A combined -omics and bioinformatics approach to generate the atlas of Greek-oriented sweet cherry genetic resources (SweetBiOmics)

Background Natural products are not only positioned in the heart of traditional medicine but also in modern medicine as many current drugs are coming from natural sources. Apart from the field of medicine and therapeutics, natural products are broadly used in other industrial fields such as nutrition, skincare products and nanotechnology. Methods and results The aim of this study was to assess the effects of sweet cherry (Prunus avium L.) fruit extract from the Greek native cultivar ‘Vasiliadi’, on the human 2D and 3D in vitro models in order to investigate its potential impact on skin. We focused on 2D culture of primary normal human epidermal keratinocytes (NHEK) that were treated with sweet cherry fruit extract. In the first place, we targeted fruit extract potential cytotoxicity by determining ATP intracellular levels. Furthermore, we assessed its potential skin irritability by using 3D skin model. To better understand the bioactivity of sweet cherry fruit. extract, we used qPCR to study the expression of various genes that are implicated in the skin functions. Our experiments showed that sweet cherry fruit extract is non-toxic in 2D keratinocytes culture as well as non-irritant in 3D skin model. Our results revealed that the extract mediated important pathways for the optimum epidermis function such as cell proliferation, immune and inflammatory response. Conclusion The sweet cherry fruit extracts possesses significant activity in epidermis function without any potential of cytotoxicity or skin irritability, which makes it a rather promising active agent for skincare.

Genome-wide transcriptome analysis is a method that produces important data on plant biology at a systemic level. The lack of understanding of the relationships between proteins and genes in plants, necessitates a further thorough analysis at a proteogenomic level. Recently, our group generated a quantitative proteogenomic atlas of 15 sweet cherry tissues represented by 29,247 genes and 7,584 proteins. The aim in the current study was to perform a targeted analysis at a gene/protein level to assess the structure of their relation, and the biological implications. Weighted correlation network analysis and causal modeling were employed to, respectively, cluster the gene/protein pairs, and reveal their cause–effect relations, aiming to assess the associated biological functions. To the best of our knowledge, this is the first time that causal modeling is employed within the proteogenomics concept in plants. The analysis revealed the complex nature of causal relations among genes/proteins that are important for traits of interest in perennial fruit trees, particularly regarding the fruit softening and ripening process in sweet cherry. Causal discovery could be used to highlight persistent relations at a gene/protein level with stimulating biological interpretation, and facilitate further studying the proteogenomic atlas in plants.

Genome-wide transcriptome analysis provides systems-level insights into plant biology. Due to the limited depth of quantitative proteomics our understanding of gene-protein-complex stoichiometry is largely unknown in plants. Recently, the complexity of the proteome and its cell/tissue specific distribution boost the research community to the integration of transcriptomics and proteomics landscapes in a proteogenomic approach. Herein, we generated a quantitative proteome and transcriptome abundance atlas of 15 major sweet cherry tissues represented by 29,247 genes and 7,584 proteins. Additionally, 199,984 alternative splicing events, particularly exon skipping and alternative 3′ splice site, were identified in 23,383 transcribed regions of the analyzed tissues. Common signatures as well as differences between mRNA and protein quantities, including genes encoding transcription factors and allergens, within and across the different tissues are reported. Using our integrated data set, we identified key putative regulators of fruit development, notably genes involved in the biosynthesis of the anthocyanins and flavonoids. We also provide proteogenomic-based evidence for the involvement of ethylene signaling and pectin degradation in cherry fruit ripening. Moreover, clusters of genes and proteins with similar and different expression and suppression trends across diverse tissues and developmental stages revealed a relatively low RNA abundance-to-protein correlation. The present proteogenomic analysis allows us to identify 17 novel sweet cherry proteins without prior protein-level annotation evidenced in the currently available databases. To facilitate use by the community, we also developed the Sweet Cherry Atlas Database (https://grcherrydb.com/) for viewing and data-mining these resources. This work provides new insights into the proteogenomics workflow in plants and a rich knowledge resource for future investigation of gene and protein functions in Prunus species.

There is a persistent interest in innovative and multifunctional ingredients in biology research. With regards to this, natural sources have an important role due to their multiple benefits. Thus, this study aims to present the pleiotropic activity of Prunus avium L. extract on human primary fibroblasts for proving its efficacy in dermis-related processes. We focused on the safety and efficacy assessments based on cytotoxicity and gene expression analysis under oxidative stress. Specifically, Prunus avium L. extract was proved non-cytotoxic in human fibroblasts. The gene expression analysis unveiled that this extract has in vitro protective properties on human dermal fibroblasts under oxidative stress related to antioxidant activity, anti-inflammatory response, cell proliferation and cell-aging. Our study demonstrated for the very first time that the Prunus avium L. extract is a multifunctional ingredient as it mediates several human dermis-related in vitro processes highlighting its potential to be used as an active ingredient in skin care products.

Sweet cherry germplasm contains a high variety of phenotypes, which are associated to fruit size and shape, sugar content etc. High phenotypic variation can be a result of genetic or epigenetic diversity, that may interact through time. Recent studies provide evidence that besides allelic variation, epi-allelic variation can establish new heritable phenotypes. Herein we conducted a genetic and an epigenetic study (using AFLP and MSAP markers respectively), accompanied with phenotypic traits correlation analysis in sweet cherry gene pools. The mean genetic diversity was higher than epigenetic diversity (hgen = 0.193; hepi = 0.185), whereas no significant relationship was found between genetic and epigenetic distance according to a Mantel test. Furthermore, ac-cording to correlation analyses our results provided evidence that epigenetic diversity in predefined populations of sweet cherry had a stronger impact on phenotypic traits rather than their rich genetic diversity.

