Strawberry phenotypic plasticity in flowering time is driven by interaction between genetic loci and temperature.

The flowering time (FT), which determines when fruits or seeds can be harvested, is subject to phenotypic plasticity, i.e. the ability of a genotype to display different phenotypes in response to environmental variations. Here, we investigated how the environment affects the genetic architecture of FT in cultivated strawberry (Fragaria ×ananassa) and modifies its QTL effects. To this end, we used a bi-parental segregating population grown for two years at widely divergent latitudes (5 European countries) and combined climatic variables with genomic data (Affymetrix® SNP array). Examination, using different phenological models, of the response of FT to photoperiod, temperature and global radiation, indicated that temperature is the main driver of FT in strawberry. We next characterized in the segregating population the phenotypic plasticity of FT by using three statistical approaches that generated plasticity parameters including reaction norm parameters. We detected 25 FT QTL summarized into 10 unique QTL. Mean values and plasticity parameters QTL were co-localized in three of them, including the major 6D_M QTL whose effect is strongly modulated by temperature. The design and validation of a genetic marker for the 6D_M QTL offers great potential for breeding programs, for example for selecting early-flowering strawberry varieties well adapted to different environmental conditions.

Towards smart and sustainable development of modern berry cultivars in Europe.

Fresh berries are a popular and important component of the human diet. The demand for high-quality berries and sustainable production methods is increasing globally, challenging breeders to develop modern berry cultivars that fulfill all desired characteristics. Since 1994, research projects have characterized genetic resources, developed modern tools for high-throughput screening, and published data in publicly available repositories. However, the key findings of different disciplines are rarely linked together, and only a limited range of traits and genotypes has been investigated. The Horizon2020 project BreedingValue will address these challenges by studying a broader panel of strawberry, raspberry and blueberry genotypes in detail, in order to recover the lost genetic diversity that has limited the aroma and flavor intensity of recent cultivars. We will combine metabolic analysis with sensory panel tests and surveys to identify the key components of taste, flavor and aroma in berries across Europe, leading to a high-resolution map of quality requirements for future berry cultivars. Traits linked to berry yields and the effect of environmental stress will be investigated using modern image analysis methods and modeling. We will also use genetic analysis to determine the genetic basis of complex traits for the development and optimization of modern breeding technologies, such as molecular marker arrays, genomic selection and genome-wide association studies. Finally, the results, raw data and metadata will be made publicly available on the open platform Germinate in order to meet FAIR data principles and provide the basis for sustainable research in the future.

Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit.

The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsy-transposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

Contrasting dynamics in abscisic acid metabolism in different Fragaria spp. during fruit ripening and identification of the enzymes involved.

Abscisic acid (ABA) is a key hormone in non-climacteric Fragaria spp, regulating multiple physiological processes throughout fruit ripening. Its concentration increases during ripening, and it promotes fruit (receptacle) development. However, its metabolism in the fruit is largely unknown. We analyzed the concentrations of ABA and its catabolites at different developmental stages of strawberry ripening in diploid and octoploid genotypes and identified two functional ABA-glucosyltransferases (FvUGT71A49 and FvUGT73AC3) and two regiospecific ABA-8'-hydroxylases (FaCYP707A4a and FaCYP707A1/3). ABA-glucose ester content increased during ripening in diploid F. vesca varieties but decreased in octoploid F.×ananassa. Dihydrophaseic acid content increased throughout ripening in all analyzed receptacles, while 7'-hydroxy-ABA and neo-phaseic acid did not show significant changes during ripening. In the studied F. vesca varieties, the receptacle seems to be the main tissue for ABA metabolism, as the concentration of ABA and its metabolites in the receptacle was generally 100 times higher than in achenes. The accumulation patterns of ABA catabolites and transcriptomic data from the literature show that all strawberry fruits produce and metabolize considerable amounts of the plant hormone ABA during ripening, which is therefore a conserved process, but also illustrate the diversity of this metabolic pathway which is species, variety, and tissue dependent.

Variation in Volatile Organic Compounds in native, synanthropic accessions and cultivars of the musk strawberry (Fragaria moschata Duchesne ex Weston).

