Molecular Authentication, Propagation Trials and Field Establishment of Greek Native Genotypes of Sambucus nigra L. (Caprifoliaceae): Setting the Basis for Domestication and Sustainable Utilization

In the context of sustainable utilisation of valuable but neglected plant resources, a multifaceted study is presented herein for Greek native genotypes of elderberry (Sambucus nigra L., Caprifoliaceae), a species with an appreciated pharmaceutical and nutritional value. At the first phase, fresh plant materials (leaves, softwood cuttings) were collected from nine geographically separated genotypes originating from wild-growing Greek native germplasm of S. nigra. The leaf samples were genetically authenticated using DNA barcoding (ITS2). The next phase included the development of an asexual propagation protocol via cuttings which included screening of the collected genotypes in terms of propagation success, and further experimentation over a two-year period on a prioritised genotype. The propagation results highlighted the importance of external application of indole-3-butyric acid (IBA) rooting hormone at levels of 2000–4000 ppm, which consistently presented high rooting rates (100%) of summer softwood, leafy cuttings of apical or sub-apical type. At the same time, rooting quality in terms of root number and length as well as early plant growth after rooting, were improved by external hormone application resulting in high rates of plant survival. This study reports first-time data on multifaceted assessment of Greek native S. nigra genotypes on molecular authentication and asexual propagation, thus ultimately setting the basis for domestication and sustainable utilization of this species.

Garcinia intermedia, a little-known fruit tree in the American tropics

Garcinia intermedia (Pittier) Hammel, known as the lemon drop mangosteen in English and by a variety of names including limoncillo and toronjil in Mexico, belongs to the family Clusiaceae, and is distributed in the warm tropical regions of Mexico and Central America at elevations of 300 to 1,000 m. It is an underutilized fruit tree that reaches up to 20 m in height and produces yellow fruits with up to four seeds. The pulp is bittersweet and is highly valued by the rural population of the regions where it grows, where bats and spider monkeys also consume it. The fruit has medicinal properties; it is rich in benzophenones, which attack colon cancer cells. The fruit of G. intermedia has higher antioxidant capacity than the fruit of the mangosteen (G. mangostana Linn.); however, little is known about the horticultural management of this species, and basic knowledge, such as asexual propagation or postharvest conservation, has not been reported. There is no information on ex situ conservation of this species in Mexico and Central America, and no selection of outstanding specimens with high quality fruits has been made. Most of the information reported so far for this species is about its ecology and medicinal properties.

Dynamic Changes of DNA Methylation During Wild Strawberry (Fragaria nilgerrensis) Tissue Culture

Tissue culture is an important tool for asexual propagation and genetic transformation of strawberry plants. In plant tissue culture, variation of DNA methylation is a potential source of phenotypic variation in regenerated plants. However, the genome wide dynamic methylation patterns of strawberry tissue culture remain unclear. In this study, we used whole-genome bisulfite sequencing (WGBS) to study genomic DNA methylation changes of a wild strawberry Fragaria nilgerrensis at six stages: from explants of shoot tips to outplanting and acclimation. Global methylation levels showed that CG sites exhibited the highest methylation level in all stages with an average of 49.5%, followed by CHG (33.2%) and CHH (12.4%). Although CHH accounted for the lowest proportion of total cytosine methylation, it showed the most obvious methylation change and the most of these changes occurred in the transposable element regions. The overall methylation levels alternately decreased and increased during the entire tissue culture process and the distribution of DNA methylation was non-uniform among different genetic regions. Furthermore, much more differentially methylated regions (DMRs) were detected in dedifferentiation and redifferentiation stages and most of them were transposable elements, suggesting these processes involved activating or silencing of amounts of transposons. The functional enrichment of the DMR-related genes indicated that genes involved in hormone metabolic processes, plant development and the stress response changed methylation throughout the tissue culture process. Finally, the quantitative real-time PCR (qRT-PCR) was conducted to examine the association of methylation and gene expression of a set of different methylated genes. Our findings give deeper insight into the epigenetic regulation of gene expression during the plant tissue cultures process, which will be useful in the efficient control of somaclonal variations and in crop improvement.

Molecular Authentication, Phytochemical Evaluation and Asexual Propagation of Wild-Growing Rosa canina L. (Rosaceae) Genotypes of Northern Greece for Sustainable Exploitation

Dogroses belong to a taxonomically difficult genus and family and represent important phytogenetic resources associated with high ornamental, pharmaceutical-cosmetic and nutritional values, thus suggesting a potentially high exploitation merit. Triggered by these prospects, wild-growing Rosa canina populations of Greece were selected for investigation and evaluation of their potential for integrated domestication. We collected ripe rosehips from Greek native wild-growing populations (samples from seven genotypes) for phytochemical analysis (total phenolics, total flavonoids, antioxidant activity and vitamin C content), leaf samples for DNA analysis using the ITS2 sequence (nine genotypes) and fresh soft-wood stem cuttings for propagation trials (seven genotypes). After evaluation of these materials, this study reports for the first-time distinct DNA-fingerprinted genotypes from Greece with interesting phytochemical profiles mainly in terms of Vitamic C content (up to 500.22 ± 0.15 mg of ascorbic acid equivalents/100 g of sample) as well as effective asexual propagation protocols for prioritized R. canina genotypes via cuttings. The latter highlights the importance of the levels of external hormone application (2000 ppm of indole-3-butyric acid), the effect of season (highly-effective spring trials) and genotype-specific differences in rooting capacities of the studied genotypes. All inclusive, this study offers new artificially selected material of Greek native R. canina with a consolidated identity and interesting phytochemical profile. These materials are currently under ex-situ conservation for further evaluation and characterization in pilot field studies, thus facilitating its sustainable exploitation for applications in the agro-alimentary, medicinal-cosmetic, and ornamental sectors.

