Abstract
The genus Urochloa P. Beauv. presents a prominent role in the tropical agricultural scenario being composed of species with different ploidy levels. Studies on the genomic relationship within this genus as well as specific analysis involving epigenetic marks are limited. The aim of the present study was to identify the cytosine methylation (5-mCyt) and histone H3 lysine 9 dimethylation (H3k9me2) in the different modulations of 45S ribosomal DNA (rDNA) sites in interphase nuclei and to associate these results with gene expression analysis in Urochloa ruziziensis (2n = 4x = 36), Urochloa brizantha cv. Marandu (2n = 4x = 36), and their respective interspecific hybrid H1863 (2n = 4x = 36). Immunolocalization techniques were performed in combination with Fluorescence in situ hybridization (FISH) for the location of the 45S rDNA sites. Predominantly, we observed intra- and perinucleolar sites, mostly hypomethylated and/or hyper/hypomethylated, decondensed or partially condensed. The gene expression analysis was performed qualitatively through the conventional PCR using complementary DNA and confirmed by the RT-qPCR technique and primers designed for the ITS-1 region of U. brizantha and U. ruziziensis. The molecular analyses performed on leaves showed that there is dominance of U. brizantha 45S rDNA gene expression on U. ruziziensis in the H1863 hybrid. In roots, the analyses showed that the 45S rDNA genes of the two parents are expressed in the hybrid genome. Thus, it is plausible to infer a tissue-specific nuclear dominance model in which the pattern of hypermethylated cytosine sites with heterochromatic marks and, therefore, silenced were mostly inherited from U. ruziziensis, whereas the rDNA originated from U. brizantha was characterized by cytosine and H3k9 hypomethylation.
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The authors thank the support of the Foundation for Research Support of the State of Minas Gerais (FAPEMIG), the Coordination for the Improvement of Higher Education Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq) for financial support for the development of this study.
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VHT conceived the study. YS and CP carried out the immunolocalization and FISH techniques. GR participated in the prepation and evaluation of the slides. YS, VHT, and WP analyzed and interpreted the data. WP carried out the experiment of transcript analysis. AL and AC-J contributed to the qPCR data. FS responsible for the breeding program of Lolium; provide seeds and reviewed the data. YS and VHT drafted the manuscript. WP, AL, and FS helped to draft the manuscript. All authors read and approved the manuscript to the final submission.
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Key Message
DNA methylation marks (5-mCyt) and H3k9me2 are directly associated to the different condensation status of the rDNA 45S sites in all evaluated genotypes. The genic expression study of rDNA 45S sites from 1863 hybrid characterized a scenario of tissue specific nucleolar dominance.
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Fig. S1
Agarose gel with the results of PCR on genomic DNA (gDNA). Primers used: Bz_Fwd, Rz_Fwd, and BzRz_Rvs. Legend: NC Negative Control, Bz U. brizantha, Hb Hybrid H1863, Rz U. ruziziensis (PNG 45 kb)
Fig. S2
Agarose gels with the results of PCRs on the complementary DNA (cDNA). Primers used: Bz_Fwd, Rz_Fwd, and BzRz_Rvs; cDNAs used: U. brizantha (Bz), U. ruzizienis (Rz), and hybrid H1863 (Hb). Legend: CN Negative Control, Bz U. brizantha, Hb Hybrid H1863, Rz U. ruziziensis (PNG 321 kb)
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Santos, Y.D., Pereira, W.A., de Paula, C.M.P. et al. Epigenetic Marks Associated to the Study of Nucleolar Dominance in Urochloa P. Beauv.. Plant Mol Biol Rep 38, 380–393 (2020). https://doi.org/10.1007/s11105-020-01203-4
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DOI: https://doi.org/10.1007/s11105-020-01203-4