Prior to the world-wide dominance of F. ×ananassa in strawberry production, native species had been cultivated in European gardens for centuries. Especially the musk strawberry (F. moschata) had been highly appreciated due to its fruit size and extraordinary aroma. Detailed studies on the diversity of the species' fruit aroma are lacking, although breeding aims to improve strawberry aroma by complex crossings during recent years. Today a few cultivars, abandoned synanthropic occurrences and native populations of this species exist in Germany. Here we characterised aroma profiles of F. moschata accessions by analysing Volatile Organic Compounds. In particular, differences between native and cultivated accessions as well as the diversity in aroma profiles of native populations were investigated. Profiles of Volatile Organic Compounds were analysed by immersion stir bar sorptive extraction-gas chromatography-quadrupol mass spectrometry (imm-SBSE-GC-qMS). These data were compared with a genetic characterisation of samples based on eight microsatellite loci using univariate and multivariate statistical analyses. High amounts of furanones and the key compound methyl anthranilate were characteristic for the aroma profile of F. moschata. We detected a considerable diversity of Volatile Organic Compounds among accessions of F. moschata, particularly among genetically distinct samples from the same population. Native accessions contained more terpenoids but less esters and were moderately differentiated from cultivated samples. The observed patterns of Volatile Organic Compounds indicate that cultivated accessions had been selected for favourable aroma profiles and thus showing traces of domestication. Moreover, native populations harbour a great diversity of Volatile Organic Compounds, which could be also considered for future breeding efforts.

QTL analysis for ascorbic acid content in strawberry fruit reveals a complex genetic architecture and association with GDP-L-galactose phosphorylase.

Strawberry (Fragaria × ananassa) fruits are an excellent source of L-ascorbic acid (AsA), a powerful antioxidant for plants and humans. Identifying the genetic components underlying AsA accumulation is crucial for enhancing strawberry nutritional quality. Here, we unravel the genetic architecture of AsA accumulation using an F1 population derived from parental lines 'Candonga' and 'Senga Sengana', adapted to distinct Southern and Northern European areas. To account for environmental effects, the F1 and parental lines were grown and phenotyped in five locations across Europe (France, Germany, Italy, Poland and Spain). Fruit AsA content displayed normal distribution typical of quantitative traits and ranged five-fold, with significant differences among genotypes and environments. AsA content in each country and the average in all of them was used in combination with 6,974 markers for quantitative trait locus (QTL) analysis. Environmentally stable QTLs for AsA content were detected in linkage group (LG) 3A, LG 5A, LG 5B, LG 6B and LG 7C. Candidate genes were identified within stable QTL intervals and expression analysis in lines with contrasting AsA content suggested that GDP-L-Galactose Phosphorylase FaGGP(3A), and the chloroplast-located AsA transporter gene FaPHT4;4(7C) might be the underlying genetic factors for QTLs on LG 3A and 7C, respectively. We show that recessive alleles of FaGGP(3A) inherited from both parental lines increase fruit AsA content. Furthermore, expression of FaGGP(3A) was two-fold higher in lines with high AsA. Markers here identified represent a useful resource for efficient selection of new strawberry cultivars with increased AsA content.

Natural Variation in the Control of Flowering and Shoot Architecture in Diploid Fragaria Species.

In perennial fruit and berry crops of the Rosaceae family, flower initiation occurs in late summer or autumn after downregulation of a strong repressor TERMINAL FLOWER1 (TFL1), and flowering and fruiting takes place the following growing season. Rosaceous fruit trees typically form two types of axillary shoots, short flower-bearing shoots called spurs and long shoots that are, respectively, analogous to branch crowns and stolons in strawberry. However, regulation of flowering and shoot architecture differs between species, and environmental and endogenous controlling mechanisms have just started to emerge. In woodland strawberry (Fragaria vesca L.), long days maintain vegetative meristems and promote stolon formation by activating TFL1 and GIBBERELLIN 20-OXIDASE4 (GA20ox4), respectively, while silencing of these factors by short days and cool temperatures induces flowering and branch crown formation. We characterized flowering responses of 14 accessions of seven diploid Fragaria species native to diverse habitats in the northern hemisphere and selected two species with contrasting environmental responses, Fragaria bucharica Losinsk. and Fragaria nilgerrensis Schlecht. ex J. Gay for detailed studies together with Fragaria vesca. Similar to F. vesca, short days at 18°C promoted flowering in F. bucharica, and the species was induced to flower regardless of photoperiod at 11°C after silencing of TFL1. F. nilgerrensis maintained higher TFL1 expression level and likely required cooler temperatures or longer exposure to inductive treatments to flower. We also found that high expression of GA20ox4 was associated with stolon formation in all three species, and its downregulation by short days and cool temperature coincided with branch crown formation in F. vesca and F. nilgerrensis, although the latter did not flower. F. bucharica, in contrast, rarely formed branch crowns, regardless of flowering or GA20ox4 expression level. Our findings highlighted diploid Fragaria species as rich sources of genetic variation controlling flowering and plant architecture, with potential applications in breeding of Rosaceous crops.

