Dissecting the impact of environment, season and genotype on blackcurrant fruit quality traits.

This work aims to determine the effect of genotype x environment (GxE) interaction that influence blackcurrant (Ribes nigrum) fruit quality. We applied metabolomics-driven analysis on fruits from four cultivars grown in contrasting European-locations over two seasons. By integrating metabolomics and sensory analysis, we also defined specific metabolic signatures associated with consumer acceptance. Our results showed that rainfall is a crucial factor associated with accumulation of delphinidin- and cyanidin-3-O-glucoside, the two mayor blackcurrant pigments meanwhile temperature affects the main organic acid levels which can be decisive for fruit taste. Sensorial analysis showed that increases in terpenoid and acetate ester volatiles were strongly associated with higher appreciation score, while proacacipetalin, a cyanogenic-glycoside, was positively associated to bitter taste. Our results pave the way for the selection of high-quality cultivars and suitable production sites for blackcurrant cultivation.

Quantitative trait loci mapping of polyphenol metabolites in blackcurrant (Ribes nigrum L.).

INTRODUCTION: Commercially, blackcurrants (Ribes nigrum L.) are grown mainly for processing, especially for juice production. They are valued for their high levels of polyphenols, especially anthocyanins, which contribute to their characteristic deep colour, but also as a good source of vitamin C. Recently, evidence has accrued that polyphenols, such as anthocyanins, may have specific human health benefits. OBJECTIVE: The aims of this study were to investigate the genetic control of polyphenols and other key juice processing traits in blackcurrants. METHODS: The levels, over 2 years, of vitamin C, citrate, malate, succinate, total organic acids, total anthocyanins and total phenolics together with 46 mainly polyphenol metabolites were measured in a blackcurrant biparental mapping population. Quantitative trait loci (QTLs) for these traits were mapped onto a high-density SNP linkage map. RESULTS: At least one QTL was detected for each trait, with good consistency between the 2 years. Clusters of QTLs were found on each of the eight linkage groups (LG). For example, QTLs for the major anthocyanidin glucosides, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, co-localised with a QTL for total anthocyanin content on LG3 whereas the major anthocyanidin rutinosides, delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, had QTLs on LG1 and LG2. Many of the QTLs explained a high proportion of the trait variation, with the most significant region, on LG3 at ~ 35 cM, explaining more than 60% of the variation in the coumaroylated metabolites, Cyanidin-coumaroyl-glucose, Delphinidin-coumaroyl-glucose, Kaempferol-coumaroyl-glucose and Myricetin-coumaroyl-glucose. CONCLUSION: The identification of robust QTLs for key polyphenol classes and individual polyphenols in blackcurrant provides great potential for marker-assisted breeding for improved levels of key components.

A Transcript and Metabolite Atlas of Blackcurrant Fruit Development Highlights Hormonal Regulation and Reveals the Role of Key Transcription Factors.

Blackcurrant fruit collected at six stages of development were assessed for changes in gene expression using custom whole transcriptome microarrays and for variation in metabolite content using a combination of liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Principal components analysis demonstrated that fruit development could be clearly defined according to their transcript or metabolite profiles. During early developmental stages, metabolite profiles were dominated by amino acids and tannins, whilst transcript profiles were enriched in functions associated with cell division, anatomical structure morphogenesis and cell wall metabolism. During mid fruit development, fatty acids accumulated and transcript profiles were consistent with seed and embryo development. At the later stages, sugars and anthocyanins accumulated consistent with transcript profiles that were associated with secondary metabolism. Transcript data also indicated active signaling during later stages of fruit development. A targeted analysis of signaling networks revealed a dynamic activation and repression of almost 60 different transcripts encoding transcription factors across the course of fruit development, many of which have been demonstrated as pivotal to controlling such processes in other species. Transcripts associated with cytokinin and gibberellin were highly abundant at early fruit development, whilst those associated with ABA and ethylene tended to be more abundant at later stages. The data presented here provides an insight into fruit development in blackcurrant and provides a foundation for further work in the elucidation of the genetic basis of fruit quality.

Phytochemical Composition and Cytotoxic Effects on Liver Hepatocellular Carcinoma Cells of Different Berries Following a Simulated In Vitro Gastrointestinal Digestion.

