The first complete mitochondrial genome of Grossulariaceae: Molecular features, structure recombination, and genetic evolution.

BACKGROUND: Mitochondria play crucial roles in the growth, development, and adaptation of plants. Blackcurrant (Ribes nigrum L.) stands out as a significant berry species due to its rich nutritional profile, medicinal properties, and health benefits. Despite its importance, the mitochondrial genome of blackcurrant remains unassembled. RESULTS: This study presents the first assembly of the mitochondrial genome of R. nigrum in the Grossulariaceae family. The genome spans 450,227 base pairs (bp) and encompasses 39 protein-coding genes (PCGs), 19 transfer RNAs (tRNAs), and three ribosomal RNAs (rRNAs). Protein-coding regions constitute 8.88% of the entire genome. Additionally, we identified 180 simple sequence repeats, 12 tandem repeats, and 432 pairs of dispersed repeats. Notably, the dispersed sequence R1 (cotig3, 1,129 bp) mediated genome recombination, resulting in the formation of two major conformations, namely master and double circles. Furthermore, we identified 731 C-to-U RNA editing sites within the PCGs. Among these, cox1-2, nad1-2, and nad4L-2 were associated with the creation of start codons, whereas atp6-718 and rps10-391 were linked to termination codons. We also detected fourteen plastome fragments within the mitogenome, constituting 1.11% of the total length. Phylogenetic analysis suggests that R. nigrum might have undergone multiple genomic reorganization and/or gene transfer events, resulting in the loss of two PCGs (rps2 and rps11) during its evolutionary history. CONCLUSIONS: This investigation unveils the molecular characteristics of the R. nigrum mitogenome, shedding light on its evolutionary trajectory and phylogenetic implications. Furthermore, it serves as a valuable reference for evolutionary research and germplasm identification within the genus.

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.

Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit.

INTRODUCTION: Blackcurrant (Ribes nigrum L.) is an excellent example of a "super fruit" with potential health benefits. Both genotype and cultivation environment are known to affect the chemical composition of blackcurrant, especially ascorbic acid and various phenolic compounds. Environmental conditions, like temperature, solar radiation and precipitation can also have significant impact on fruit chemical composition. The relevance of the study is further accentuated by the predicted and ongoing changes in global climate. OBJECTIVES: The aim of the present study was to provide new knowledge and a deeper understanding of the effects of post flowering environmental conditions, namely temperature and day length, on fruit quality and chemical composition of blackcurrant using an untargeted high performance liquid chromatography-photo diode array-mass spectrometry (HPLC-PDA-MS) metabolomics approach. METHODS: A phytotron experiment with cultivation of single-stemmed potted plants of blackcurrant cv. Narve Viking was conducted using constant temperatures of 12, 18 or 24 °C and three different photoperiods (short day, short day with night interruption, and natural summer daylight conditions). Plants were also grown under ambient outdoor conditions. Ripe berries were analysed using an untargeted HPLC-PDA-MS metabolomics approach to detect the presence and concentration of molecules as affected by controlled climatic factors. RESULTS: The untargeted metabolomics dataset contained a total of 7274 deconvolved retention time-m/z pairs across both electrospray ionisation (ESI) positive and negative polarities, from which 549 metabolites were identified or minimally annotated based upon accurate mass MS. Conventional principal component analysis (PCA) in combination with the Friedman significance test were applied to first identify which metabolites responded to temperature in a linear fashion. Multi-block hierarchical PCA in combination with the Friedman significance test was secondly applied to identify metabolites that were responsive to different day length conditions. Temperature had significant effect on a total of 365 metabolites representing a diverse range of chemical classes. It was observed that ripening of the blackcurrant berries under ambient conditions, compared to controlled conditions, resulted in an increased accumulation of 34 annotated metabolites, mainly anthocyanins and flavonoids. 18 metabolites were found to be regulated differentially under the different daylength conditions. Moreover, based upon the most abundant anthocyanins, a comparison between targeted and untargeted analyses, revealed a close convergence of the two analytical methods. Therefore, the study not just illustrates the value of non-targeted metabolomics approaches with respect to the huge diversity and numbers of significantly changed metabolites detected (and which would be missed by conventional targeted analyses), but also shows the validity of the non-targeted approach with respect to its precision compared to targeted analyses. CONCLUSIONS: Blackcurrant maturation under controlled ambient conditions revealed a number of insightful relationships between environment and chemical composition of the fruit. A prominent reduction of the most abundant anthocyanins under the highest temperature treatments indicated that blackcurrant berries in general may accumulate lower total anthocyanins in years with extreme hot summer conditions. HPLC-PDA-MS metabolomics is an excellent method for broad analysis of chemical composition of berries rich in phenolic compounds. Moreover, the experiment in controlled phytotron conditions provided additional knowledge concerning plant interactions with the environment.

