Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

Hybridization and polyploidization effects on LTR-retrotransposon activation in potato genome.

Hybridization and polyploidization are major forces in plant evolution and potatoes are not an exception. It is proposed that the proliferation of Long Terminal Repeat-retrotransposons (LTR-RT) is related to genome reorganization caused by hybridization and/or polyploidization. The main purpose of the present work was to evaluate the effect of interspecific hybridization and polyploidization on the activation of LTR-RT. We evaluated the proliferation of putative active LTR-RT in a diploid hybrid between the cultivated potato Solanum tuberosum and the wild diploid potato species S. kurtzianum, allotetraploid lines derived from this interspecific hybrid and S. kurtzianum autotetraploid lines (ktz-autotetraploid) using the S-SAP (sequence-specific amplified polymorphism) technique and normalized copy number determination by qPCR. Twenty-nine LTR-RT copies were activated in the hybrid and present in the allotetraploid lines. Major LTR-RT activity was detected in Copia-27, Copia-12, Copia-14 and, Gypsy-22. According to our results, LTR-RT copies were activated principally in the hybrid, there was no activation in allotetraploid lines and only one copy was activated in the autotetraploid.

Response to water deficit of semi-desert wild potato Solanum kurtzianum genotypes collected from different altitudes.

Drought-sensitive crops are threatened as a consequence of limited available water due to climate change. The cultivated potato (Solanum tuberosum) is susceptible to drought and within its wild relative species, Solanum kurtzianum is the Argentinian wild potato species best adapted to arid conditions. However, its physiological responses to water deficit (WD) are still missing. Within the distribution of S. kurtzianum, genotypes could be adapted to differential precipitation regimes. The aim of this work was to evaluate responses of three S. kurtzianum genotypes collected at 1100 (G1), 1900 (G2) and 2100 m a.s.l. (G3) to moderate and severe WD. Treatments were imposed since flowering and lasted 36 days. Yield components, morpho-physiological and biochemical responses; and phenotypic plasticity were evaluated. The three genotypes presented mechanisms to tolerate both WD treatments. G1 presented the lowest yield reduction under moderate WD, mainly through a rapid stomatal closure and a modest vegetative growth. The differences among genotypes suggest that local adaptation is taking place within its natural habitat. Also, G2 presented environmentally induced shifts in plasticity for stomatal length and carotenoids, suggesting that phenotypic plasticity has a role in acclimation of plants to WD until selection works.

Environmentally induced phenotypic plasticity and DNA methylation changes in a wild potato growing in two contrasting Andean experimental gardens.

DNA methylation can be environmentally modulated and plays a role in phenotypic plasticity. To understand the role of environmentally induced epigenetic variation and its dynamics in natural populations and ecosystems, it is relevant to place studies in a real-world context. Our experimental model is the wild potato Solanum kurtzianum, a close relative of the cultivated potato S. tuberosum. It was evaluated in its natural habitat, an arid Andean region in Argentina characterised by spatial and temporal environmental fluctuations. The dynamics of phenotypic and epigenetic variability (with Methyl Sensitive Amplified Polymorphism markers, MSAP) were assayed in three genotypes across three growing seasons. These genotypes were cultivated permanently and also reciprocally transplanted between experimental gardens (EG) differing in ca. 1000 m of altitude. In two seasons, the genotypes presented differential methylation patterns associated to the EG. In the reciprocal transplants, a rapid epigenomic remodelling occurred according to the growing season. Phenotypic plasticity, both spatial (between EGs within season) and temporal (between seasons), was detected. The epigenetic and phenotypic variability was positively correlated. The lack of an evident mitotic epigenetic memory would be a common response to short-term environmental fluctuations. Thus, the environmentally induced phenotypic and epigenetic variation could contribute to populations persistence through time. These results have implications for understanding the great ecological diversity of wild potatoes.

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.

Genomic re-assessment of the transposable element landscape of the potato genome.

KEY MESSAGE: We provide a comprehensive and reliable potato TE landscape, based on a wide variety of identification tools and integrative approaches, producing clear and ready-to-use outputs for the scientific community. Transposable elements (TEs) are DNA sequences with the ability to autoreplicate and move throughout the host genome. TEs are major drivers in stress response and genome evolution. Given their significance, the development of clear and efficient TE annotation pipelines has become essential for many species. The latest de novo TE discovery tools, along with available TEs from Repbase and sRNA-seq data, allowed us to perform a reliable potato TEs detection, classification and annotation through an open-source and freely available pipeline ( https://github.com/DiegoZavallo/TE_Discovery ). Using a variety of tools, approaches and rules, we were able to provide a clearly annotated of characterized TEs landscape. Additionally, we described the distribution of the different types of TEs across the genome, where LTRs and MITEs present a clear clustering pattern in pericentromeric and subtelomeric/telomeric regions respectively. Finally, we analyzed the insertion age and distribution of LTR retrotransposon families which display a distinct pattern between the two major superfamilies. While older Gypsy elements concentrated around heterochromatic regions, younger Copia elements located predominantly on euchromatic regions. Overall, we delivered not only a reliable, ready-to-use potato TE annotation files, but also all the necessary steps to perform de novo detection for other species.

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 consequences of interploidal hybridisation in synthetic and natural interspecific potato hybrids.