Berry fruits are rich in nutrients and polyphenols, providing potential health benefits. Understanding the factors that affect their bioavailability is becoming of utmost importance for evaluating their biological significance and efficacy as functional food. In this study, the phytochemical composition and the total antioxidant capacity of different varieties of five berries (blackberry, blackcurrant, blueberry, raspberry, and strawberry) were evaluated after an in vitro gastrointestinal digestion process. The cultivar of each berry that showed the higher content of total phenols and flavonoids was selected to study its cytotoxic effect on human hepatoma cells. Digestion resulted in a high reduction (p ˂ 0.05) of total phenolic, flavonoid and anthocyanin contents and total antioxidant capacity, in the "IN" samples compared to the "OUT" extracts, which represent the "serum-available" and the "colon-available" fractions, respectively. Incubation of the digested fraction for 24 h didn't exert any effect on cellular viability, while a dose- and time-dependent cytotoxicity was observed after 48 h and 72 h of incubation for all the berries analyzed. Our results suggest that the approach proposed in this work may represent a rapid tool for evaluating and identifying new berries with increased phytochemical bioavailability, highlighting their antiproliferative agents after an in vitro digestion.

Plant Defense against Herbivorous Pests: Exploiting Resistance and Tolerance Traits for Sustainable Crop Protection.

Interactions between plants and insect herbivores are important determinants of plant productivity in managed and natural vegetation. In response to attack, plants have evolved a range of defenses to reduce the threat of injury and loss of productivity. Crop losses from damage caused by arthropod pests can exceed 15% annually. Crop domestication and selection for improved yield and quality can alter the defensive capability of the crop, increasing reliance on artificial crop protection. Sustainable agriculture, however, depends on reduced chemical inputs. There is an urgent need, therefore, to identify plant defensive traits for crop improvement. Plant defense can be divided into resistance and tolerance strategies. Plant traits that confer herbivore resistance typically prevent or reduce herbivore damage through expression of traits that deter pests from settling, attaching to surfaces, feeding and reproducing, or that reduce palatability. Plant tolerance of herbivory involves expression of traits that limit the negative impact of herbivore damage on productivity and yield. Identifying the defensive traits expressed by plants to deter herbivores or limit herbivore damage, and understanding the underlying defense mechanisms, is crucial for crop scientists to exploit plant defensive traits in crop breeding. In this review, we assess the traits and mechanisms underpinning herbivore resistance and tolerance, and conclude that physical defense traits, plant vigor and herbivore-induced plant volatiles show considerable utility in pest control, along with mixed species crops. We highlight emerging approaches for accelerating the identification of plant defensive traits and facilitating their deployment to improve the future sustainability of crop protection.

Chilling requirement of Ribes cultivars.

It is usually thought that adequate winter chill is required for the full flowering of many temperate woody species. This paper investigates the sensitivity of blackcurrant bud burst and flowering to natural weather fluctuations in a temperate maritime climate, and compares a range of chill models that have been proposed for assessing the accumulation of winter chill. Bud break for four contrasting cultivars are compared in an exceptionally cold and in a mild winter in Eastern Scotland. The results confirm the importance of chilling at temperatures lower than 0°C and demonstrate that no single chilling function applies equally to all blackcurrant cultivars. There is a pressing need for further model development to take into account the relationship between chilling temperatures and warming temperatures occurring both during and after the chill accumulation period.

Over-seasons analysis of quantitative trait loci affecting phenolic content and antioxidant capacity in raspberry.

This study examined the total phenol content (TPC) and total anthocyanin content (TAC) in ripe fruit of progeny of a mapping population generated from a cross between the European red raspberry cv. Glen Moy ( Rubus ideaus var. idaeus) and the North American red raspberry cv. Latham ( Rubus ideaus var. strigosus) over five seasons in two different growing environments. Measurements of antioxidant capacity (FRAP and TEAC) were also carried out. TPC was highly correlated with TEAC and FRAP across the entire data set. The subset of anthocyanin content was genotype-dependent but also correlated with TPC, although the proportion of anthocyanin compounds varied between progeny. Quantitative trait locus (QTL) analysis was carried out, and key markers were tested for consistency of effects over sites and years. Four regions, on linkage groups 2, 3, 5, and 6, were identified. These agree with QTLs from a previous study over a single season and indicate that QTL effects were robust over seasons.

Identification, utilisation and mapping of novel transcriptome-based markers from blackcurrant (Ribes nigrum).