Key Regulatory Pathways, MicroRNAs, and Target Genes Participate in Adventitious Root Formation of Acer Rubrum L

BackgroundAcer rubrum L. is a colorful ornamental tree with great economic value. Because this tree is difficult to root under natural conditions and the seedling survival rate is low, vegetative propagation methods are often used. Because the formation of adventitious roots (ARs) is essential for the survival of asexual propagation of A. rubrum, it is necessary to investigate the molecular regulatory mechanisms in the formation of ARs of A. ruburm. To address this knowledge gap, we sequenced the transcriptome and sRNA of the A. rubrum variety ‘Autumn Fantasy’ using high-throughput sequencing and explored changes in gene and microRNA (miRNA) expression in response to exogenous auxin treatment. ResultsWe identified 82,468 differentially expressed genes between the treated and untreated ARs, as well as 48 known and 95 novel miRNAs. We also identified 172 target genes of the known miRNAs using degradome sequencing. Two regulatory pathways (ubiquitin mediated proteolysis and plant hormone signal transduction), Ar-miR160a and the target gene ArARF10 were shown to be involved in the auxin response. We further investigated the expression patterns and regulatory roles of ArARF10 through subcellular localization, transcriptional activation, plant transformation, qRT-PCR analysis, and GUS staining. ConclusionsDifferential expression patterns indicated the Ar-miR160a-ArARF10 interaction might play a significant role in the regulation of AR formation in A. rubrum. Our study provided new insights into mechanisms underlying the regulation of AR formation in A. rubrum.

Revealing the Systematic Nature of Coral Attachment to Reef Substrates

Reef-building coral colonies propagate by periodic sexual reproduction and continuous asexual fragmentation. The latter depends on successful attachment to the reef substrate through modification of soft tissues and skeletal growth. Despite decades of research examining coral sexual and asexual propagation, the contact response, tissue motion, and cellular reorganisation responsible for attaching to the substrate via a newly formed skeleton have not been documented. Here, we correlated fluorescence and electron microscopy image data with ‘live’ microscopic time-lapse of the coral tissue biomechanics and developed a multiscale imaging approach to establish the first “coral attachment model” (CAM) - identifying three distinct phases that determine the timing and success of attachment during asexual propagation: (i) an initial immune response, followed by (ii) fragment stabilisation through anchoring by the soft tissue and (iii) formation of a “lappet appendage” structure leading to substrate bonding of the tissue for encrustation through the onset of skeletal calcification. In developing CAM, we provide new frameworks and metrics that enable reef researchers, managers and coral restoration practitioners to evaluate attachment effectiveness needed to optimise species-substrate compatibility.

Genome-wide analysis illustrates the effect of polyploidization in Phragmites australis

Polyploidization is a common event in plant evolution, and it plays an important role in plant speciation and adaptation. To address the role of polyploidization in grass diversification, we studied Phragmites australis, a species with intraspecific variation of chromosome numbers ranging from 2n=36 to 144. A combined analysis of genome structure, phylogeny and population genetics were used to study the evolution of P. australis. Whole-genome sequencing of three representative lineages revealed the allopolyploid origin of the species, with subgenome divergence dating back to approximately 29 million years ago, and the genomes showed hallmarks of relaxed selection associated with asexual propagation. Genome-wide analysis of 88 individuals from different populations around the world using restriction site associated DNA sequencing (RAD-seq) identified seven main intraspecific lineages with extensive genetic admixture. Each lineage was dominated by a distinct ploidy level, mostly tetraploid or octoploid, suggesting several polyploid events. Furthermore, we observed octoploid and hexaploid lineages at contact zones in Romania, Hungary and South Africa, suggestively due to genomic conflicts of allotetraploid parental lineages. Polyploidy may have evolved as a strategy to escape from the evolutionary dead-end of asexual propagation and the resulting decrease in genomic plasticity.

The use of shared haplotype length information for pedigree reconstruction in asexually propagated outbreeding crops, demonstrated for apple and sweet cherry

Pedigree information is of fundamental importance in breeding programs and related genetics efforts. However, many individuals have unknown pedigrees. While methods to identify and confirm direct parent–offspring relationships are routine, those for other types of close relationships have yet to be effectively and widely implemented with plants, due to complications such as asexual propagation and extensive inbreeding. The objective of this study was to develop and demonstrate methods that support complex pedigree reconstruction via the total length of identical by state haplotypes (referred to in this study as “summed potential lengths of shared haplotypes”, SPLoSH). A custom Python script, HapShared, was developed to generate SPLoSH data in apple and sweet cherry. HapShared was used to establish empirical distributions of SPLoSH data for known relationships in these crops. These distributions were then used to estimate previously unknown relationships. Case studies in each crop demonstrated various pedigree reconstruction scenarios using SPLoSH data. For cherry, a full-sib relationship was deduced for ‘Emperor Francis, and ‘Schmidt’, a half-sib relationship for ‘Van’ and ‘Windsor’, and the paternal grandparents of ‘Stella’ were confirmed. For apple, 29 cultivars were found to share an unknown parent, the pedigree of the unknown parent of ‘Cox’s Pomona’ was reconstructed, and ‘Fameuse’ was deduced to be a likely grandparent of ‘McIntosh’. Key genetic resources that enabled this empirical study were large genome-wide SNP array datasets, integrated genetic maps, and previously identified pedigree relationships. Crops with similar resources are also expected to benefit from using HapShared for empowering pedigree reconstruction.