Molecular characterization and detection of a new closterovirus identified from blackcurrant by high-throughput sequencing.

Two large contigs with high sequence similarities to several closteroviruses were identified by high-throughput sequencing from a blackcurrant plant. The complete genome of this new virus was determined to be 17,320 nucleotides. Its genome contains ten open reading frames (ORF) that include, in the 5'-3' direction, a large ORF encoding a putative viral polyprotein (ORF 1a) and nine ORFs that encode RNA-dependent RNA polymerase (RdRp, ORF 1b), p6 (ORF 2), heat shock protein 70-like protein (Hsp70h, ORF 3), Hsp-90-like protein (p61, ORF 4), CP minor (ORF 5), CP (ORF 6), p17 (ORF 7), p11 (ORF 8), and p26 (ORF 9), respectively. BCCV-1 shares nucleotide sequence identities of 43-45% with other 9 closteroviruses at genome sequences. The amino acid sequence identities between BCCV-1 and the closteroviruses were 49-55% (RdRp), 37-41% (Hsp70h), 19-33% (p61), 26-38% (CPm), and 19-28% (CP), respectively. Phylogenetic analysis of Hsp70h sequences placed the new virus with members of genus Closterovirus in the same group. The results indicate that this new virus, which is provisionally named as Blackcurrant closterovirus 1, should represent a new species of the genus Closterovirus. A RT-PCR was developed and used to detect BCCV-1 in more germplasm accessions of Ribes spp.

Polyphenol metabolism in differently colored cultivars of red currant (Ribes rubrum L.) through fruit ripening.

MAIN CONCLUSION: Rare red currants colors caused by low anthocyanin content in the pink and a lack of anthocyanins in the white cultivar correlated with low ANS gene expression, enzyme activity, and increased sugar/acid ratios. Changes in the contents of polyphenols, sugars, and organic acids in berries of the three differently colored Ribes rubrum L. cultivars ('Jonkheer van Tets', 'Pink Champagne', and 'Zitavia') were determined by LC-MS and HPLC at 4 sampling times during the last month of fruit ripening. The activities of the main flavonoid enzymes, chalcone synthase/chalcone isomerase (CHS/CHI), flavanone 3-hydroxylase (FHT), and dihydroflavonol 4-reductase (DFR), and the expression of anthocyanidin synthase (ANS) were additionally measured. Despite many attempts, activities of flavonol synthase and glycosyltransferase did not show reliable results, the reason of which they could not be demonstrated in this study. The pink fruited cultivar 'Pink Champagne' showed generally lower enzyme activity than the red cultivar 'Jonkheer van Tets'. The white cultivar 'Zitavia' showed very low CHS/CHI activity and ANS expression and no FHT and DFR activities were detected. The DFR of R. rubrum L. clearly preferred dihydromyricetin as substrate although no 3',4',5'-hydroxylated anthocyanins were present. The anthocyanin content of the red cultivar slightly increased during the last three weeks of ripening and reached a maximum of 890 mg kg-1 FW. Contrary to this, the pink cultivar showed low accumulation of anthocyanins; however, in the last three weeks of ripening, their content increased from 14 to 105 mg kg-1 FW. Simultaneously, the content of polyphenols slightly decreased in all 3 cultivars, while the sugar/acid ratio increased. The white cultivar had no anthocyanins, but the sugar/acid ratios were the highest. In the white and pink cultivars, reduction/lack of anthocyanins was mainly compensated by increased relative concentrations of hydroxycinnamic acids and flavonols.

Ascorbate pool, sugars and organic acids in black currant (Ribes nigrum L.) berries are strongly influenced by genotype and post-flowering temperature.

