Three-year study of DNA cytosine methylation dynamics in transplanted Malbec grapevines.

DNA cytosine methylation, an epigenetic mechanism involved in gene regulation and genome stability, remains poorly understood in terms of its role under changing environmental conditions. Previous research using methylation-sensitive amplified polymorphism (MSAP) markers in a Vitis vinifera L. cv. Malbec clone showed vineyard-specific DNA methylation polymorphism, but no change in overall methylation levels. To complement these findings, the present study investigates the intra-seasonal epigenetic dynamics between genetically identical plants grown in different vineyards through a transplanting experiment. Cuttings of the same clone, showing differential methylation patterns imposed by the vineyard of origin (Agrelo and Gualtallary), were cultivated in a common vineyard (Lunlunta). Using high-performance liquid chromatography-ultraviolet detection, the quantification of global DNA 5-methylcytosine (5-mC) levels revealed relatively low overall 5-mC percentages in grapevines, with higher levels in Agrelo (5.8%) compared to Gualtallary plants (3.7%). The transplanted plants maintained the 5-mC levels differences between vineyards (9.8% vs 6.2%), which equalized in subsequent seasons (7.5% vs 7%). Additionally, the study examined 5-mC polymorphism using MSAP markers in Lunlunta transplanted plants over three seasons. The observed differences between vineyards in MSAP patterns during the initial growing season gradually diminished, suggesting a reprogramming of the hemimethylated pattern following implantation in the common vineyard. In contrast, the non-methylated pattern exhibited greater stability, indicating a potential memory effect. Overall, this study provides valuable insights into the dynamic nature of DNA methylation in grapevines under changing environmental conditions, with potential implications for crop management and breeding strategies.

Vineyard environments influence Malbec grapevine phenotypic traits and DNA methylation patterns in a clone-dependent way.

KEY MESSAGE: By studying three cv. Malbec clones cultivated in two vineyards with contrasting environmental conditions, we demonstrated that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent. Clonal selection and vegetative propagation determine low genetic variability in grapevine cultivars, although it is common to observe diverse phenotypes. Environmental signals may induce epigenetic changes altering gene expression and phenotype. The range of phenotypes that a genotype expresses in different environments is known as phenotypic plasticity. DNA methylation is the most studied epigenetic mechanism, but only few works evaluated this novel source of variability in grapevines. In the present study, we analyzed the effects on phenotypic traits and epigenome of three Vitis vinifera cv. Malbec clones cultivated in two contrasting vineyards of Mendoza, Argentina. Anonymous genome regions were analyzed using methylation-sensitive amplified polymorphism (MSAP) markers. Clone-dependent phenotypic and epigenetic variability between vineyards were found. The clone that presented the clearer MSAP differentiation between vineyards was selected and analyzed through reduced representation bisulfite sequencing. Twenty-nine differentially methylated regions between vineyards were identified and associated to genes and/or promoters. We discuss about a group of genes related to hormones homeostasis and sensing that could provide a hint of the epigenetic role in the determination of the different phenotypes observed between vineyards and conclude that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent.

Variation in the amino acids, volatile organic compounds and terpenes profiles in induced polyploids and in Solanum tuberosum varieties.

Polyploids often display a variety of phenotypic novelties when compared to their diploid progenitors, some of which may represent ecological advantages, especially regarding tolerance to biotic and abiotic factors. Plants cope with environmental factors by producing chemicals such as volatile organic compounds (VOCs) and specific amino acids (AAs). In potato, the third most important food crop in the world, gene introgression from diploid wild relative species into the genetic pool of the cultivated species (tetraploid) would be of great agronomical interest. The consequences of allopolyploidization on the potato VOCs and AAs profiles have not been yet analyzed. In this work, the effects of whole genome duplication on VOCs and AAs contents in leaves of potato allo- and autotetraploids and cultivated varieties were studied. The polyploids were obtained by chromosomal duplication of a genotype of the wild diploid species S. kurtzianum (autopolyploid model), and a diploid interspecific hybrid between the cultivated species S. tuberosum and S. kurtzianum (allopolyploid model). Almost all compounds levels varied greatly among these tetraploid lines; while all tetraploids showed higher contents of non-isoprenoids compounds than diploids, we found either increments or reductions in terpenes and AAs content. The results support the idea that genome duplication is a stochastic source of variability, which might be directly used for introgression in the 4x gene pool of the cultivated potato by sexual hybridization.

Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids.

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum.

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.

Changes in micro RNA expression in a wild tuber-bearing Solanum species induced by 5-Azacytidine treatment.

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species.

Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii.

BACKGROUND: The wild potato Solanum ruiz-lealii Brüch. (2n = 2x = 24), a species of hybrid origin, is endemic to Mendoza province, Argentina. Recurrent flower malformations, which varied among inflorescences of the same plant, were observed in a natural population. These abnormalities could be the result of genomic instabilities, nucleus-cytoplasmic incompatibility or epigenetic changes. To shed some light on their origin, nuclear and mitochondrial DNA of plants with normal and plants with both normal and malformed flowers (from here on designated as plants with normal and plants with abnormal flower phenotypes, respectively) were analyzed by AFLP and restriction analyses, respectively. Also, the wide genome methylation status and the level of methylation of a repetitive sequence were studied by MSAP and Southern blots analyses, respectively. RESULTS: AFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes. However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes. The abnormal flower phenotype was obtained by treating a normal plant with 5-Azacytidine, a demethylating agent, giving support to the idea of the role of DNA methylation in the origin of flower abnormalities. In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence. CONCLUSION: The epigenetic nature of the observed flower abnormalities is consistent with the results and indicates that in the diploid hybrid studied, natural variation in methylation profiles of anonymous DNA sequences could be of biological significance.