Power and Weakness of Repetition - Evaluating the Phylogenetic Signal From Repeatomes in the Family Rosaceae With Two Case Studies From Genera Prone to Polyploidy and Hybridization (Rosa and Fragaria).

Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a "repeatomic fingerprint," and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa, at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete repeatomes with those from ribosomal DNA clusters only, because they represent one of the most widely used barcoding markers. Our results demonstrated that repeatome data contained a clear phylogenetic signal supporting the current subfamilial classification within Rosaceae. Accordingly, the well-accepted major evolutionary lineages within Fragaria were distinguished, and hybrids showed intermediate positions between parental species in data sets retrieved from both complete repeatomes and rDNA clusters. Within the taxonomically more complicated and particularly frequently hybridizing genus Rosa, we detected rather weak phylogenetic signals but surprisingly found a geographic pattern at a population scale. In sum, our method revealed promising results at larger taxonomic scales as well as within taxa with manageable levels of reticulation, but success remained rather taxon specific. Since repeatomes can be technically easy and comparably inexpensively retrieved even from samples of rather poor DNA quality, our phylogenomic method serves as a valuable alternative when high-quality genomes are unavailable, for example, in the case of old museum specimens.

A strawberry accession with elevated methyl anthranilate fruit concentration is naturally resistant to the pest fly Drosophila suzukii.

During the past decade, Drosophila suzukii has established itself as a global invasive fruit pest, enabled by its ability to lay eggs into fresh, ripening fruit. In a previous study, we investigated the impact of different strawberry accessions on the development of D. suzukii eggs, in the search of natural resistance. We identified several accessions that significantly reduced adult fly emergence from infested fruit. In the present study, we aimed at understanding the chemical basis of this effect. We first noted that one of the more resistant accessions showed an unusual enrichment of methyl anthranilate within its fruit, prompting us to investigate this fruit compound as a possible cause limiting fly development. We found that methyl anthranilate alone triggers embryo lethality in a concentration-dependent manner, unlike another comparable organic fruit compound. We also showed that a chemical fraction of the resistant strawberry accession that contains methyl anthranilate carries some activity toward the egg hatching rate. Surprisingly, in spite of the lethal effect of this compound to their eggs, adult females are not only attracted to methyl anthranilate at certain concentrations, but they also display a concentration-dependent preference to lay on substrates enriched in methyl anthranilate. This study demonstrates that methyl anthranilate is a potent agonist molecule against D. suzukii egg development. Its elevated concentration in a specific strawberry accession proven to reduce the fly development may explain, at least in part the fruit resistance. It further illustrates how a single, natural compound, non-toxic to humans could be exploited for biological control of a pest species.

What Do We Know about the Chemistry of Strawberry Aroma?

The strawberry, with its unique aroma, is one of the most popular fruits worldwide. The demand for specific knowledge of metabolism in strawberries is increasing. This knowledge is applicable for genetic studies, plant breeding, resistance research, nutritional science, and the processing industry. The molecular basis of strawberry aroma has been studied for more than 80 years. Thus far, hundreds of volatile organic compounds (VOC) have been identified. The qualitative composition of the strawberry volatilome remains controversial though considerable progress has been made during the past several decades. Between 1997 and 2016, 25 significant analytical studies were published. Qualitative VOC data were harmonized and digitized. In total, 979 VOC were identified, 590 of which were found since 1997. However, 659 VOC (67%) were only listed once (single entries). Interestingly, none of the identified compounds were consistently reported in all of the studies analyzed. The present need of data exchange between "omic" technologies requires high quality and robust metabolic data. Such data are unavailable for the strawberry volatilome thus far. This review discusses the divergence of published data regarding both the biological material and the analytical methods. The VOC extraction method is an essential step that restricts interlaboratory comparability. Finally, standardization of sample preparation and data documentation are suggested to improve consistency for VOC quantification and measurement.

Effect of the Strawberry Genotype, Cultivation and Processing on the Fra a 1 Allergen Content.