BACKGROUND: Marked effects of the climatic environment on fruit chemical composition have often been demonstrated in field experiments. However, complex covariations of several climatic factors in the natural environment complicate the interpretation of such experiments and the identification of the causal factors. This can be better achieved in a phytotron where the various climatic factors can be varied systematically. Therefore, we grew four black currant cultivars of contrasting origin in a phytotron under controlled post-flowering temperature and photoperiod conditions and analysed the berries for their ascorbic acid, sugar and organic acid contents. RESULTS: The analyses revealed significant effects of genotype on all investigated compounds. Particularly large cultivar differences were observed in the concentrations of l-ascorbic acid (AA) and sucrose. The concentrations of both AA and dehydroascorbic acid (DHAA), as well as the concentrations of all major sugars, decreased consistently with an increasing temperature over the temperature range 12-24 °C. Fructose and glucose were the predominant sugars with concentrations several fold higher than that for sucrose. AA was the main contributor to the total ascorbate pool in black currant berries. The AA/DHAA ratio varied from 5.6 to 10.3 among the studied cultivars. The concentration of citric acid, which was the predominant organic acid in black currant berries, increased with an increasing temperature, whereas the opposite trend was observed for malic and shikimic acid. Quninic acid was always present at relatively low concentrations. By contrast, photoperiod had no significant effect on berry content of any of the investigated compounds. CONCLUSION: It is concluded that the post-flowering temperature has marked effects on the concentration of important chemical compounds responsible for taste and nutritional value of black currant berries, whereas photoperiod has no such effect in the studied cultivars. © 2016 Society of Chemical Industry.

Cloning of an anthocyanidin synthase gene homolog from blackcurrant (Ribes nigrum L.) and its expression at different fruit stages.

Anthocyanidin synthase (ANS), a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, catalyzes the penultimate step in anthocyanin biosynthesis, from leucoanthocyanidins to anthocyanidins, the first colored compound in the anthocyanin pathway. In this study, a full-length, 1427-bp long cDNA named RnANS1, which is homologous to the anthocyanidin synthase gene, was cloned from blackcurrant using a homologous cloning strategy. RnANS1 is highly homologous to other plant ANS genes at both the nucleotide and amino acid sequence levels. The deduced protein contains domains conserved in the 2OG and Fe(II)-dependent oxygenase, and is phylogenetically closely related to Paeonia suffruticosa and Paeonia lactiflora. The expression of RnANS1 was upregulated during fruit maturation, and correlated with the accumulation of anthocyanins and soluble carbohydrates in the fruit. Further characterization of the structure and expression patterns of RnANS1 will clarify our understanding of anthocyanin biosynthesis in blackcurrant, and support the development of molecular approaches to manipulate anthocyanin production in this plant.

Berry antioxidants: small fruits providing large benefits.

Small berry fruits are consumed because of their attractive colour and special taste, and are considered one of the richest sources of natural antioxidants. Their consumption has been linked to the prevention of some chronic and degenerative diseases. The term 'berry fruits' encompasses the so-called 'soft fruits', primarily strawberry, currants, gooseberry, blackberry, raspberry, blueberry and cranberry. The objective of this review is to highlight the nutraceutical value of berries and to summarize the factors affecting berry fruit antioxidants. Particular attention is given to postharvest and processing operation factors that may affect fruit phytochemical content. The structure-antioxidant relationships for phenolic compounds - the main group of antioxidants in this fruit group - are presented and major areas for future research are identified.

A new strategy for identification of currant (Ribes nigrum L.) cultivars using RAPD markers.

We developed a new approach using RAPD fingerprints to distinguish 37 currant cultivars from northeastern China based on optimization of RAPD by choosing 11 nucleotide primers and strict screening PCR annealing temperature. We found that the manual cultivar identification diagram (MCID) approach clearly developed fingerprints from 8 different primers that were useful for cultivar identification; a cultivar identification diagram (CID) was readily constructed. This CID allows efficient currant cultivar identification, providing information to separate all the currant cultivars from each other, based on the detail polymorphic bands from the corresponding primers, which were marked in the correct positions on the currant CID. According to the CID, 10 currant cultivars in 5 groups were randomly selected for the referable and workable identification of this strategy. The results proved the workability and efficiency of the MCID method, facilitating the identification of fruit cultivars with DNA markers. This MCID approach will be useful for early identification of seedlings in the nursery industry and protection of cultivar rights.

