Comparative Pangenomic Insights into the Distinct Evolution of Virulence Factors Among Grapevine Trunk Pathogens.

The permanent organs of grapevines (Vitis vinifera L.), like those of other woody perennials, are colonized by various unrelated pathogenic ascomycete fungi secreting cell wall-degrading enzymes and phytotoxic secondary metabolites that contribute to host damage and disease symptoms. Trunk pathogens differ in the symptoms they induce and the extent and speed of damage. Isolates of the same species often display a wide virulence range, even within the same vineyard. This study focuses on Eutypa lata, Neofusicoccum parvum, and Phaeoacremonium minimum, causal agents of Eutypa dieback, Botryosphaeria dieback, and Esca, respectively. We sequenced 50 isolates from viticulture regions worldwide and built nucleotide-level, reference-free pangenomes for each species. Through examination of genomic diversity and pangenome structure, we analyzed intraspecific conservation and variability of putative virulence factors, focusing on functions under positive selection and recent gene family dynamics of contraction and expansion. Our findings reveal contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. For example, carbohydrate active enzymes (CAZymes) were prevalent in the core genomes of each pangenome, whereas biosynthetic gene clusters were prevalent in the dispensable genomes of E. lata and P. minimum. The dispensable fractions were also enriched in Gypsy transposable elements and virulence factors under positive selection (polyketide synthase genes in E. lata and P. minimum, glycosyltransferases in N. parvum). Our findings underscore the complexity of the genomic architecture in each species and provide insights into their adaptive strategies, enhancing our understanding of the underlying mechanisms of virulence. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A super-pangenome of the North American wild grape species.

BACKGROUND: Capturing the genetic diversity of wild relatives is crucial for improving crops because wild species are valuable sources of agronomic traits that are essential to enhance the sustainability and adaptability of domesticated cultivars. Genetic diversity across a genus can be captured in super-pangenomes, which provide a framework for interpreting genomic variations. RESULTS: Here we report the sequencing, assembly, and annotation of nine wild North American grape genomes, which are phased and scaffolded at chromosome scale. We generate a reference-unbiased super-pangenome using pairwise whole-genome alignment methods, revealing the extent of the genomic diversity among wild grape species from sequence to gene level. The pangenome graph captures genomic variation between haplotypes within a species and across the different species, and it accurately assesses the similarity of hybrids to their parents. The species selected to build the pangenome are a great representation of the genus, as illustrated by capturing known allelic variants in the sex-determining region and for Pierce's disease resistance loci. Using pangenome-wide association analysis, we demonstrate the utility of the super-pangenome by effectively mapping short reads from genus-wide samples and identifying loci associated with salt tolerance in natural populations of grapes. CONCLUSIONS: This study highlights how a reference-unbiased super-pangenome can reveal the genetic basis of adaptive traits from wild relatives and accelerate crop breeding research.

A multitiered haplotype strategy to enhance phased assembly and fine mapping of a disease resistance locus.

Fine mapping of quantitative trait loci (QTL) to dissect the genetic basis of traits of interest is essential to modern breeding practice. Here, we employed a multitiered haplotypic marker system to increase fine mapping accuracy by constructing a chromosome-level, haplotype-resolved parental genome, accurate detection of recombination sites, and allele-specific characterization of the transcriptome. In the first tier of this system, we applied the preexisting panel of 2,000 rhAmpSeq core genome markers that is transferable across the entire Vitis genus and provides a genomic resolution of 200 kb to 1 Mb. The second tier consisted of high-density haplotypic markers generated from Illumina skim sequencing data for samples enriched for relevant recombinations, increasing the potential resolution to hundreds of base pairs. We used this approach to dissect a novel Resistance to Plasmopara viticola-33 (RPV33) locus conferring resistance to grapevine downy mildew, narrowing the candidate region to only 0.46 Mb. In the third tier, we used allele-specific RNA-seq analysis to identify a cluster of 3 putative disease resistance RPP13-like protein 2 genes located tandemly in a nonsyntenic insertion as candidates for the disease resistance trait. In addition, combining the rhAmpSeq core genome haplotype markers and skim sequencing-derived high-density haplotype markers enabled chromosomal-level scaffolding and phasing of the grape Vitis × doaniana 'PI 588149' assembly, initially built solely from Pacific Biosciences (PacBio) high-fidelity (HiFi) reads, leading to the correction of 16 large-scale phasing errors. Our mapping strategy integrates high-density, phased genetic information with individual reference genomes to pinpoint the genetic basis of QTLs and will likely be widely adopted in highly heterozygous species.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

HiFi chromosome-scale diploid assemblies of the grape rootstocks 110R, Kober 5BB, and 101-14 Mgt.