Interploidal hybridisation can generate changes in plant chromosome numbers, which might exert effects additional to the expected due to genome merger per se (that is genetic, epigenetic and phenotypic novelties). Wild potatoes are suitable to address this question in an evolutionary context. To this end, we performed genetic (AFLP and single sequence repeart (SSR)), epigenetic (MSAP), and cytological comparisons in: (1) natural populations of the diploid cytotype of the hybrid taxonomic species Solanum × rechei (2n = 2×, 3×) and its parental species, the triploid cytotype of Solanum microdontum (2n = 2×, 3×) and Solanum kurtzianum (2n = 2×); and (2) newly synthesised intraploidal (2× × 2×) and interploidal (3× × 2×) S. microdontum × S. kurtzianum hybrids. Aneuploidy was detected in S. × rechei and the synthetic interploidal progeny; this phenomenon might have originated the significantly higher number of methylation changes observed in the interploidal vs the intraploidal hybrids. The wide epigenetic variability induced by interploidal hybridisation is consistent with the novel epigenetic pattern established in S. × rechei compared to its parental species in nature. These results suggest that aneuploid potato lineages can persist throughout the short term, and possibly medium term, and that differences in parental ploidy resulting in aneuploidy are an additional source of epigenetic variation.

Changes in grapevine DNA methylation and polyphenols content induced by solar ultraviolet-B radiation, water deficit and abscisic acid spray treatments.

Environment and crop management shape plant's phenotype. Argentinean high-altitude vineyards are characterized by elevated solar ultraviolet-B radiation (UVB) and water deficit (D) that enhance enological quality for red winemaking. These signals promote phenolics accumulation in leaves and berries, being the responses mediated by abscisic acid (ABA). DNA methylation is an epigenetic mechanism that regulates gene expression and may affect grapevine growth, development and acclimation, since methylation patterns are mitotically heritable. Berry skins low molecular weight polyphenols (LMWP) were characterized in field grown Vitis vinifera L. cv. Malbec plants exposed to contrasting UV-B, D, and ABA treatments during one season. The next season early fruit shoots were epigenetically (methylation-sensitive amplification polymorphism; MSAP) and biochemically (LMWP) characterized. Unstable epigenetic patterns and/or stochastic stress-induced methylation changes were observed. UV-B and D were the treatments that induced greater number of DNA methylation changes respect to Control; and UV-B promoted global hypermethylation of MSAP epiloci. Sequenced MSAP fragments associated with UV-B and ABA showed similarities with transcriptional regulators and ubiquitin ligases proteins activated by light. UV-B was associated with flavonols accumulation in berries and with hydroxycinnamic acids in the next season fruit shoots, suggesting that DNA methylation could regulate the LMWP accumulation and participate in acclimation mechanisms.

Dissimilar evolutionary histories of two resistance gene families in the genus Solanum.

Genomic analyses have shown that most genes in eukaryotic lineages belong to families. Gene families vary in terms of number of members, nucleotide similarity, gene integrity, expression, and function. Often, the members of gene families are arranged in clusters, which contribute to maintaining similarity among gene copies and also to generate duplicates through replication errors. Gene families offer us an opportunity to examine the forces involved in the evolution of the genomes and to study recombination events and genomic rearrangements. In this work, we focused on the evolution of two plant resistance gene families, Sw5 and Mi-1, and analyzed the completely sequenced nuclear genomes of potato and tomato. We first noticed that the potato genome carries larger resistance gene families than tomato, but all gene copies are pseudogenes. Second, phylogenetic analyses indicated that Sw5 and Mi-1 gene families had dissimilar evolutionary histories. In contrast to Sw5, Mi-1 homologues suffered repeated gene conversion events among the gene copies, particularly in the tomato genome.

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.

Evolution of nematode-resistant Mi-1 gene homologs in three species of Solanum.

Plants have evolved several defense mechanisms, including resistance genes. Resistance to the root-knot nematode Meloidogyne incognita has been found in wild plant species. The molecular basis for this resistance has been best studied in the wild tomato Solanum peruvianum and it is based on a single dominant gene, Mi-1.2, which is found in a cluster of seven genes. This nematode attacks fiercely several crops, including potatoes. The genomic arrangement, number of copies, function and evolution of Mi-1 homologs in potatoes remain unknown. In this study, we analyzed partial genome sequences of the cultivated potato species S. tuberosum and S. phureja and identified 59 Mi-1 homologs. Mi-1 homologs in S. tuberosum seem to be arranged in clusters and located on chromosome 6 of the potato genome. Previous studies have suggested that Mi-1 genes in tomato evolved rapidly by frequent sequence exchanges among gene copies within the same cluster, losing orthologous relationships. In contrast, Mi-1 homologs from cultivated potato species (S. tuberosum and S. phureja) seem to have evolved by a birth-and-death process, in which genes evolve mostly by mutations and interallelic recombinations in addition to sequence exchanges.

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.

Centromeric localization and adaptive evolution of an Arabidopsis histone H3 variant.

Centromeric H3-like histones, which replace histone H3 in the centromeric chromatin of animals and fungi, have not been reported in plants. We identified a histone H3 variant from Arabidopsis thaliana that encodes a centromere-identifying protein designated HTR12. By immunological detection, HTR12 localized at centromeres in both mitotic and meiotic cells. HTR12 signal revealed tissue- and stage-specific differences in centromere morphology, including a distended bead-like structure in interphase root tip cells. The anti-HTR12 antibody also detected spherical organelles in meiotic cells. Although the antibody does not label centromeres in the closely related species Arabidopsis arenosa, HTR12 signal was found on all centromeres in allopolyploids of these two species. Comparison of the HTR12 genes of A. thaliana and A. arenosa revealed striking adaptive evolution in the N-terminal tail of the protein, similar to the pattern seen in its counterpart in Drosophila. This finding suggests that the same evolutionary forces shape centromeric chromatin in both animals and plants.