A major QTL controlling apple skin russeting maps on the linkage group 12 of 'Renetta Grigia di Torriana'.

BACKGROUND: Russeting is a disorder developed by apple fruits that consists of cuticle cracking followed by the replacement of the epidermis by a corky layer that protects the fruit surface from water loss and pathogens. Although influenced by many environmental conditions and orchard management practices, russeting is under genetic control. The difficulty in classifying offspring and consequent variable segregation ratios have led several authors to conclude that more than one genetic determinant could be involved, although some evidence favours a major gene (Ru). RESULTS: In this study we report the mapping of a major genetic russeting determinant on linkage group 12 of apple as inferred from the phenotypic observation in a segregating progeny derived from 'Renetta Grigia di Torriana', the construction of a 20 K Illumina SNP chip based genetic map, and QTL analysis. Recombination analysis in two mapping populations restricted the region of interest to approximately 400 Kb. Of the 58 genes predicted from the Golden Delicious sequence, a putative ABCG family transporter has been identified. Within a small set of russeted cultivars tested with markers of the region, only six showed the same haplotype of 'Renetta Grigia di Torriana'. CONCLUSIONS: A major determinant (Ru_RGT) for russeting development putatively involved in cuticle organization is proposed as a candidate for controlling the trait. SNP and SSR markers tightly co-segregating with the Ru_RGT locus may assist the breeder selection. The observed segregations and the analysis of the 'Renetta Grigia di Torriana' haplotypic region in a panel of russeted and non-russeted cultivars may suggest the presence of other determinants for russeting in apple.

Expression of flavonoid genes in the red grape berry of 'Alicante Bouschet' varies with the histological distribution of anthocyanins and their chemical composition.

The mature berry of Vitis vinifera 'Alicante Bouschet' is entirely red, but anthocyanin metabolism discloses elements of histological discontinuity. This provides an experimental system amenable to studies of compartmentalised secondary metabolism in a fleshly fruit. We compared microscopy of fixed berry sections and chemical composition of anthocyanin extracts with the expression of 41 flavonoid genes in three berry tissues. In the pericarp, anthocyanins formed membrane-encased spherical coalescences that gradually enlarged and were shuttled into the vacuolar system. The size and the intensity of in situ pigmentation and of colour extracts of anthocyanin vesicles all decreased with depth beneath the epidermis. Shades of red colour, and the quantity and types of anthocyanins in skin, flesh, and seed extracts were correlated with differences in the expression of flavonoid 3',5'-hydroxylases and anthocyanin genes encoding transcription factors, enzymatic proteins, and transporters. Fine adjustments in the global transcriptional modulation of the pathway occurred distinctively in each tissue, within four groups of co-expressed genes that were more associated with either the pericarp or the seed, and with either early or late-ripening stages. All structural genes controlling early steps of the flavonoid pathway exist in the grapevine genome in multiple copies that were recruited by antagonistic branches of the pathway in the 'Alicante Bouschet' berry. Expression patterns of individual paralogs were spatiotemporally distinct from one another, in step with either anthocyanin genes or proanthocyanidin genes.

Evaluation of sensitivity and specificity in RNA-Seq-based detection of grapevine viral pathogens.

Virus detection is a crucial step for the implementation of clean stock programs that preserve healthy crop species. Viral infections in grapevine, a vegetatively propagated perennial crop, cannot be eradicated from the vineyards by the application of agrochemicals and must be curtailed at the stage of nursery production during the propagation of planting material. Viral detection is routinely performed using enzyme-linked immunosorbent assays (ELISA) or Reverse Transcription-quantitative Polymerase Chain Reactions (RT-qPCR). High throughput sequencing (HTS) approaches have the potential to detect all viral pathogens in a plant specimen. However, to date, no published HTS-based study has used threshold selection based on ROC curves for discriminating positive from negative samples. To fill this gap, we assessed the specificity and sensitivity of different sequencing and bioinformatics approaches for nine common viruses, which were tested in the same specimens using ELISA and/or RT-qPCR. The normalized detection thresholds giving the best results were 19.28 Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for alignment-based total RNA-Seq approaches, 386 Reads Per Million mapped reads (RPM) for metagenomics-based total RNA-Seq, 1572 FPKM for alignment-based small RNA-Seq analysis and 0.97 % of contigs for de novo analysis of small RNA-Seq data. Validation of the proposed thresholds using independent specimens collected over time from the same stocks and other specimens collected from nearby stocks that had derived from the same propagating material showed that HTS approaches are accurate, with RNA-Seq approaches showing better performance than small RNA-Seq.