Cultivated grapevines are commonly grafted on closely related species to cope with specific biotic and abiotic stress conditions. The three North American Vitis species V. riparia, V. rupestris, and V. berlandieri, are the main species used for breeding grape rootstocks. Here, we report the diploid chromosome-scale assembly of three widely used rootstocks derived from these species: Richter 110 (110R), Kober 5BB, and 101-14 Millardet et de Grasset (Mgt). Draft genomes of the three hybrids were assembled using PacBio HiFi sequences at an average coverage of 53.1 X-fold. Using the tool suite HaploSync, we reconstructed the two sets of nineteen chromosome-scale pseudomolecules for each genome with an average haploid genome size of 494.5 Mbp. Residual haplotype switches were resolved using shared-haplotype information. These three reference genomes represent a valuable resource for studying the genetic basis of grape adaption to biotic and abiotic stresses, and designing trait-associated markers for rootstock breeding programs.

Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

Glutathione S-transferase: a candidate gene for berry color in muscadine grapes (Vitis rotundifolia).

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n = 40) belong to the Muscadinia subgenus of Vitis, while other cultivated grape species belong to the subgenus Euvitis (2n = 38). The muscadine berry color locus was mapped to a 0.8 Mbp region syntenic with chromosome 4 of Vitis vinifera. In this study, we identified glutathione S-transferase4 as a likely candidate gene for anthocyanin transport within the berry color locus. PCR and Kompetitive allele-specific PCR genotyping identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine mutation within the muscadine glutathione S-transferase4 (VrGST4) that differentiated black (CC and CT) from bronze (TT) muscadines in 126 breeding selections, 76 cultivars, and 359 progeny from 3 mapping populations. Anthocyanin profiling on a subset of the progeny indicated a dominant VrGST4 action. VrGST4 was expressed in skins of both black and bronze muscadines at similar levels. While nonsynonymous polymorphisms between black and bronze muscadines were discovered in VrGSTF12, another Type I GST-coding gene in the muscadine color locus, this gene was ruled out as a possible candidate for berry color because RNA sequencing indicated it is not expressed in berry skins at véraison from black or bronze genotypes. These results suggest that the bronze phenotype in muscadines is regulated by a mechanism distinct from the MybA gene cluster responsible for berry color variation in Vitis vinifera.

The grape powdery mildew resistance loci Ren2, Ren3, Ren4D, Ren4U, Run1, Run1.2b, Run2.1, and Run2.2 activate different transcriptional responses to Erysiphe necator.

Multiple grape powdery mildew (PM) genetic resistance (R) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia (Run1, Run1.2b, Run2.1, Run2.2), Vitis cinerea (Ren2), V. romanetii (Ren4D and Ren4U), and the interspecific hybrid Villard blanc (Ren3). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post-inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 +, Ren4D +, and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 +, Ren4D +, and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

Rootstock influences the effect of grapevine leafroll-associated viruses on berry development and metabolism via abscisic acid signalling.

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Transcriptomics Provides a Genetic Signature of Vineyard Site and Offers Insight into Vintage-Independent Inoculated Fermentation Outcomes.

Ribosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur nonrandomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. Here, ribosomal DNA amplicon sequencing from grape musts and RNA sequencing of eukaryotic transcripts from primary fermentations inoculated with the wine yeast Saccharomyces cerevisiae RC212 were used to profile fermentations from 15 vineyards in California and Oregon across two vintages. These data demonstrate that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing correlated with neither transcript abundance from those same organisms within the RNA sequencing data nor gene expression of the inoculated RC212 yeast strain. These data suggest that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing were not active during the primary stage of these inoculated fermentations and were not a major factor in determining RC212 gene expression. However, unique genetic signatures were detected within the ribosomal DNA amplicon and eukaryotic transcriptomic sequencing that were predictive of vineyard site and region. These signatures included S. cerevisiae gene expression patterns linked to nitrogen, sulfur, and thiamine metabolism. These genetic signatures of site offer insight into specific environmental factors to consider with respect to fermentation outcomes and vineyard site and regional wine characteristics.IMPORTANCE The wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. In this work, as in previous studies, it is demonstrated that specific bacteria and fungi are associated with individual wine regions and vineyard sites. However, this work further shows that their presence is not associated with detectable fungal gene expression during the primary fermentation or the expression of specific genes by the inoculate Saccharomyces cerevisiae strain RC212. The detected RC212 gene expression signatures associated with region and vineyard site also allowed the identification of flavor-associated metabolic processes and environmental factors that could impact primary fermentation outcomes. These data offer novel insights into the complexities and subtleties of vineyard-specific inoculated wine fermentation and starting points for future investigations into factors that contribute to regional wine distinctiveness.

