High-density linkage mapping and QTL identification of black rot resistance towards marker-assisted breeding in grapevine

Today sustainability is a pivotal objective for viticulture, but it also presents us with new challenges. The decrease in treatments has in fact changed grapevine-pathogen interactions and dynamics causing the reaffirmation of diseases previously considered secondary. This is the case of black rot (BR, caused by Phyllosticta ampelicida), which is spreading with increasing pressure in warm-humid regions causing the loss of entire crops. The interinstitutional DAMAGE project between Edmund Mach Foundation (FEM) and the Institute for Grapevine Breeding (JKI-Geilweilerhof) aims to characterize BR resistance and to develop a toolkit of molecular markers (MM) to be routinely used in marker-assisted breeding for the introgression of this trait into mildew resistant backgrounds. The first fungal strain was isolated from infected leaves collected in Trentino-Italy and mixed with a second strain isolated in southwestern Germany. This inoculum has been used to screen a segregating population derived from 'Merzling' (V. rupestris × V. lincecumii, mid-resistant) × 'Teroldego' (V. vinifera, susceptible). BR resistance phenotyping consisted of a newly developed inoculation protocol on potted plants. The F1 individuals were genotyped with the Vitis18KSNP chip and a high-density genetic map has been constructed, following the integration of 190 informative SSRs. Moreover, explorative QTL analyzes have been conducted on phenotypic data of 2020 and 2021 seasons. Finally, upon the genomic interval characterization, new MM will be designed, tested and validated on various segregating populations with different genetic backgrounds

Temporal and spatial change of exergy and ascendency in different benthic marine ecosystems

Holistic indicators such as exergy and ascendency have been widely employed to assess the health of ecosystems given by their structure, function and organization. In this study we calculate the exergy, specific exergy and ascendency for the microbenthic loop that represents a major sub-system within the marine food chain. The analysis of the microbenthic loop investigated in terms of organic matter, bacteria, microphytobenthos and meiofauna reflected changes occurring in the trophic state of benthic ecosystems and provided a tool for comparison between different environments. Temporal and spatial variability of the holistic indicators were evaluated using benthic measures collected at different times for different environments in the Mediterranean Sea. Exergy was strongly correlated with the organic contents of the sediments, and did not provide a useful description of the investigated system. In contrast, specific exergy resulted related to the microbenthic loop structure and complexity while Ascendency mostly reflected its activity and organization. Temporal analysis showed that in natural ecosystems specific exergy and ascendency showed convergence and follow similar seasonal trends. On the contrary in strongly eutrophicated systems an uncoupling between the two indicators occurred indicating a malfunctioning of the microbenthic loop that become strongly dissipative.http://www.sciencedirect.com/science/article/B6V2S-4CC7RP3-D/1/49cf1c6b40bddddc9a3bb98c5072e51

Towards Marker-Assisted Breeding for Black Rot Bunch Resistance: Identification of a Major QTL in the Grapevine Cultivar 'Merzling'

Black rot (BR), caused by Guignardia bidwellii, is an emergent fungal disease threatening viticulture and affecting several mildew-tolerant varieties. However, its genetic bases are not fully dissected yet. For this purpose, a segregating population derived from the cross 'Merzling' (hybrid, resistant) × 'Teroldego' (V. vinifera, susceptible) was evaluated for BR resistance at the shoot and bunch level. The progeny was genotyped with the GrapeReSeq Illumina 20K SNPchip, and 7175 SNPs were combined with 194 SSRs to generate a high-density linkage map of 1677 cM. The QTL analysis based on shoot trials confirmed the previously identified Resistance to Guignardia bidwellii (Rgb)1 locus on chromosome 14, which explained up to 29.2% of the phenotypic variance, reducing the genomic interval from 2.4 to 0.7 Mb. Upstream of Rgb1, this study revealed a new QTL explaining up to 79.9% of the variance for bunch resistance, designated Rgb3. The physical region encompassing the two QTLs does not underlie annotated resistance (R)-genes. The Rgb1 locus resulted enriched in genes belonging to phloem dynamics and mitochondrial proton transfer, while Rgb3 presented a cluster of pathogenesis-related Germin-like protein genes, promoters of the programmed cell death. These outcomes suggest a strong involvement of mitochondrial oxidative burst and phloem occlusion in BR resistance mechanisms and provide new molecular tools for grapevine marker-assisted breeding

