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Differential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species


Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect ‘genetic’ information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.

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The authors are indebted to Ing. Agr. F. Cano, G. Benito, and G. Barreiro from “Jardín Botánico Carlos Thays de Buenos Aires” and to Dr. P.A. Sansberro (IBONE-UNNE), who kindly provided specimens from living collections. We are also grateful to Drs. L. Babino, D. Kachanovsky, J.C. Vilardi, M.S. Rodriguero, and G.R. Cueto (all from FCEN, UBA) for their helpful and kind technical assistance, and we thank the anonymous reviewers whose suggestions helped to improve this manuscript. Grants from the Universidad de Buenos Aires (UBACYT 20020170100614BA) and the Agencia Nacional de Investigaciones Científicas y Técnicas (PICT 2015–2525) are gratefully acknowledged.

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JC and AMG conceived and designed the experiments and wrote the manuscript; RMA helped in qPCR assays and results interpretation; JC performed the experiments; JC and DIP analyzed the data; reagents were provided through grants awarded to AMG. All authors read and approved the manuscript.

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Correspondence to Alexandra Marina Gottlieb.

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The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Cascales, J., Acevedo, R.M., Paiva, D.I. et al. Differential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species. J Plant Res 134, 559–575 (2021).

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  • Dioecy
  • DNA methylation
  • Molecular markers
  • MSAP
  • Multivariate analyses