BACKGROUND: Deep-level second generation sequencing (2GS) technologies are now being applied to non-model species as a viable and favourable alternative to Sanger sequencing. Large-scale SNP discovery was undertaken in blackcurrant (Ribes nigrum L.) using transcriptome-based 2GS 454 sequencing on the parental genotypes of a reference mapping population, to generate large numbers of novel markers for the construction of a high-density linkage map. RESULTS: Over 700,000 reads were produced, from which a total of 7,000 SNPs were found. A subset of polymorphic SNPs was selected to develop a 384-SNP OPA assay using the Illumina BeadXpress platform. Additionally, the data enabled identification of 3,000 novel EST-SSRs. The selected SNPs and SSRs were validated across diverse Ribes germplasm, including mapping populations and other selected Ribes species.SNP-based maps were developed from two blackcurrant mapping populations, incorporating 48% and 27% of assayed SNPs respectively. A relatively high proportion of visually monomorphic SNPs were investigated further by quantitative trait mapping of theta score outputs from BeadStudio analysis, and this enabled additional SNPs to be placed on the two maps. CONCLUSIONS: The use of 2GS technology for the development of markers is superior to previously described methods, in both numbers of markers and biological informativeness of those markers. Whilst the numbers of reads and assembled contigs were comparable to similar sized studies of other non-model species, here a high proportion of novel genes were discovered across a wide range of putative function and localisation. The potential utility of markers developed using the 2GS approach in downstream breeding applications is discussed.

Candidate genes associated with bud dormancy release in blackcurrant (Ribes nigrum L.).

BACKGROUND: The detrimental effects of mild winter temperatures on the consistency of cropping of blackcurrant (Ribes nigrum L.) in parts of Europe have led to increasing interest in the genetic control of dormancy release in this species. This study examined patterns of gene expression in leaf buds of blackcurrant to identify key differential changes in these profiles around the time of budbreak. RESULTS: Using leaf bud tissue of blackcurrant, a cDNA library was generated as a source of blackcurrant ESTs for construction of a custom microarray, which was used to identify differential gene expression during dormancy release. Gene activity was lowest in early stages of dormancy, increasing to reach a maximum around the time of budbreak. Genes with significantly changing expression profiles were clustered and evidence is provided for the transient activity of genes previously associated with dormancy processes in other species. Expression profiling identified candidate genes which were mapped onto a blackcurrant genetic linkage map containing budbreak-related QTL. Three genes, which putatively encode calmodulin-binding protein, beta tubulin and acetyl CoA carboxylase respectively, were found to co-localise with budbreak QTL. CONCLUSIONS: This study provides insight into the genetic control of dormancy transition in blackcurrant, identifying key changes in gene expression around budbreak. Genetic mapping of ESTs enabled the identification of genes which co-localise with previously-characterised blackcurrant QTL, and it is concluded that these genes have probable roles in release of dormancy and can therefore provide a basis for the development of genetic markers for future breeding deployment.

The development of a PCR-based marker linked to resistance to the blackcurrant gall mite (Cecidophyopsis ribis Acari: Eriophyidae).

Gall mite (Cecidophyopsis ribis) is the most serious pest of blackcurrant (Ribes nigrum L.), causing the damaging condition known as 'big bud' and also transmitting blackcurrant reversion virus (BRV) within and between plantations. The identification of resistant germplasm is at present a time-consuming and expensive process, dependent on field infestation plots. Resistance based on gene Ce introgressed from gooseberry has been used in UK breeding programmes for blackcurrant. Using a bulked segregant analysis, 90 AFLP primer combinations were screened and a linkage map constructed around the resistance locus controlled by Ce. Sixteen of the primer combinations produced a fragment in the resistant bulked progeny and the gall mite-resistant parent, but not in the susceptible bulked progeny and parent; subsequent testing on individual progeny identified an AFLP fragment closely linked to gall mite resistance. This fragment, designated E41M88-280, was converted to a PCR-based marker based on sequence-specific primers, amplifying only in resistant individuals. Validation of this marker across a range of susceptible and resistant blackcurrant germplasm with different genetic backgrounds confirmed its reliability in the identification of mite-resistant germplasm containing gene Ce. The conversion of an AFLP fragment to a sequence-based PCR marker simplifies its application and therefore increases its utility for selection of mite-resistant germplasm in high-throughput breeding programmes for blackcurrant.

L-Ascorbic acid accumulation in fruit of Ribes nigrum occurs by in situ biosynthesis via the L-galactose pathway.

Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high vitamin C (l-ascorbic acid, AsA) content. In the present study, a systematic analysis of the mechanism of fruit AsA accumulation was undertaken. AsA accumulation occurred during fruit expansion and was associated with high in situ biosynthetic capacity via the l-galactose pathway and low rates of turnover. Cessation of AsA accumulation was associated with reduced biosynthesis and increased turnover. Translocation of AsA from photosynthetic or vegetative tissues contributed little to fruit AsA accumulation. Manipulation of substrate availability by defoliation had no effect on fruit AsA concentration but significantly reduced fruit yields. Supply of the AsA precursor l-galactono-1,4-lactone to intact, attached fruit transiently increased fruit AsA concentration which rapidly returned to control levels after removal of the compound. These data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.

Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds.