Diploid chromosome-scale assembly of the Muscadinia rotundifolia genome supports chromosome fusion and disease resistance gene expansion during Vitis and Muscadinia divergence.

Muscadinia rotundifolia, the muscadine grape, has been cultivated for centuries in the southeastern United States. M. rotundifolia is resistant to many of the pathogens that detrimentally affect Vitis vinifera, the grape species commonly used for winemaking. For this reason, M. rotundifolia is a valuable genetic resource for breeding. Single-molecule real-time reads were combined with optical maps to reconstruct the two haplotypes of each of the 20 M. rotundifolia cv. Trayshed chromosomes. The completeness and accuracy of the assembly were confirmed using a high-density linkage map. Protein-coding genes were annotated using an integrated and comprehensive approach. This included using full-length cDNA sequencing (Iso-Seq) to improve gene structure and hypothetical spliced variant predictions. Our data strongly support that Muscadinia chromosomes 7 and 20 are fused in Vitis and pinpoint the location of the fusion in Cabernet Sauvignon and PN40024 chromosome 7. Disease-related gene numbers in Trayshed and Cabernet Sauvignon were similar, but their clustering locations were different. A dramatic expansion of the Toll/Interleukin-1 Receptor-like Nucleotide-Binding Site Leucine-Rich Repeat (TIR-NBS-LRR) class was detected on Trayshed chromosome 12 at the Resistance to Uncinula necator 1 (RUN1)/Resistance to Plasmopara viticola 1 (RPV1) locus, which confers strong dominant resistance to powdery and downy mildews. A genome browser, annotation, and Blast tool for Trayshed are available at www.grapegenomics.com.

Fungal and bacterial communities of 'Pinot noir' must: effects of vintage, growing region, climate, and basic must chemistry.

BACKGROUND: The geographic and temporal distributions of bacterial and fungal populations are poorly understood within the same wine grape cultivar. In this work, we describe the microbial composition from 'Pinot noir' must with respect to vintage, growing region, climate, and must chemistry across the states of California and Oregon, USA. MATERIALS AND METHODS: We sampled 'Pinot noir' clone 667 clusters from 15 vineyards existing in a latitudinal gradient spanning nearly 1,200 km in California and Oregon for two vintages (2016 and 2017). Regions included five American Viticultural Areas (AVA). In order from southern California to Oregon, these AVAs were Santa Barbara, Monterey, Sonoma, Mendocino, and Willamette Valley. Uninoculated grape musts were subjected to 16S rRNA gene and ITS-1 amplicon sequencing to assess composition of microbial communities. We also measured grape maturity metrics. Finally, to describe regions by precipitation and growing degree days, we queried the Parameter-elevation Regressions on Independent Slopes Model (PRISM) spatial climate dataset. RESULTS: Most of the dominant bacterial taxa in must samples were in the family Enterobacteriaceae, notably the lactic acid bacteria or the acetic acid bacteria groups, but some, like the betaproteobacterial genus Massilia, belonged to groups not commonly found in grape musts. Fungal communities were dominated by Hanseniaspora uvarum (Saccharomycetaceae). We detected relationships between covariates (e.g., vintage, precipitation during the growing season, pH, titratable acidity, and total soluble solids) and bacterial genera Gluconobacter and Tatumella in the family Enterobacteraceae, Sphingomonas (Sphingomonodaceae), Lactobacillus (Lactobacillaceae), and Massilia (Oxalobacteraceae), as well as fungal genera in Hanseniaspora, Kazachstania, Lachancea, Torulaspora in the family Saccharomycetaceae, as well as Alternaria (Pleosporaceae), Erysiphe (Erysiphaceae), and Udeniomyces (Cystofilobasidiaceae). Fungal community distances were significantly correlated with geographic distances, but this was not observed for bacterial communities. Climate varied across regions and vintages, with growing season precipitation ranging from 11 mm to 285 mm and growing degree days ranging from 1,245 to 1,846. DISCUSSION: We determined that (1) bacterial beta diversity is structured by growing season precipitation, (2) fungal beta diversity reflects growing season precipitation and growing degree days, and (3) microbial differential abundances of specific genera vary with vintage, growing season precipitation, and fruit maturity metrics. Further, the correlation between fungal community dissimilarities and geographic distance suggests dispersal limitation and the vineyard as a source for abundant fungal taxa. Contrasting this observation, the lack of correlation between bacterial community dissimilarity and geographic distance suggests that environmental filtering is shaping these communities.