The current study characterizes the physicochemical, sensory and bioactive compound traits of twenty-two sweet cherry accessions, namely breeding lines, landraces and modern cultivars, embodying the majority of Greek germplasm. The evaluated accessions differ in several quality traits including colour parameters and textural properties as well as sensory attributes, such as taste intensity and overall acceptance. Significant differences in primary metabolites, including fructose, glucose, sorbitol, malic acid were recorded among tested accessions. All genotypes were rich in polyphenols, primarily in quercetin-3,4-O-diglucoside, esculetin, rutin and neochlorogenic acid. An anthocyanins-related discrimination among accessions was also obtained based on cyanidin-3-O-rutinoside and peonidin glycosides content. Overall, the cultivars ‘Tsolakeika’ and ‘Bakirtzeika’ exhibited the higher consumer acceptance while the cultivars ‘Vasiliadi’ and ‘Tragana Edessis-Naousis’ and especially the breeding line ‘TxAg33’ contained high polyphenol levels. These results represent a valuable resource for future breeding efforts for sweet cherry cultivars with improved nutritional quality traits.

The recent sequencing of many Rosaceae complete genomes, including that of sweet cherry (Prunus avium L.), along with the availability of high-throughput resources offers new challenges and opportunities for cherry breeders in the genomic era towards improvement of climate-smart traits and diseases resistance against the main pathogens, which are consistently plagued the crop. Conventional breeding approaches are laborious, time-consuming and inefficient to fulfil increasing demands, especially in terms of climate change. The advances both in marker-assisted and genomics-assisted breeding, high-throughput sequencing technologies and bioinformatics tools should enable the sweet cherry breeding at a faster pace. These genomics technologies will certainly generate a large amount of data, and this new knowledge might be efficiently employed in cherry breeding towards the development of varieties with elevated adaptation to climatic challenges, including disease resistance against pathogens. The rapidly accumulating genomic resources will enable the development of molecular markers associated with many important quantitative trait loci, deciphering the genomic variations in various germplasms towards the development of climate-smart and disease resistant sweet cherries. Furthermore, an integrated approach based on a full range of plant omics sciences and their outcomes would result in the development of efficient genomics-based trait selection and identification of allelic variations involved in flowering time, dormancy and defence reactions against pathogens. Especially climate change alters dramatically the susceptibility of sweet cherry cultivars to rapidly evolved pathogens, and although the recent advances in genomics resources, there are still only a few reports of genomics applications for diseases resistance evaluation in germplasm collections. In this chapter, we discuss and summarize the advances through genomics-assisted breeding towards improvement of climate-smart traits and diseases resistance in sweet cherry.

Sweet cherries, Prunus avium L. (Rosaceae), are gaining importance due to their perenniallity and nutritional attributes beneficial for human health. Interestingly, sweet cherry cultivars exhibit a wide range of phenotypic diversity in important agronomic traits, such as flowering time and defense reactions against pathogens. In this study, whole-genome resequencing (WGRS) was employed to characterize genetic variation, population structure and allelic variants in a panel of 20 sweet cherry and one wild cherry genotypes, embodying the majority of cultivated Greek germplasm and a representative of a local wild cherry elite phenotype. The 21 genotypes were sequenced in an average depth of coverage of 33.91x. and effective mapping depth, to the genomic reference sequence of ‘Satonishiki’ cultivar, between 22.21X to 36.62X. Discriminant analysis of principal components (DAPC) with SNPs revealed two clusters of genotypes. There was a rapid linkage disequilibrium decay, as the majority of SNP pairs with r2 in near complete disequilibrium (>0.8) were found at physical distances less than 10kb. Functional analysis of the variants showed that the genomic ratio of non-synonymous/synonymous (dN/dS) changes was 1.78. The higher dN frequency in the Greek cohort of sweet cherry could be the result of artificial selection pressure imposed by breeding, in combination with the vegetative propagation of domesticated cultivars through grafting. The majority of SNPs with high impact (e.g., stop codon gaining, frameshift), were identified in genes involved in flowering time, dormancy and defense reactions against pathogens, providing promising resources for future breeding programs. Our study has established the foundation for further large scale characterization of sweet cherry germplasm, enabling breeders to incorporate diverse germplasm and allelic variants to fine tune flowering and maturity time and disease resistance in sweet cherry cultivars.

An increasing demand for cherry production (Prunus avium L.) in Greece led to the development of new high quality sweet cherry cultivars. Self-incompatibility in cherry is one of the most challenging issues for the species' cultivation and top breeding priority. Τhe present study focuses on the development of new hybrids with improved traits such as productivity, fruit size, organoleptic characteristics and self compatibility. For this purpose, thirty different cultivars were crossed and produced hybrids that were evaluated according to 34 morpho-physiological characteristics. The results were analyzed using the XLSTAT (version 2014.1) software and a dendrogram was constructed using the Agglomerative Hierarchical Clustering (AHC) method. Optimal hybrid clustering was achieved when characteristics of great economic importance such as fruit shape and size, growth habit and days to blooming were included in the analysis. Based on the results, new sweet cherry hybrids with the special character of self-compatibility were developed. Our findings provide crucial new information for sweet cherry future breeding programs and cultivation.