Analysis of Wine-Producing Vitis vinifera L. Biotypes, Autochthonous to Crete (Greece), Employing Ampelographic and Microsatellite Markers.

Vitis vinifera ssp. vinifera (domesticated grapevine) includes thousands of cultivars, which are classified according to their main uses, as wines, fresh fruits or dried raisins and sultanas since ancient times. Evidence showed that Crete grapevine cultivars and winemaking date back to 2300 BC. In this study, fifty-one genotypes belonging to seven different traditional Vitis vinifera cultivars, presumed autochthonous to the island of Crete, were selected for their wine-producing potential and classified by 51 ampelographic descriptors. In addition, five genotypes belonging to two non-autochthonous cultivars were included as out-group controls. Subsequently, in order to characterize genetic diversity, establish genetic relationships within and between cultivars and solve accession-labeling problems, genotypes were fingerprinted employing Simple Sequence Repeat (SSR or microsatellite) markers. Four of the autochthonous cultivars namely 'Vidiano', 'Vilana', 'Plyto', and 'Moschato Spinas' are used in the local economy for blanc (white) wine production while the rest, namely 'Kotsifali', 'Liatiko' and 'Mantilari' for Noir (red) wines. The two cultivars employed as out-group were 'Moschato Samou' and 'Moschato Alexandrias': both white wine producers. Ampelography-based clustering grouped the majority of genotypes along cultivar-specific clusters. All three Moschato cultivars formed a distinct clade pointing to the non-autochthonous origin of 'Moschato Spinas'. A total of one hundred and thirteen (113) SSR alleles were amplified from thirteen (13) SSR loci, with an average number of alleles per locus equal to 10.23 revealing ample genetic polymorphism. The cumulative probability of identity was also quite high (3.389 × 10-16). The overall observed heterozygosity was 0.837 while for twenty-nine of the examined genotypes, at least one private SSR allele was detected. The majority of genotypes were grouped in cultivar-specific clusters. The results of this paper pave the way for the certification and registration of clones of some of the most important wine-producing cultivars in Crete.

Elucidation of the Origin of the Monumental Olive Tree of Vouves in Crete, Greece.

The olive tree of Vouves in Crete, is considered the oldest producing olive tree in the world with an estimated age exceeding 4000 years. In the present study, we sequenced two samples (from the bottom and the top of the tree) to elucidate the genetic relation of this ancient tree with other olive cvs as well as to gain some insights about its origin. Our results showed that both samples have different genetic origins, proving that this ancient tree has been grafted at least one time. On the basis of whole genome sequences the sample from the top of the Vouves tree showed relation of the same order than half-siblings to one accession corresponding to the present-day Greek cv 'Mastoidis'. Nevertheless, in the framework of a microsatellite analysis it was found to cluster with the 'Mastoidis' samples. The Vouves rootstock (bottom sample) showed a clear grouping with the oleaster samples in a similar way to that of 'Megaritiki' Greek cv although it does not show any signal of introgression from them. The genomic analyses did not show a strong relation of this sample with the present-day Greek cvs analyzed in this study so it cannot be proved that it has been used as a source for cultivated olive tree populations represented by available genome sequences. Nevertheless, on the basis of microsatellite analyses, the Vouves rootstock showed affinity with two present-day Greek cvs, one "ancient" rootstock from continental Greece as well as monumental trees from Cyprus. The analysis of the impact of the variants in the gene space revealed an enrichment of genes associated to pathways related with carbohydrate and amino acid metabolism. This is in agreement with what has been found before in the sweep regions related with the process of domestication. The absence of oleaster gene flow, its old age and its variant profile, similar to other cultivated populations, makes it an excellent reference point for domestication studies.

Characterization of the F Locus Responsible for Floral Anthocyanin Production in Potato.

Anthocyanins are pigmented secondary metabolites produced via the flavonoid biosynthetic pathway and play important roles in plant stress responses, pollinator attraction, and consumer preference. Using RNA-sequencing analysis of a cross between diploid potato (Solanum tuberosum L.) lines segregating for flower color, we identified a homolog of the ANTHOCYANIN 2 (AN2) gene family that encodes a MYB transcription factor, herein termed StFlAN2, as the regulator of anthocyanin production in potato corollas. Transgenic introduction of StFlAN2 in white-flowered homozygous doubled-monoploid plants resulted in a recovery of purple flowers. RNA-sequencing revealed the specific anthocyanin biosynthetic genes activated by StFlAN2 as well as expression differences in genes within pathways involved in fruit ripening, senescence, and primary metabolism. Closer examination of the locus using genomic sequence analysis revealed a duplication in the StFlAN2 locus closely associated with gene expression that is likely attributable to nearby genetic elements. Taken together, this research provides insight into the regulation of anthocyanin biosynthesis in potato while also highlighting how the dynamic nature of the StFlAN2 locus may affect expression.