The genetic basis of sex determination in grapes.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

Independent Whole-Genome Duplications Define the Architecture of the Genomes of the Devastating West African Cacao Black Pod Pathogen Phytophthora megakarya and Its Close Relative Phytophthora palmivora.

Phytophthora megakarya and P. palmivora are oomycete pathogens that cause black pod rot of cacao (Theobroma cacao), the most economically important disease on cacao globally. While P. palmivora is a cosmopolitan pathogen, P. megakarya, which is more aggressive on cacao than P. palmivora, has been reported only in West and Central Africa where it has been spreading and devastating cacao farms since the 1950s. In this study, we reconstructed the complete diploid genomes of multiple isolates of both species using single-molecule real-time sequencing. Thirty-one additional genotypes were sequenced to analyze inter- and intra-species genomic diversity. The P. megakarya genome is exceptionally large (222 Mbp) and nearly twice the size of P. palmivora (135 Mbp) and most known Phytophthora species (∼100 Mbp on average). Previous reports pointed toward a whole-genome duplication (WGD) in P. palmivora In this study, we demonstrate that both species underwent independent and relatively recent WGD events. In P. megakarya we identified a unique combination of WGD and large-scale transposable element driven genome expansion, which places this genome in the upper range of Phytophthora genome sizes, as well as effector pools with 1,382 predicted RxLR effectors. Finally, this study provides evidence of adaptive evolution of effectors like RxLRs and Crinklers, and discusses the implications of effector expansion and diversification.

Regulation of monocot and dicot plant development with constitutively active alleles of phytochrome B.

The constitutively active missense allele of Arabidopsis phytochrome B, AtPHYBY276H or AtYHB, encodes a polypeptide that adopts a light-insensitive, physiologically active conformation capable of sustaining photomorphogenesis in darkness. Here, we show that the orthologous OsYHB allele of rice phytochrome B (OsPHYBY283H ) also encodes a dominant "constitutively active" photoreceptor through comparative phenotypic analyses of AtYHB and OsYHB transgenic lines of four eudicot species, Arabidopsis thaliana, Nicotiana tabacum (tobacco), Nicotiana sylvestris and Solanum lycopersicum cv. MicroTom (tomato), and of two monocot species, Oryza sativa ssp. japonica and Brachypodium distachyon. Reciprocal transformation experiments show that the gain-of-function constitutive photomorphogenic (cop) phenotypes by YHB expression are stronger in host plants within the same class than across classes. Our studies also reveal additional YHB-dependent traits in adult plants, which include extreme shade tolerance, both early and late flowering behaviors, delayed leaf senescence, reduced tillering, and even viviparous seed germination. However, the strength of these gain-of-function phenotypes depends on the specific combination of YHB allele and species/cultivar transformed. Flowering and tillering of OsYHB- and OsPHYB-expressing lines of rice Nipponbare and Kitaake cultivars were compared, also revealing differences in YHB/PHYB allele versus genotype interaction on the phenotypic behavior of the two rice cultivars. In view of recent evidence that the regulatory activity of AtYHB is not only light insensitive but also temperature insensitive, selective YHB expression is expected to yield improved agronomic performance of both dicot and monocot crop plant species not possible with wild-type PHYB alleles.

The genomic diversification of grapevine clones.

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

Diploid Genome Assembly of the Wine Grape Carménère.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.

Iso-Seq Allows Genome-Independent Transcriptome Profiling of Grape Berry Development.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding.

BACKGROUND: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology. RESULTS: We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers. CONCLUSIONS: Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.