Differential gene expression in Phaseolus vulgaris I locus NILs challenged with Bean common mosaic virus

The Phaseolus vulgaris I locus-Bean common mosaic virus (BCMV; Potyviridae) pathosystem is of critical importance to bean geneticists, breeders and pathologists because of the worldwide distribution of both the virus and germplasm containing this resistance gene. In order to learn more about the molecular responses characteristic of this resistance gene, a cDNA-AFLP screen was conducted on homozygous NILs of P. vulgaris variety ‘Black Turtle Soup' (BT), containing either the I locus allele for resistance (BTII) or susceptibility (BTii) to BCMV. Eight conditions were compared in a factorial analysis: BTII versus BTii; mock inoculated versus BCMV inoculated; 26 versus 34°C. Transcripts induced in response to viral infection and that were further responsive to temperature, genotype or both were isolated and cloned. Sequence analysis of the resultant clones revealed several classes of putative genes, including transcription-related and signal transduction-related genes. Review of disease resistance literature suggests further avenues of research involving the candidates isolated in this scree

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

Computational Analysis of AmpSeq Data for Targeted, High-Throughput Genotyping of Amplicons

Amplicon sequencing (AmpSeq) is a practical, intuitive strategy with a semi-automated computational pipeline for analysis of highly multiplexed PCR-derived sequences. This genotyping platform is particularly cost-effective when multiplexing 96 or more samples with a few amplicons up to thousands of amplicons. Amplicons can target from a single nucleotide to the upper limit of the sequencing platform. The flexibility of AmpSeq’s wet lab methods make it a tool of broad interest for diverse species, and AmpSeq excels in flexibility, high-throughput, low-cost, accuracy, and semi-automated analysis. Here we provide an open science framework procedure to output data out of an AmpSeq project, with an emphasis on the bioinformatics pipeline to generate SNPs, haplotypes and presence/absence variants in a set of diverse genotypes. Open-access tutorial datasets with actual data and a containerization open source software instance are provided to enable training in each of these genotyping applications. The pipelines presented here should be applicable to the analysis of various target-enriched (e.g., amplicon or sequence capture) Illumina sequence data

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.

On the abundance of non-cometary HCN on Jupiter

Using one-dimensional thermochemical/photochemical kinetics and transport models, we examine the chemistry of nitrogen-bearing species in the Jovian troposphere in an attempt to explain the low observational upper limit for HCN. We track the dominant mechanisms for interconversion of N2-NH3 and HCN-NH3 in the deep, hightemperature troposphere and predict the rate-limiting step for the quenching of HCN at cooler tropospheric altitudes. Consistent with other investigations that were based solely on time-scale arguments, our models suggest that transport-induced quenching of thermochemically derived HCN leads to very small predicted mole fractions of hydrogen cyanide in Jupiter's upper troposphere. By the same token, photochemical production of HCN is ineffective in Jupiter's troposphere: CH4-NH3 coupling is inhibited by the physical separation of the CH4 photolysis region in the upper stratosphere from the NH3 photolysis and condensation region in the troposphere, and C2H2-NH3 coupling is inhibited by the low tropospheric abundance of C2H2. The upper limits from infrared and submillimeter observations can be used to place constraints on the production of HCN and other species from lightning and thundershock sources.Comment: 56 pages, 0 tables, 6 figures. Submitted to Faraday Discussions [in press

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

Dissecting foliar physiology and chemical properties with integrated highthroughput phenotyping and molecular markers in grape improvement

An advanced phenotyping protocol using a hyperspectral spectrometer to better understand foliar chemical composition and physiological processes in relation to grapevine yield, quality, biotic and abiotic resistance was initiated with in-house and public spectral resources. The initial result for foliar pigments calibration was promising for classification and measurements to support breeding. We demonstrated the potential of adapting public spectral resources in supporting modern phenotyping in programs with limited resources and when combined with our current effort in deploying marker-assisted selection, the dual innovations provide new information to fast-tracking grapevine research and trait improvement

Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map

Improved efficacy and durability of powdery mildew resistance can be enhanced via knowledge of the genetics of resistance and susceptibility coupled with the development of high-resolution maps to facilitate the stacking of multiple resistance genes and other desirable traits. We studied the inheritance of powdery mildew (Erysiphe necator) resistance and susceptibility of wild Vitis rupestris B38 and cultivated V. vinifera ‘Chardonnay', finding evidence for quantitative variation. Molecular markers were identified using genotyping-by-sequencing, resulting in 16,833 single nucleotide polymorphisms (SNPs) based on alignment to the V. vinifera ‘PN40024' reference genome sequence. With an average density of 36 SNPs/Mbp and uniform coverage of the genome, this 17K set was used to identify 11 SNPs on chromosome 7 associated with a resistance locus from V. rupestris B38 and ten SNPs on chromosome 9 associated with a locus for susceptibility from ‘Chardonnay' using single marker association and linkage disequilibrium analysis. Linkage maps for V. rupestris B38 (1,146 SNPs) and ‘Chardonnay' (1,215 SNPs) were constructed and used to corroborate the ‘Chardonnay' locus named Sen1 (Susceptibility to Erysiphe necator 1), providing the first insight into the genetics of susceptibility to powdery mildew from V. vinifera. The identification of markers associated with a susceptibility locus in a V. vinifera background can be used for negative selection among breeding progenies. This work improves our understanding of the nature of powdery mildew resistance in V. rupestris B38 and ‘Chardonnay', while applying next-generation sequencing tools to advance grapevine genomics and breeding

Toward the Elucidation of Cytoplasmic Diversity in North American Grape Breeding Programs

Plants have an intriguing tripartite genetic system: Nuclear genome 9 Mitochondria 9 Plastids and their interactions may impact germplasm breeding. In grapevine, the study of cytoplasmic genomes has been limited, and their role with respect to grapevine germplasm diversity has yet to be elucidated. In the present study, the results of an analysis of the cytoplasmic diversity among 6073 individuals (comprising cultivars, interspecific hybrids and segregating progenies) are presented. Genotyping by sequencing (GBS) was used to elucidate plastid and mitochondrial DNA sequences, and results were analyzed using multivariate techniques. Single nucleotide polymorphism (SNP) effects were annotated in reference to plastid and mitochondrial genome sequences. The cytoplasmic diversity identified was structured according to synthetic domestication groups (wine and raisin/table gr.ape types) and interspecific-hybridization-driven groups with introgression from North American Vitis species, identifying five cytoplasmic groups and four major clusters. Fifty-two SNP markers were used to describe the diversity of the germplasm. Ten organelle genes showed distinct SNP annotations and effect predictions, of which six were chloroplast-derived and three were mitochondrial genes, in addition to one mitochondrial SNP affecting a nonannotated open reading frame. The results suggest that the application of GBS will aid in the study of cytoplasmic genomes in grapevine, which will enable further studies on the role of cytoplasmic genomes in grapevine germplasm, and then allow the exploitation of these sources of diversity in 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