Complete chloroplast genome assembly and phylogenetic analysis of blackcurrant (Ribes nigrum), red and white currant (Ribes rubrum), and gooseberry (Ribes uva-crispa) provide new insights into the phylogeny of Grossulariaceae.

Background: Blackcurrant (Ribes nigrum), red currant (R. rubrum), white currant (R. rubrum), and gooseberry (R. uva-crispa) belong to Grossulariaceae and are popular small-berry crops worldwide. The lack of genomic data has severely limited their systematic classification and molecular breeding. Methods: The complete chloroplast (cp) genomes of these four taxa were assembled for the first time using MGI-DNBSEQ reads, and their genome structures, repeat elements and protein-coding genes were annotated. By genomic comparison of the present four and previous released five Ribes cp genomes, the genomic variations were identified. By phylogenetic analysis based on maximum-likelihood and Bayesian methods, the phylogeny of Grossulariaceae and the infrageneric relationships of the Ribes were revealed. Results: The four cp genomes have lengths ranging from 157,450 to 157,802 bp and 131 shared genes. A total of 3,322 SNPs and 485 Indels were identified from the nine released Ribes cp genomes. Red currant and white currant have 100% identical cp genomes partially supporting the hypothesis that white currant (R. rubrum) is a fruit color variant of red currant (R. rubrum). The most polymorphic genic and intergenic region is ycf1 and trnT-psbD, respectively. The phylogenetic analysis demonstrated the monophyly of Grossulariaceae in Saxifragales and the paraphyletic relationship between Saxifragaceae and Grossulariaceae. Notably, the Grossularia subgenus is well nested within the Ribes subgenus and shows a paraphyletic relationship with the co-ancestor of Calobotrya and Coreosma sections, which challenges the dichotomous subclassification of the Ribes genus based on morphology (subgenus Ribes and subgenus Grossularia). These data, results, and insights lay a foundation for the phylogenetic research and breeding of Ribes species.

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.

[Cloning, structure analysis and functional verification of MYB10 in Ribes L.]

This study aims to investigate the molecular mechanism of the transcription factor MYB10, which is involved in anthocyanin biosynthesis, in different colors of Ribes L. fruitification. Rapid amplification of cDNA ends (RACE) was used to clone the MYB10 genes from Ribes nigrum L. (RnMYB10), Ribes rubrum L. (RrMYB10), and Ribes album L. (RaMYB10), respectively. Phylogenetic analysis showed that RnMYB10 and RrMYB10 were evolutionarily homologous. Real-time quantitative PCR (RT-qPCR) showed that the expression of MYB10 in the fruits of Ribes nigrum L. was higher than that of Ribes rubrum L. and much higher than that of Ribes album L. The expression of RnMYB10 and RrMYB10 increased at first and then decreased as the fruit diameter increased and the fruit color deepened (the maximum expression level was reached at 75% of the fruit color change), while the expression level of RaMYB10 was very low. Overexpression of RnMYB10 and RrMYB10 in Arabidopsis thaliana resulted in purple petioles and leaves, whereas overexpression of RaMYB10 resulted in no significant color changes. This indicates that MYB10 gene plays an important role in the coloration of Ribes L. fruit.

Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog.

In a large-scale screening effort, we identified the gene gooseberry (gsb) as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. Here, we define a new postembryonic role for gooseberry. We show that gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and we present initial evidence that this activity is conserved during synaptic homeostasis. Thus, we have identified a gene (gsb) that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release.

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.

Nontargeted 1 H NMR fingerprinting and multivariate statistical analysis for traceability of Greek PDO Vostizza currants.