Rgb3: an organ-specific QTL for bunch resistance to black rot identified in the hybrid cultivar ‘Merzling’ Hands-on demo of the potential of Grapedia

Crops are continuously exposed to the onset of emerging diseases and the exploitation of resistance donors in breeding programs is a widely used valid strategy. In the last two decades black rot (BR), caused by the ascomycete Phyllosticta ampelicida (syn. Guignardia bidwellii), has begun to threaten European viticulture in humid continental areas due to the advent of higher temperatures which favor the pathogenic cycle of the fungus. For this reason, a program has been established for the dissection of BR resistance trait and its introgression both in Vitis vinifera varieties and mildew resistant hybrids. A preliminary study was carried out to improve germplasm screening through different approaches. Historical phenotypic and pedigree information of resistance donors were gathered to support the decision-making process in breeding. A new propagation and inoculation strategy was developed to optimize and fasten inoculation experiments. Isolates of P. ampelicida were genetically characterized and combined for the assessment of resistance independent of race-specificity. Finally, these improvements were employed first for the identification of new BR resistant parental lines and breeding selections, and then for the quantitative trait locus (QTL) analysis in a segregating population derived from the cross ‘Merzling’ (hybrid, resistant) × ‘Teroldego’ (V. vinifera, susceptible). The screening of this progeny, under green-house and field conditions, allowed the discrimination between two distinct organ-specific QTLs on chromosome 14. The previously identified Resistance to G. bidwellii (Rgb)1 locus was confirmed associated with leaf/shoot resistance, while upstream a new QTL designated Rgb3 was discovered linked to bunch resistance. Driven and inspired by the INTEGRAPE community effort that led to GRAPEDIA (GRAPEvine -omics encyclopDIA), all the available tools and resources have been exploited for the exploration of the physical region of the two QTLs, providing a hands-on demo of the potential of this portal

Highly dense linkage mapping and identification of a major QTL associated with BLACK ROT resistance in the grapevine cultivar ‘Merzling’

When the urgency of a sustainable transition comes to agriculture, one of the key areas of improvement involves the decrease of chemicals. Although vineyards cover less than 5% of agricultural land in Europe, viticulture is responsible for the use of more than 60% of all fungicides. The exploitation of grapevine varieties resistant to mildews is an efficient strategy already implemented in integrated/organic farming to reduce treatments. However, from the beginning of this century, European viticulture has been threatened by severe outbreaks of black rot (BR), an emergent and destructive disease caused by the ascomycete Phyllosticta ampelicida (sexual morph Guignardia bidwellii). These events introduced the urgent need for the introgression of BR resistance in mildew-tolerant genotypes. For this purpose, a set of parental lines and breeding selections of the Fondazione Edmund Mach has been screened for BR resistance in a growing chamber with in vivo produced spores using an optimized artificial infection protocol. Given the good performance of ‘Merzling’ (a complex genotype derived from V. vinifera and V. rupestris × V. aestivalis var. lincecumii), this cultivar was used for a cross with the susceptible variety ‘Teroldego’ (V. vinifera) and the segregating offspring was genetically characterized by means of the GrapeReSeq 18K Vitis SNP chip. Five phenotypic experiments were carried out under controlled conditions on leaves of potted plants, and three on bunches in the field. A dense genetic map was constructed combining 7,175 SNP with 194 SSR markers of a previous map. All QTL analyses revealed the presence of a strong major BR resistance locus on chromosome 14. It explains up to 45% of the trait variability (LOD 10.5) and spans a genomic region of 1.36 Mb. A specific SNP marker was found robustly associated with the resistance trait. No minor QTLs were detected. The genes underlying this region are currently under investigation via bioinformatic analysis, and microscopic inspections of disease progression are in place to understand the biological causes of the resistance trait. Finally, new molecular markers will be developed and validated on segregating populations with different genetic backgrounds, to be implemented in marker-assisted selection for BR resistance in grapevin

