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

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

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

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 Genetic Variation between Obligate Plant Pathogens Pseudoperonospora cubensis and P-humuli Using RNA Sequencing and Genotyping-By-Sequencing

RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) were used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. Twenty isolates of P. cubensis and 19 isolates of P. humuli were genotyped using RNA-seq and GBS. Principle components analysis (PCA) of each data set showed genetic separation between the two species. Additionally, results supported previous findings that P. cubensis isolates from squash are genetically distinct from cucumber and cantaloupe isolates. A PCA-based procedure was used to identify SNPs correlated with the separation of the two species, with 994 and 4,231 PCA-correlated SNPs found within the RNA-seq and GBS data, respectively. The corresponding unigenes (n = 800) containing these potential species-specific SNPs were then annotated and 135 putative pathogenicity genes, including 3 effectors, were identified. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways

A Next-generation Marker Genotyping Platform (AmpSeq) in Heterozygous Crops: A Case Study for Marker-assisted Selection in Grapevine

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates traitassociated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads ( ⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had o10% missing data, and read depth had a median of 220–244 × . Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, lowcost and easily automated analysis

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

Foreword: Special issue on fungal grapevine diseases

An impressively large proportion of fungicides applied in European, North American and Australian agriculture has been used to manage grapevine powdery mildew (Erysiphe necator), grapevine downy mildew (Plasmopara viticola), and botrytis bunch rot (Botrytis cinerea). These fungal and oomycetous plant pathogens remain amongst the most important plant pathological problems worldwide, despite extensive research on pathogenesis, epidemiology and management