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.

Pollen-mediated gene flow from a commercial potato cultivar to the wild relative S. chacoense Bitter under experimental field conditions in Argentina.

The common potato, Solanum tuberosum ssp. tuberosum (tbr, 2n = 4x = 48; 4EBN), has many closely related wild tuber-bearing species. Around 28 to 35 of them spontaneously grow in Argentina overlapping, in some areas, with the crop and/or experimental transgenic potatoes. Although it is well proven that hybridization barriers in potatoes can be incomplete, information on gene flow between cultivated and wild germplasm is scarce. Thus, a gene flow field experiment with a circular array was set up in Balcarce, Argentina, in 2009, and evaluated over two seasons. The tetraploid tbr cultivar Huinkul MAG and one compatible cloned genotype of the related wild potato S. chacoense Bitter (chc, 2n = 2x = 24; 2EBN), which produced 2n eggs, were used, respectively, as pollen donor and receptor. Berries with hybrid seeds - as revealed by ploidy and RAPD profiles - were obtained in one season, at 30 m from the pollen donor. These results reinforce others previously obtained with the same pollen donor and a male sterile tbr cultivar in a similar array, pointing out to the need of increasing isolation distances in areas of overlap between cultivated and wild potato germplasm to prevent or minimize undesirable pollen-mediated gene flow.

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.

Molecular relationships between Saccharomyces cerevisiae strains involved in winemaking from Mendoza, Argentina.

Three molecular typing techniques were applied to assess the molecular relationships of Saccharomyces cerevisiae strains isolated from winery equipment, grapes, and spontaneous fermentation in a cellar located in "Zona Alta del Río Mendoza" (Argentina). In addition, commercial Saccharomyces strains widely used in this region were also included. Interdelta PCR typing, mtDNA restriction analysis, and microsatellite (SSR) genotyping were applied. Dendrograms were constructed based on similarity among different patterns of bands. The combination of the three techniques discriminated 34 strains among the 35 isolates. The results of this study show the complex relationships found at molecular level among the isolates that share the same ecological environment, i.e., the winemaking process. With a few exceptions, the yeast isolates were generally clustered in different ways, depending on the typing technique employed. Three clusters were conserved independently of the molecular method applied. These groups of yeasts always clustered together and had high degree of similarity. Furthermore, the dendrograms mostly showed clusters combining strains from winery and fermentation simultaneously. Most of the commercial strains included in this study were clustered separately from the other isolates analyzed, and just a few of them grouped with the strains mainly isolated from spontaneous fermentation. Only one commercial strain was clustered repetitively with a noncommercial strain isolated from spontaneous fermentation in the three dendrograms. On the other hand, this study has demonstrated the importance of selecting an appropriate molecular method according to the main objectives of the research.

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.

Genetic diversity among varieties of the native forage grass Trichloris crinita based on AFLP markers, morphological characters, and quantitative agronomic traits.

We assessed the genetic diversity in Trichloris crinita (Poaceae) varieties from South America, using AFLPs, morphological characters, and quantitative agronomic traits. Owing to the importance of this species for range grazing, we first characterized the varieties based on forage productivity. Biomass production varied 9 fold among the materials evaluated. Analysis of AFLP fingerprints allowed the discrimination of all varieties with a few selected primer combinations. Pair-wise genetic similarities, using marker data, ranged from 0.31 to 0.92 (Jaccard coefficients). Marker-based unweighted pair group method with arithmetic averaging (UPGMA) cluster analysis did not show geographical clustering, but rather grouped the varieties according to their biomass production. We identified 18 markers associated with biomass production, of which 8 showed complete correlation (r = 1.00) with this trait. These DNA markers can be used to assist selection for high forage productivity in T. crinita. Cluster analysis using morphological and quantitative characters revealed 4 distinct groups of varieties, clearly separated according to their biomass yield. The variables foliage height and basal diameter were strongly correlated with biomass production and these phenotypic markers can be used to select productive plants. The relations among the varieties based on AFLP data were significantly correlated with those based on agronomic and morphological characters, suggesting that the 2 systems give similar estimates of genetic relations among the varieties.

Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids.

The joining of different genomes in allotetraploids played a major role in plant evolution, but the molecular implications of this event are poorly understood. In synthetic allotetraploids of Arabidopsis and Cardaminopsis arenosa, we previously demonstrated the occurrence of frequent gene silencing. To explore the involvement of epigenetic phenomena, we investigated the occurrence and effects of DNA methylation changes. Changes in DNA methylation patterns were more frequent in synthetic allotetraploids than in the parents. Treatment with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in the development of altered morphologies in the synthetic allotetraploids, but not in the parents. We profiled mRNAs in control and 5-aza-2'-deoxycytidine-treated parents and allotetraploids by amplified fragment length polymorphism-cDNA. We show that DNA demethylation induced and repressed two different transcriptomes. Our results are consistent with the hypothesis that synthetic allotetraploids have compromised mechanisms of epigenetic gene regulation.