Epigenetic Changes and Transcriptional Reprogramming Upon Woody Plant Grafting for Crop Sustainability in a Changing Environment.

Plant grafting is an ancient agricultural practice widely employed in crops such as woody fruit trees, grapes, and vegetables, in order to improve plant performance. Successful grafting requires the interaction of compatible scion and rootstock genotypes. This involves an intricate network of molecular mechanisms operating at the graft junction and associated with the development and the physiology of the scion, ultimately leading to improved agricultural characteristics such as fruit quality and increased tolerance/resistance to abiotic and biotic factors. Bidirectional transfer of molecular signals such as hormones, nutrients, proteins, and nucleic acids from the rootstock to the scion and vice versa have been well documented. In recent years, studies on rootstock-scion interactions have proposed the existence of an epigenetic component in grafting reactions. Epigenetic changes such as DNA methylation, histone modification, and the action of small RNA molecules are known to modulate chromatin architecture, leading to gene expression changes and impacting cellular function. Mobile small RNAs (siRNAs) migrating across the graft union from the rootstock to the scion and vice versa mediate modifications in the DNA methylation pattern of the recipient partner, leading to altered chromatin structure and transcriptional reprogramming. Moreover, graft-induced DNA methylation changes and gene expression shifts in the scion have been associated with variations in graft performance. If these changes are heritable they can lead to stably altered phenotypes and affect important agricultural traits, making grafting an alternative to breeding for the production of superior plants with improved traits. However, most reviews on the molecular mechanisms underlying this process comprise studies related to vegetable grafting. In this review we will provide a comprehensive presentation of the current knowledge on the epigenetic changes and transcriptional reprogramming associated with the rootstock-scion interaction focusing on woody plant species, including the recent findings arising from the employment of advanced-omics technologies as well as transgrafting methodologies and their potential exploitation for generating superior quality grafts in woody species. Furthermore, will discuss graft-induced heritable epigenetic changes leading to novel plant phenotypes and their implication to woody crop improvement for yield, quality, and stress resilience, within the context of climate change.

Genetic Diversity and Structure Analysis Assessed by SSR Markers in a Large Collection of Vitis Cultivars from the Island of Crete, Greece.

The grape (Vitis vinifera L.) cultivars in the island of Crete, Greece represent one of the oldest populations of the species; nevertheless, very scarce information is available about its genetic structure. In this study, Vitis cultivars collected from the island of Crete were characterized using microsatellite markers. A broad germplasm collection representing 44 inferred Vitis cultivars, a total of 163 accessions, from the area of Crete including 37 wine and 7 table cultivars were fingerprinted employing thirteen (13) standardized simple sequence repeat (SSR, microsatellite) loci. SSR allelic analysis and a similarity dendrogram construction (cluster analysis) was followed by a hierarchical STRUCTURE analysis. The mean observed (Ho) and expected heterozygosity (He) were 0.7372 and 0.7686, respectively. The cumulative probability of identity was very low with a value of 3.18 × 10e-15. According to the cluster analysis, twenty-nine of the 44 Vitis cultivars were presented in single clusters and five cultivars were presented as distinct single accessions. In addition, ten (10) cases of synonyms and ten (10) groups of homonyms were also identified. STRUCTURE analysis provided evidence for three genetic groups (putative ancestry groups). Hierarchical STRUCTURE analysis revealed further stratification within each of the three ancestry groups. This work provides the molecular fingerprinting of 44 Vitis cultivars and an initial proposal in their ancestry. In the future, molecular genetic information along with morphological (ampelographic) data will provide an intergraded characterization of existing diversity and will allow for its use in breeding efforts and in commercial viticulture.

Lichen as Micro-Ecosystem: Extremophilic Behavior with Astrobiotechnological Applications.

This work demonstrates the tolerance of lichen Pleurosticta acetabulum under extreme conditions similar to those encountered in extraterrestrial environments. Specifically, the impact of three extreme Mars-like conditions-complete dehydration, extremely low temperature (-196°C/77K), and oxygen depletion-on lichens was investigated. The symbiosis of mycobiont and photobiont partners creates a micro-ecosystem that ensures viability of both symbiotic partners under prolonged desiccation and extremely low temperatures without any cultivation care. Changes in the molecular structure and function of the photosynthetic apparatus, in the level of chlorophylls, polyamines, fatty acids, carbohydrates, ergosterol, efflux of K+, and DNA methylation ensure the ecological integrity of the system and offer resistance of lichens to above-mentioned extreme environmental conditions. For the first time, we also demonstrate that the unprecedented polyextremophilic characteristic of lichens could be linked to biotechnological applications, following exposure to these extreme conditions, such that their ability to produce a high yield of hydrogen was unchanged. All these support that lichens are (a) ideal model systems for a space mission to inhabit other planets, supporting also the aspect that the panspermia theory could be extended to incorporate in the traveling entities not only single organisms but micro-ecosystems like lichens, and (b) ideal model systems for astrobiotechnological applications (hydrogen production), such as in the development of bioregeneration systems for extraterrestrial environments.