In this study, non-targeted 1 H NMR fingerprinting was used in combination with multivariate statistical analyses for the classification of Greek currants based on their geographical origins (Aeghion, Nemea, Kalamata, Zante, and Amaliada). As classification techniques, Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were carried out. To elucidate different components according to PDO (Protected Designation of Origin), products from Aeghion (Vostizza) were statistically compared with each one of the four other regions. PLS-DA plots ensure that currants from Kalamata, Nemea, Zante, and Amaliada are well classified with respect to the PDO currants, according to differences observed in metabolites. Results suggest that composition differences in carbohydrates, amino, and organic acids of currants are sufficient to discriminate them in correlation to their geographical origin. In conclusion, currants metabolites which mostly contribute to classification performance of such discriminant analysis model present a suitable alternative technique for currants traceability. The study results contribute information to the currants' metabolite fingerprinting by NMR spectroscopy and their geographical origin. PRACTICAL APPLICATION: This study presents an analytical approach for a high nutritional value Greek PDO product, Vostizza currant. A further research and implementation of this method in food industry, can be the key to food fraud incidents. Thus, application of this work opens up posibilities to "farm to table" mission.

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.

Identification and functional analysis of the genes encoding Delta6-desaturase from Ribes nigrum.

Gamma-linolenic acid (gamma-linolenic acid, GLA; C18:3 Delta(6, 9, 12)) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Delta(6)-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Delta(6)-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Delta(6)-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Delta(6)-desaturases that could use both alpha-linolenic acids (ALA; C18:3 Delta(9,12,15)) and linoleic acid (LA; C18:2 Delta(9,12)) precursors in vivo, whereas RnD8A/B were Delta(8)-sphingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Delta(6)-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Delta(6,9,12,15)) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Multi-environment QTL mapping in blackcurrant (Ribes nigrum L.) using mixed models.

The first genetic linkage map of blackcurrant, published by Brennan et al. (Euphytica 161:19-34, 2008), identified regions where quantitative trait loci (QTLs) for some important traits were located. The analysis was complicated by the fact that the mapping population was found to contain two subgroups, with segregation ratios consistent with these being crossed and selfed offspring. The QTL analysis was based on the trait mean over 3 years and focused on the crossed offspring. Here we proposed a mixed model multi-environment approach for this population. The 3 years are considered as three separate environments, the data from both the selfed and crossed offspring are combined and different residual terms are explored to model the correlation between the years. This permits tests for interactions between the QTLs, the year and the type of offspring (selfed or crossed). This is applied to re-analyse two important traits, anthocyanin concentration and budbreak. Several additional QTLs were identified, some affecting the trait in both the selfed and crossed offspring, others in just one.

Detection of 8-hydroxy-2-deoxyguanosine as a marker of oxidative damage in DNA and germplasm exposed to cryogenic treatments.

An HPLC method has been optimised to measure 8-hydroxy-2-deoxyguanosine (8OHdG) in DNA and germplasm with the objective of using the adduct as a marker of cryostorage stability. The encapsulation-dehydration cryopreservation protocol was adapted as a model system for assessing the formation of 8OHdG from alginate-encapsulated DNA of calf thymus (CT) and currant species (Ribes) exposed to temperatures of -20 and -196 degree C. The presence of H2O2 exacerbated the formation of 8OHdG in encapsulated CT and Ribes DNA. Production of the oxidized adduct was lower in the plant system. A reduction in residual water following osmotic dehydration and evaporative desiccation was associated with reduced adduct formation in encapsulated DNA. No significant differences in 8OHdG adduct formation were observed in plants regenerated from cryopreserved Ribes meristems derived from genotypes known to have differential tolerance to cryopreservation.

Cellular biological activity and regulation of gene expression of antioxidant dietary fibre fraction isolated from blackcurrant incorporated in the wholemeal cereals cookies.

Free and bound phenolics were extracted from the fibre fraction of wholemeal (W) wheat and barley (B) cookies which had been fortified with 15% blackcurrant powder. Blackcurrant enriched cookies contained between 55 and 66 % higher total phenolics respectively compared control cookies. Ferulic acid in wheat and barley cookie extracts, and quercetin and kaempferol-3-glucoside in wheat cookies with 15% blackcurrant were the dominant phenolic acids. Cellular antioxidant activity was higher in samples with blackcurrant inclusion when evaluated in a cancer cell HepG2 model. Inhibition of cell proliferation was lower for the phenolic samples from cookies with blackcurrant addition. These samples suppressed the regulation of inflammatory cytokine IL-1ß (about 3 to 4-fold), IL-6 (about 2-fold) and transcription signalling factor NF-kB (about 2-fold) and showed an up-regulation of the satiety gene NUCB-2/nesfatin-1 (about 4-fold) in compared with control samples.