenario, being composed of species with different levels of ploidy and reproduction’s types. Studies on the genomic relationship within this genus are rare, as the researches involving epigenetic marks, directly involved with the genome organization. The aim of this work was to identify the pattern of cytosine methylation (5-mCyt) related to gene silencing and the dimethylation of lysine 9 in histone H3 (H3K9me2), associated with the heterochromatic structure, in the different conformations of the 45S rDNA sites, in the interphase nuclei and to associate these results with the analysis of the gene expression in Urochloa ruziziensis (genome B 2 B 2 B 2 B 2 ), Urochloa brizantha cv. Marandu (genome BBB 1 B 1 ) and its interspecific hybrid – H1863 (genome BB 1 B 2 B 2 ). Immunostaining techniques were performed in combination with FISH (fluorescence in situ hybridization) for the localization of the 45S rDNA sites. The slides were made with meristematic cells obtained from the root tips fixed in Carnoy (ethanol:acetic acid, 3:1 v/v). The indirect immunodetection was performed with the primary antibodies anti-H3K9me2 mouse monoclonal and anti-5-methyl cytosine mouse monoclonal. The detection was done using secondary antibodies mouse polyclonal TRITC-conjugated. FISH Technique was performed sequentially on immunostaining using rDNA probes obtained from Triticum aestivum L. From the combination of these two techniques it was possible to perceive a direct relation between the epigenetic marks and the different conformations of the nucleolar organizing regions (NORs). It was noticed predominantly intra and perinucleolar sites, which were mostly hypomethylated and/or hyper/hypomethylated, decondensed or partially condensed. Gene expression analysis was performed qualitatively through conventional PCR using complementary DNA (cDNA) and it was confirmed by the RT-qPCR technique, using primers designed for the ITS-1 region of U. brizantha and U. ruziziensis. The molecular analyzes showed differential expression between the 45S rDNA sites of the different genomes within the genome of hybrid 1863, characterizing a scenario of nucleolar dominance, in which happens the preferential expression of the 45S rDNA genes inherited from U. Brizantha, while the sites from U. ruziziensis are silenced through epigenetic mechanisms. These results allowed to infer that those sites that were hypermethylated in cytosine and H3k9me2 marks were inherited from U. ruziziensis, whereas the portion of rDNA characterized by hypomethylation of cytosine and H3k9me2 was originated from U. brizantha.