Defence responses in Rpv3-dependent resistance to grapevine downy mildew.

The Rpv3 locus determines the ability to operate an isolate-specific hypersensitive response (HR) against Plasmopara viticola in grapevines that carry a resistant Rpv3 (+) haplotype. Artificial infection was performed on leaf discs of Rpv3 (+) and Rpv3 (-) grapevines with two distinct isolates of the pathogen (avrRpv3 (+) and avrRpv3 (-)). The plant response, including the establishment of HR and changes in expression of 33 genes, was compared to the development of the pathogen. HR was induced exclusively in the Rpv3 (+) host upon inoculation with the avrRpv3 (+) isolate of the pathogen, which is assumed to use avrRpv3 (+) effectors that are recognised by/through the plant Rpv3 (+) gene product. The limitation imposed on pathogen growth was the result of inducible responses elicited by the Rpv3 (+)-avrRpv3 (+) interaction. This host reaction relied on transcriptional induction of the HR-associated gene HSR1 and salicylic acid-induced pathogenesis-related (PR) genes PR-1 and PR-2 during the initial 24-48 h post-inoculation. These events had no parallel in the Rpv3 (-) host or upon infection with the avrRpv3 (-) isolate. The emerging model for Rpv3-mediated defence, which is dependent upon race-specific recognition, associated with up-regulation of PR-1 and PR-2 genes, and enforced by localised HR-type necrosis, is compatible with the cascade of events initiated by the products of NB-LRR and LRR-kinase receptor-like genes, such as those residing in the Rpv3 locus.

Differential regulation of triterpene biosynthesis induced by an early failure in cuticle formation in apple.

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of 'Golden Delicious' (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

Expansion and subfunctionalisation of flavonoid 3',5'-hydroxylases in the grapevine lineage.

BACKGROUND: Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant. RESULTS: The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation of F3'5'Hs has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'Hs are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent. CONCLUSIONS: These results suggest that expansion and subfunctionalisation of F3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.

Day Temperature Has a Stronger Effect Than Night Temperature on Anthocyanin and Flavonol Accumulation in 'Merlot' (Vitis vinifera L.) Grapes During Ripening.

Flavonoids impart color and mouthfeel to grapes and wine and are very sensitive to environmental conditions. Growth chamber experiments were performed to investigate the effect of temperature regimes and the differences between day/night temperatures on anthocyanins and flavonols in Merlot grapes. Among the regimes tested, the ones with diurnal 20°C determined the highest levels of anthocyanins and flavonols. Higher diurnal temperatures decreased those levels but increased the proportion of methoxylated and acylated species. When regimes with the same day temperature but different night temperatures were compared, differences between day/night temperatures did not affect anthocyanins, unless a difference of 25°C between day and night temperatures was imposed. When regimes with the same night temperature but different day temperatures were compared, the regime with higher day temperature had a lower anthocyanin level. No relationships were observed between the effects of temperature regimes on anthocyanin level and the expression of key anthocyanin genes. However, the effects on anthocyanin acylation level were consistent with the effects on the acyltransferase expression, and the effects on flavonol level were consistent with the effects on the expression of key flavonol genes. This study indicates that, in Merlot grapes, anthocyanins and flavonols are mostly sensitive to day temperatures.

Historical introgression of the downy mildew resistance gene Rpv12 from the Asian species Vitis amurensis into grapevine varieties.

The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance-a hypersensitive response in leaves challenged with P. viticola-was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12(+) haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12(+) haplotype is shared by 15 varieties, the most ancestral of which are the century-old 'Zarja severa' and 'Michurinets'. Before this knowledge, the chromosome segment around Rpv12(+) became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12(+) has an additive effect with Rpv3(+) to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3(+) plants.