Secondary and primary metabolites reveal putative resistance-associated biomarkers against Erysiphe necator in resistant grapevine genotypes

Numerous fungicide applications are required to control Erysiphe necator, the causative agent of powdery mildew. This increased demand for cultivars with strong and long-lasting field resistance to diseases and pests. In comparison to the susceptible cultivar ‘Teroldego’, the current study provides information on some promising disease-resistant varieties (mono-locus) carrying one E. necator-resistant locus: BC4 and ‘Kishmish vatkana’, as well as resistant genotypes carrying several E. necator resistant loci (pyramided): ‘Bianca’, F26P92, F13P71, and NY42. A clear picture of the metabolites’ alterations in response to the pathogen is shown by profiling the main and secondary metabolism: primary compounds and lipids; volatile organic compounds and phenolic compounds at 0, 12, and 48 hours after pathogen inoculation. We identified several compounds whose metabolic modulation indicated that resistant plants initiate defense upon pathogen inoculation, which, while similar to the susceptible genotype in some cases, did not imply that the plants were not resistant, but rather that their resistance was modulated at different percentages of metabolite accumulation and with different effect sizes. As a result, we discovered ten up-accumulated metabolites that distinguished resistant from susceptible varieties in response to powdery mildew inoculation, three of which have already been proposed as resistance biomarkers due to their role in activating the plant defense response

Black rot resistance of grapevine: from organ-specific QTL mapping to the sequencing of the donor towards candidate gene identification

Black rot is considered one of the most problematic emerging fungal diseases in Europe due to the increasing pressure favored by climate change, its destructiveness – which can lead up to 80% of fruit loss – and the susceptibility of a large part of the mildew resistant varieties, so-called PIWI. Therefore, in the perspective of the genomic designing of the cultivars of the future, modern breeding has to focus on the combination of multiple beneficial alleles both for disease resistance and abiotic stress resilience. Doubtless, the genetic dissection of the traits of interest is a necessary starting point. To this objective, the segregation of black rot resistance has been studied in a population (N=147) derived from the cross between the resistant Vitis hybrid ‘Merzling’ and the susceptible V. vinifera ‘Teroldego’. The progeny was genotyped with the GrapeReSeq Illumina 20K SNPchip, and 7,175 SNPs were combined with 194 SSRs to generate a high-density linkage map of 1,677 cM. The outcomes uncovered two organ-specific QTLs on chromosome 14. One was associated with the resistance of shoot and co-segregated with the previously mapped Rgb1 locus. A second newly discovered QTL explained almost 70% of the phenotypic variance of bunch resistance and was designated Rgb3. The genes underlying the QTLs have been firstly studied based on the reference vinifera genome, PN40024.v4, revealing the absence of resistance (R)-genes and the enrichment of two clusters of 15 pathogenesis-related Germin-like protein 3, and five phloem Sieve occlusion element type B genes associated with bunch and shoot resistance, respectively. A molecular toolkit has been therefore designed for marker-assisted breeding applications. Finally, to propose reliable candidate genes for biotechnological approaches, the genome of the resistance donor ‘Merzling’ has been sequenced through PacBio HiFi technology. This data has been assembled into a haplotype-resolved and phased genome sequence. Preliminary results will be discussed regarding significant differences with the reference vinifera genome, which confirms the high value of exploring the genomes of hybrids. These findings corroborate the need for the scientific community to engage in building a pangenome to unveil and exploit the genetic variability and informativeness of the highly heterozygous Vitis genus

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V . cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V . cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V . cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V . cholerae and failed to amplify strains of the closely-related species Vibrio mimicus . The sensitivity of the assay applied to environmental and stool samples spiked with V . cholerae ATCC 39315 was comparable to that of pure cultures and was of 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V . cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V . cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies

Whole-genome enrichment provides deep insights into Vibrio cholerae metagenome from an African river

The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (<50 genome unit/L) from natural water collected in the Morogoro river (Tanzania). The protocol is based on the use of biotinylated RNA baits for target enrichment of V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on a Illumina MiSeq platform. Up to 1.8X107 bp (4.5x mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices this study provides a \u201cproof of concept\u201d on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments