Transcriptome analysis of grapevine powdery mildew to elucidate the molecular basis of resistance to metrafenone

Grapevine powdery mildew, caused by Erysiphe necator, is a serious disease widespread in all grape growing areas. The pathogen can infect all green parts of the plant, causing the major economic damage on bunches. Metrafenone is a valuable fungicide used in the powdery mildew disease management since 2006 especially in rotation with other chemistries on grapevine and cereals. Its exact mechanism of action is still not known, but based on cross-resistance studies it is different from other fungicides used in powdery mildew management. Early studies on barley and wheat powdery mildews suggest that it may interfere with hyphal morphogenesis, polarised hyphal growth and the establishment and maintenance of cell polarity. Obtaining RNA of a good quality and sufficient quantity from obligate plant pathogens like powdery mildews can be problematic especially due to their tight adherence to the host plant epidermis and low mycelium availability at early developmental stages. Here, we searched for the best methodology for tissue collection and the RNA extraction with the aim to perform RNA transcriptome analysis to compare the expression of E. necator strains sensitive and resistant to metrafenone, identified previously by our research group. The data of gene expression from these different treatments will be compared to identify possible biosynthetic pathways/enzymatic targets involved in the interaction with metrafenone

Natural infection of Run1-positive vines by naïve genotypes of Erysiphe necator

The Run1 locus for dominant resistance to powdery mildew (Erysiphe necator) has been successfully introgressed into Euvitis from Vitis rotundifolia. In the current study, Run1 vines were hybridized with breeding lines at Cornell University, and the presence of the locus was assayed using the markers GLP1-12 and VMC8g9. Signs of powdery mildew were observed on 14 of 113 Run1-positive seedlings in October 2010 in Geneva, N.Y. Severity of infection was lower for Run1-positive than for Run1-negative seedlings. Presence of mature cleistothecia suggested infection by at least two pathogen genotypes, which since V. rotundifolia is not grown within 800+ km of Geneva, N.Y., evolved from a pathogen population naïve to Run1 resistance. Therefore, caution in the deployment of the Run1 locus in new resistant cultivars is suggested so the effectiveness of Run1 does not diminish over time.

Contributions of the VitisGen2 project to grapevine breeding and genetics

The VitisGen projects (2011-2022) have improved the tools available for breeding new grapevine cultivars with regional adaptation, high quality, and disease resistance. VitisGen2 (the second project in the series) was a multi-state collaboration (USDA-Geneva, New York; University of California, Davis; USDA-Parlier, California; Cornell University; Missouri State University; University of Minnesota; South Dakota State University; Washington State University; North Dakota State University; and E&J Gallo, California) to develop improved genetic mapping technology; to identify useful DNA marker-trait associations; and to incorporate marker-assisted selection (MAS) into breeding programs. A novel genetic mapping platform (rhAmpSeq) now provides 2000 + markers that are transferable across the Vitis genus. rhAmpSeq has been used in California, New York, Missouri, and South Dakota to identify new QTL for powdery and downy mildew resistance. In addition, fruit/flower traits that would normally take years to phenotype have been associated with predictive markers accessible from seedling DNA (e.g. malate metabolism, anthocyanin acylation, bloom phenology and flower sex). Since 2011, the project has used MAS to screen thousands of grape seedlings from public breeding programs in the United States and has produced “Ren- Stack” public domain lines to enable simultaneous access to 4 or 6 powdery mildew resistance loci from single source genotypes. High-throughput phenotyping for powdery and downy mildew resistance has been revolutionized with the Blackbird automated-imaging system powered by artificial intelligence for image analysis. Affordable DNA sequencing along with phenotyping innovations are transforming grapevine breeding

Identification of QTLs for berry acid and tannin in a Vitis aestivalis-derived 'Norton'-based population

Acidity and tannins are among the grape berry quality traits that influence wine quality. Despite advantageous environmental tolerances of Vitis aestivalis-derived 'Norton', its acidity and tannin concentrations often deviate from expectations set for V. vinifera. Identification of the genetic determinants of malic acid, tartaric acid, pH, and tannin can assist in the improvement of new hybrid cultivars. For this purpose, a 'Norton' and V. vinifera 'Cabernet Sauvignon' hybrid population containing 223 individuals was used to construct a linkage map containing 384 simple sequence repeat (SSR) and 2,084 genotyping-by-sequencing (GBS)-derived single nucleotide polymorphism (SNP) markers. The resulting map was 1,441.9 cM in length with an average inter-marker distance of 0.75 cM and spanned 19 linkage groups (LGs). Quantitative trait loci (QTLs) were detected for malic acid, tartaric acid, pH, and tannin. QTLs for malic acid (LG 8) and pH (LG 6) were observed across multiple years and explained approximately 17.7% and 18.5% of the phenotypic variation, respectively. Additionally, QTLs for tartaric acid were identified on linkage groups 1, 6, 7, 9, and 17 and tannin on LG 2 in single-year data. The QTLs for tartaric acid explained between 8.8−14.3% and tannin explained 24.7% of the phenotypic variation. The markers linked to these QTLs can be used to improve hybrid cultivar breeding through marker-assisted selection

Haplotyping the Vitis collinear core genome with rhAmpSeq improves marker transferability in a diverse genus

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent

The grapevine QTLome is ripe: QTL survey, databasing, and first applications

Overarching surveys of QTL (Quantitative Trait Loci) studies in both model plants and staple crops have facilitated the access to information and boosted the impact of existing data on plant improvement activities. Today, the grapevine community is ready to take up the challenge of making the wealth of QTL information F.A.I.R.. To ensure that all valuable published data can be used more effectively, the myriad of identified QTLs have to be captured, standardised and stored in a dedicated public database. As an outcome of the GRAPEDIA initiative, QTL-dedicated experts from around the world have gathered to compile the grapevine QTLome: the complete information (e.g., map positions, associated phenotypes) describing all experimentally supported QTLs for a specific trait. This has led to the collection of more than 150 published QTL papers and to the FAIRification of the fields relevant to the grapevine QTL database. A grapevine-QTL frontend application for uploading data has been developed to support QTL curators. For each specific trait, the QTLome will be anchored firstly to the grapevine reference PN40024.T2T(v5) genome/annotation and secondly to the published diverse genome assemblies. The generated “Grapevine QTL browser” will (i) enhance the understanding of the genetic architecture of diverse phenotypes, (ii) reveal consistent QTLs across studies (consensus genomic intervals), which are particularly valuable for marker-assisted breeding, (iii) assist the identification of candidate genes (relevant alleles) and their integration into biological/biotechnological applications. The potential of this resource will be demonstrated by a case stud

Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding

A limited genetic mapping strategy based on simple sequence repeat (SSR) marker data was used with five grape populations segregating for powdery mildew (Erysiphe necator) resistance in an effort to develop genetic markers from multiple sources and enable the pyramiding of resistance loci. Three populations derived their resistance from Muscadinia rotundifolia ‘Magnolia’. The first population (06708) had 97 progeny and was screened with 137 SSR markers from seven chromosomes (4, 7, 9, 12, 13, 15, and 18) that have been reported to be associated with powdery or downy mildew resistance. A genetic map was constructed using the pseudo-testcross strategy and QTL analysis was carried out. Only markers from chromosome 13 and 18 were mapped in the second (04327) and third (06712) populations, which had 47 and 80 progeny, respectively. Significant QTLs for powdery mildew resistance with overlapping genomic regions were identified for different tissue types (leaf, stem, rachis, and berry) on chromosome 18, which distinguishes the resistance in ‘Magnolia’ from that present in other accessions of M. rotundifolia and controlled by the Run1 gene on chromosome 12. The ‘Magnolia’ resistance locus was termed as Run2.1. Powdery mildew resistance was also mapped in a fourth population (08391), which had 255 progeny and resistance from M. rotundifolia ‘Trayshed’. A locus accounting for 50% of the phenotypic variation mapped to chromosome 18 and was named Run2.2. This locus overlapped the region found in the ‘Magnolia’-based populations, but the allele sizes of the flanking markers were different. ‘Trayshed’ and ‘Magnolia’ shared at least one allele for 68% of the tested markers, but alleles of the other 32% of the markers were not shared indicating that the two M. rotundifolia selections were very different. The last population, 08306 with 42 progeny, derived its resistance from a selection Vitis romanetii C166-043. Genetic mapping discovered a major powdery mildew resistance locus termed Ren4 on chromosome 18, which explained 70% of the phenotypic variation in the same region of chromosome 18 found in the two M. rotundifolia resistant accessions. The mapping results indicate that powdery mildew resistance genes from different backgrounds reside on chromosome 18, and that genetic markers can be used as a powerful tool to pyramid these loci and other powdery mildew resistance loci into a single line

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species