Bioenergetic strategy of microalgae for the biodegradation of tyrosol and hydroxytyrosol.

Olive mill wastewater has significant polluting properties due to its high phenolic content [mainly tyrosol (trs) and hydroxytyrosol (htrs)]. Growth kinetics and a series of fluorescence induction measurements for Scenedesmus obliquus cultures showed that microalgae can be tolerant of these phenolic compounds. Changes in the cellular energy reserves and concentration of the phenolic compounds adjust the "toxicity" of these compounds to the microalgae and are, therefore, the main parameters that affect biodegradation. Autotrophic growth conditions of microalgae and high concentrations of trs or htrs induce higher biodegradation compared with mixotrophic conditions and lower phenolic concentrations. When microalgae face trs and htrs simultaneously, biodegradation begins from htrs, the more energetically demanding compound. All these lead to the conviction that microalgae have a "rational" management of cellular energy balance. Low toxicity levels lead to higher growth and lower biodegradation, whereas higher toxicity levels lead to lower growth and higher biodegradation. The selection of appropriate conditions (compatible to the bioenergetic strategies of microalgae) seems to be the key for a successful biodegradation of a series of toxic compounds, thus paving the way for future biotechnological applications for solving complicated pollution problems, like the detoxification of olive mill wastewater.

Self-Fertility in a Cultivated Diploid Potato Population Examined with the Infinium 8303 Potato Single-Nucleotide Polymorphism Array.

Within a population of F hybrids between two genotypes ( L. Group Phureja DM 1-3 516 R44 [DM] and L. Group Tuberosum RH89-039-16 [RH]) used in the potato genome sequencing project, we observed fruit set after self-pollination on many plants. Examination of pollen tube growth in self-fertile and self-unfruitful F plants after controlled self-pollinations revealed no difference in the ability of pollen tubes to reach the ovary. To identify genomic regions linked with self-fertility, we genotyped the F population using a genome-wide single-nucleotide polymorphism (SNP) array. Polymorphic and robust SNPs were analyzed to identify allelic states segregating with the self-fertile phenotype. All 88 highly significant SNPs occurred on chromosome 12. Seeds obtained after self-pollination of self-fertile individuals were used to advance the population for four generations. Genotyping 46 self-fruitful and 46 self-unfruitful S plants on the Infinium 8303 Potato SNP array revealed eight SNPs segregating with self-fertility on chromosomes 4, 9, 11, and 12. Three times more heterozygosity than expected was found in the S generation. Estimates of heterozygosity were influenced by copy number variation (CNV) in the potato genome leading to spurious heterozygous genotyping calls. Some spurious heterozygosity could be removed by application of a CNV filter developed from alignment of additional monoploid potato genomic sequence to the DM reference genome. The genes responsible for fruit set in self-fertile plants in the F generation were restricted to chromosome 12, whereas new genomic regions contributed to the ability of S plants to set fruit after self-pollination.

Determination of epigenetic inheritance, genetic inheritance, and estimation of genome DNA methylation in a full-sib family of Cupressus sempervirens L.

Genetic inheritance and epigenetic inheritance are significant determinants of plant evolution, adaptation and plasticity. We studied inheritance of restriction site polymorphisms by the f-AFLP method and epigenetic DNA cytosine methylation inheritance by the f-MSAP technique. The study involved parents and 190 progeny of a Cupressus sempervirens L. full-sib family. Results from AFLP genetic data revealed that 71.8% of the fragments studied are under Mendelian genetic control, whereas faithful Mendelian inheritance for the MSAP fragments was low (4.29%). Further, MSAP fragment analysis showed that total methylation presented a mean of 28.2%, which was higher than the midparent value, while maternal inheritance was higher (5.65%) than paternal (3.01%). Interestingly de novo methylation in the progeny was high (19.65%) compared to parental methylation. Genetic and epigenetic distances for parents and offspring were not correlated (R(2)=0.0005). Furthermore, we studied correlation of total relative methylation and CG methylation with growth (height, diameter). We found CG/CNG methylation (N: A, C, T) to be positively correlated with height and diameter, while total relative methylation and CG methylation were positively correlated with height. Results are discussed in light of further research needed and of their potential application in breeding.