RNA-seq-based genome annotation and identification of long-noncoding RNAs in the grapevine cultivar ‘Riesling’

Background: The technological advances of RNA-seq and de novo transcriptome assembly have enabled genome annotation and transcriptome profiling in highly heterozygous species such as grapevine (Vitis vinifera L.). This work is an attempt to utilize a de novo-assembled transcriptome of the V. vinifera cultivar ‘Riesling’ to improve annotation of the grapevine reference genome sequence. Results: Here we show that the transcriptome assembly of a single V. vinifera cultivar is insufficient for a complete genome annotation of the grapevine reference genome constructed from V. vinifera PN40024. Further, we provide evidence that the gene models we identified cannot be completely anchored to the previously published V. vinifera PN40024 gene models. In addition to these findings, we present a computational pipeline for the de novo identification of lncRNAs. Our results demonstrate that, in grapevine, lncRNAs are significantly different from protein coding transcripts in such metrics as length, GC-content, minimum free energy, and length-corrected minimum free energy. Conclusions: In grapevine, high-level heterozygosity necessitates that transcriptome characterization be based on cultivar-specific reference genome sequences. Our results strengthen the hypothesis that lncRNAs have thermodynamically different properties than protein-coding RNAs. The analyses of both coding and non-coding RNAs will be instrumental in uncovering inter-cultivar variation in wild and cultivated grapevine species

Using Focus Groups to Assess Educational Programming Needs in Forestry

Extension professionals are continually faced with the challenge of effectively communicating relevant information to an ever-evolving audience with diverse interests. Using focus group data, this article highlights specific educational programming needs of nonindustrial private forest landowners (NIPFs) in Mississippi. Findings indicate NIPFs are more likely to adopt new ideas if educational programming is tailored to their specific needs, consequently indicating the need to group the audience by their interests. Data also emphasize the importance of employing new technology as means for communicating more efficiently

Economic Impact of a Large-Scale, Collaborative Forest Health Project: A Model for Making a Difference

The U.S. Department of Agriculture Forest Service, Mississippi State University (MSU) Extension, and the Mississippi Forestry Commission partnered on the Southern Pine Beetle Prevention Project, a collaboration on forest health. MSU Extension provided educational outreach to a wide audience of forest landowners and screened applications for the project\u27s tree-thinning cost-share program. From 2006 to 2016, the collaboration spent $4.5 million on educational outreach and cost sharing. Using IMPLAN, we estimated the project\u27s economic contribution to the state at$60.2 million, a value representing a benefit–cost ratio of 13:1. Collaboration is an effective means for agencies to leverage resources, and impact analysis is a useful tool for evaluating Extension program effectiveness

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

Rootstock Effects on Scion Phenotypes in a ‘Chambourcin’ Experimental Vineyard

Understanding how root systems modulate shoot system phenotypes is a fundamental question in plant biology and will be useful in developing resilient agricultural crops. Grafting is a common horticultural practice that joins the roots (rootstock) of one plant to the shoot (scion) of another, providing an excellent method for investigating how these two organ systems affect each other. In this study, we used the French-American hybrid grapevine ‘Chambourcin’ (Vitis L.) as a model to explore the rootstock–scion relationship. We examined leaf shape, ion concentrations, and gene expression in ‘Chambourcin’ grown ungrafted as well as grafted to three different rootstocks (‘SO4’, ‘1103P’ and ‘3309C’) across 2 years and three different irrigation treatments. We found that a significant amount of the variation in leaf shape could be explained by the interaction between rootstock and irrigation. For ion concentrations, the primary source of variation identified was the position of a leaf in a shoot, although rootstock and rootstock by irrigation interaction also explained a significant amount of variation for most ions. Lastly, we found rootstock-specific patterns of gene expression in grafted plants when compared to ungrafted vines. Thus, our work reveals the subtle and complex effect of grafting on ‘Chambourcin’ leaf morphology, ionomics, and gene expression

Berry Anthocyanin, Acid, and Volatile Trait Analyses in a Grapevine-Interspecific F2 Population Using an Integrated GBS and rhAmpSeq Genetic Map

Increased map density and transferability of markers are essential for the genetic analysis of fruit quality and stress tolerance in interspecific grapevine populations. We used 1449 GBS and 2000 rhAmpSeq markers to develop a dense map for an interspecific F2 population (VRS-F2) that was derived by selfing a single F1 from a Vitis riparia x ‘Seyval blanc’ cross. The resultant map contained 2519 markers spanning 1131.3 cM and was highly collinear with the Vitis vinifera ‘PN40024’ genome. Quantitative trait loci (QTL) for berry skin color and flower type were used to validate the map. Four rhAmpSeq transferable markers were identified that can be used in pairs (one pistillate and one hermaphroditic) to predict pistillate and hermaphrodite flower type with ≥99.7% accuracy. Total and individual anthocyanin diglucoside QTL mapped to chromosome 9 near a 5-O-GLUCOSYLTRANSFERASE candidate gene. Malic acid QTL were observed on chromosome 1 and 6 with two MALATE DEHYRDROGENASE CYTOPLASMIC 1 and ALUMINUM-ACTIVATED MALATE TRANSPORTER 2-LIKE (ALMT) candidate genes, respectively. Modeling malic acid identified a potential QTL on chromosome 8 with peak position in proximity of another ALMT. A first-ever reported QTL for the grassy smelling volatile (E)-2-hexenal was found on chromosome 2 with a PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE candidate gene near peak markers

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

Multi-dimensional Leaf Phenotypes Reflect Root System Genotype in Grafted Grapevine Over the Growing Season

Modern biological approaches generate volumes of multi-dimensional data, offering unprecedented opportunities to address biological questions previously beyond reach owing to small or subtle effects. A fundamental question in plant biology is the extent to which below-ground activity in the root system influences above-ground phenotypes expressed in the shoot system. Grafting, an ancient horticultural practice that fuses the root system of one individual (the rootstock) with the shoot system of a second, genetically distinct individual (the scion), is a powerful experimental system to understand below-ground effects on above-ground phenotypes. Previous studies on grafted grapevines have detected rootstock influence on scion phenotypes including physiology and berry chemistry. However, the extent of the rootstock\u27s influence on leaves, the photosynthetic engines of the vine, and how those effects change over the course of a growing season, are still largely unknown

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

A key ‘foxy’ aroma gene is regulated by homologyinduced promoter indels in the iconic juice grape ‘Concord’

‘Concord’, the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special ‘foxy’ aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key ‘foxy’ aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in ‘Concord’ and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the ‘foxy’ aroma trait and makes it genetically trackable and amenable in grapevine breeding