Birch pollen allergic patients show cross-reactivity to vegetables and fruits, including strawberries (Fragaria × ananassa). The objective of this study was to quantify the level of the Fra a 1 protein, a Bet v 1-homologous protein in strawberry fruits by a newly developed ELISA, and determine the effect of genotype, cultivation and food processing on the allergen amount. An indirect competitive ELISA using a specific polyclonal anti-Fra a 1.02 antibody was established and revealed high variability in Fra a 1 levels within 20 different genotypes ranging from 0.67 to 3.97 µg/g fresh weight. Mature fruits of red-, white- and yellow-fruited strawberry cultivars showed similar Fra a 1 concentrations. Compared to fresh strawberries, oven and solar-dried fruits contained slightly lower levels due to thermal treatment during processing. SDS-PAGE and Western blot analysis demonstrated degradation of recombinant Fra a 1.02 after prolonged (>10 min) thermal treatment at 99 °C. In conclusion, the genotype strongly determined the Fra a 1 quantity in strawberries and the color of the mature fruits does not relate to the amount of the PR10-protein. Cultivation conditions (organic and conventional farming) do not affect the Fra a 1 level, and seasonal effects were minor.

Great Cause-Small Effect: Undeclared Genetically Engineered Orange Petunias Harbor an Inefficient Dihydroflavonol 4-Reductase.

A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harboring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3'-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g., by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower color. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism-absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes-allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.

White-fruited strawberry genotypes are not per se hypoallergenic.

The strawberry fruit Fra a 1-proteins are homologues of the major birch pollen allergen Bet v 1 and have essential biological functions in pigment formation during fruit ripening. Patients affected by allergy against birch pollen tolerated fruits of a naturally occurring white-fruited F.×ananassa genotype, which showed reduced levels of Fra a 1 proteins along with enzymes of the anthocyanin pigment pathway. We evaluated the cross-reactive allergenic potential of a number of naturally occurring white- and red-fruited strawberry varieties to detect genotypes with low allergenic reactivity, whose fruit might be tolerated by patients with mild allergy. Protein extracts of 51 different strawberry varieties (Fragaria×ananassa, F. vesca, and F. nilgerensis) were screened by Western blot analysis with a polyclonal Fra a 1.02 antibody. Besides, activation of basophils of eight atopic patients allergic to birch pollen were studied using Bet v 1a and different concentrations of 15 selected strawberry protein extracts out of the 51 strawberry genotypes. Median percentages of activated basophils stimulated by extracts from white- and red-fruited genotypes ranged from 36 to 84% and 44 to 76%, respectively indicating that white-fruited strawberry are not per se hypoallergenic. Protein extracts from white-fruited F. vesca cv. Yellow Wonder showed the lowest cross-reactivity but high biological variability. The knowledge about the allergenic potential of different strawberry genotypes may help to improve food safety and can serve as starting point for the development of red-fruited hypoallergenic strawberry cultivars.

Strawberry Accessions with Reduced Drosophila suzukii Emergence From Fruits.

Drosophila suzukii is threatening soft fruit production worldwide due to the females' ability to pierce through the intact skin of ripe fruits and lay eggs inside. Larval consumption and the associated microbial infection cause rapid fruit degradation, thus drastic yield and economic loss. Cultivars that limit the proliferation of flies may be ideal to counter this pest; however, they have not yet been developed or identified. To search for potential breeding material, we investigated the rate of adult D. suzukii emergence from individual fruits (fly emergence) of 107 accessions of Fragaria species that had been exposed to egg-laying D. suzukii females. We found significant variation in fly emergence across strawberries, which correlated with accession and fruit diameter, and to a lesser extent with the strawberry species background. We identified accessions with significantly reduced fly emergence, not explained by their fruit diameter. These accessions constitute valuable breeding material for strawberry cultivars that limit D. suzukii spread.

Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.

Variation in the amounts of selected volatiles in a model population of fragaria × ananassa Duch. As influenced by harvest year.

Volatile metabolites are a basis for sensory and resistance traits of Fragaria × ananassa . Stability of expression is important for the selection of cultivars. For the first time, the stability of volatiles in a strawberry population after cross-combination of two distinct cultivars ('Mieze Schindler' × 'Elsanta') has been investigated. In this work, environmentally caused variations in the synthesis of 18 volatiles were studied over two years using a model population of 158 clones. The stability varied throughout the F1 seedling population between the two years, defining stable and unstable genotypes with respect to volatile synthesis. Most of the stable genotypes exhibited low values in relative volatile concentration. Merely 6 stable volatiles were detected in the parental cultivars, whereas about 40% of the F1 progeny had up to 11 stable volatiles. Consequently, a higher stability in volatile synthesis can be achieved by breeding.