Bud break in raspberry (Rubus idaeus L.) is often poor and uneven, with many of the subapical buds remaining in a dormant state. In order to determine the dormancy status of raspberry buds, an empirical measure of bud burst in a growth-permissive environment following exposure to chilling (4 degrees C cold storage) was developed. For cv. Glen Ample, percentage bud burst in intact canes and isolated nodes was recorded after 14 d. Isolated nodes (a measure of endodormancy) achieved 100% bud burst after approximately 1500 h chilling whereas buds on intact plants (combined endo- and paradormancy) required an additional 1000 h chilling. A microarray approach was used to follow changes in gene expression that occurred during dormancy transition. The probes for the microarrays were obtained from endodormant and paradormant raspberry bud cDNA libraries. The expression profiles of 5300 clones from these libraries were subjected to principal component analysis to determine the most significant expression patterns. Sequence analysis of these clones, in many cases, enabled their functional categorization and the development of hypotheses concerning the mechanisms of bud dormancy release. Thus a set of novel candidates for key dormancy-related genes from raspberry buds have been identified. Bud dormancy is fundamental to the study of plant developmental processes and, in addition, its regulation is of significant economic importance to fruit and horticultural industries.

A high-throughput monolithic HPLC method for rapid vitamin C phenotyping of berry fruit.

A rapid method for the quantification of L-ascorbic acid (1) in berry fruit by HPLC with photodiode array detection is presented. L-Ascorbic acid was resolved on a C18 monolithic column with aqueous buffer, after which the column was washed with acetonitrile to remove lipophilic compounds prior to re-equilibration for analysis of the next sample. Using the monolithic column format with high mobile phase flow rates, the entire separation, wash and re-equilibration were achieved in 3 min. With the exception of gooseberry (Ribes uva-crispa), for which an interfering compound co-eluted, concentrations of 1 could be determined in a wide range of berry fruits after extraction in metaphosphoric acid without further sample preparation. Using this extraction method, recoveries of 1 in excess of 85% were achieved. Fruit or juice extracts were stable in 5% metaphosphoric acid for at least 4 h and stability could be extended to longer than 150 h by the addition of the reducing agent tris(2-carboxethyl)phosphine hydrochloride. Following validation, the method was utilised for the phenotyping of fruit in a Scottish Crop Research Institute (SCRI) Ribes nigrum L. breeding population of 300 individuals. An improved extraction method allowed extraction, quantification of 1 and data analysis to be undertaken in less than one working week.

Anthocyanin-flavanol condensation products from black currant (Ribes nigrum L.).

Putative flavanol-anthocyanin condensation products were detected in a polyphenol-rich concentrate from black currant (Ribes nigrum L.). These compounds had UV-vis spectra similar to those of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, but eluted before all previously described anthocyanins on reversed phase HPLC. Mass spectrometric data indicated that they were rutinoside derivatives of novel aglycons 304 amu greater than delphinidin and cyanidin, respectively. The compounds were partly purified by semipreparative HPLC and gave MS and MS2 spectra consistent with anthocyanin rutinosides covalently linked to epigallocatechin or gallocatechin. These compounds are similar in structure to compounds thought to influence color and quality in red wines and strawberry juice products. There was also evidence for the presence of a range of other flavanol-anthocyanin condensation products. The compounds were present at differing levels in juices of 10 black currant varieties, which were roughly correlated to the content of the parent anthocyanins. The flavanol-anthocyanin products were present in polyphenol-enriched concentrates obtained by solid phase extraction, in commercially produced concentrates, and in fresh extracts of black currants. This suggests that the compounds were not artifacts formed during concentration or purification. However, differences in their comparative contents may be related to the lability of the parent anthocyanins during processing. Although present at low levels, the flavanol-anthocyanin products may influence color or quality parameters of black currant juices, and they may confer enhanced stability to the biological activities reported for their anthocyanin parents.

Fatty acid content and juice characteristics in black currant (Ribes nigrum L.) genotypes.

The fatty acid compositions of seeds from 29 black currant genotypes were determined using a rapid small-scale procedure. There was interest in alpha-linolenic, stearidonic, and, especially, gamma-linolenic acid (GLA) contents, and most samples showed values between 11.1 and 18.7%, between 2.5 and 4.5%, and between 11.6 and 17.4%, respectively. However, six genotypes exhibited gamma-linolenic contents >18%, and values >20% were recorded in four of these genotypes. The fatty acid contents of the six genotypes were also analyzed by using a conventional procedure, and only slight differences in fatty acid composition were found between the two methods. Although GLA content was not strongly correlated with juice parameters, some genotypes had both high GLA contents and desirable juice characteristics. The results obtained provide evidence that it is possible to select for GLA contents without negatively affecting juice quality, and both aspects can be combined in a single cultivar, thereby increasing the added value of the whole fruit.