A collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2–14 and 2–15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d’Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7–30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10⁻²⁷ and 9.35 × 10⁻¹² for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided.

Background: Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola. Results: In this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related. Conclusions: Our results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models. Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Individual fingerprinting based on molecular markers has become a popular tool for studies of population genetics and analysis of genetic diversity in germplasm collections, including the solution of synonymy/homonymy and analysis of paternity and kinship.

Background: Grape powdery mildew is caused by the North American native pathogenErysiphe necator. EurasianVitis viniferavarieties were all believed to be susceptible.REN1is the first resistance gene naturally found in cultivated plants ofVitis vinifera.Results: REN1is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance alleleREN1is located. TheREN1interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In theREN1locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that theREN1locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate withREN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central AsianVitis viniferaindicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues againstREN1being infused intoVitis viniferafrom a recent interspecific hybridisation.Conclusion: TheREN1gene resides in an NBS-LRR gene cluster tightly delimited by two flanking SSR markers, which can assist in the selection of this DNA block in breeding betweenVitis viniferacultivars. TheREN1locus has multiple layers of structural complexity compared with its two closely related paralogous NBS clusters, which are located some 5 Mbp upstream and 4 Mbp downstream of theREN1interval on the same chromosome.

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'Hgenes are present in low-copy number, but in grapevine they are highly redundant.Results: The first increase inF3'5'Hcopy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation ofF3'5'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 paleologousF3'5'Hhas been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. DuplicateF3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphanF3'5'Hcopy is highly expressed in vegetative organs. More recent duplicateF3'5'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 incis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in theF3'5'HmRNA pool across different cultivars. MoreF3'5'Hcopies 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%), fewerF3'5'Hcopies 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 ofF3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.