Genetic loci associated with tissue-specific resistance to powdery mildew in octoploid strawberry (Fragaria × ananassa).

Powdery mildew is one of the most problematic diseases in strawberry production. To date, few commercial strawberry cultivars are deemed to have complete resistance and as such, an extensive spray programme must be implemented to control the pathogen. Here, a large-scale field experiment was used to determine the powdery mildew resistance status of leaf and fruit tissues across a diverse panel of strawberry genotypes. This phenotypic data was used to identify Quantitative Trait Nucleotides (QTN) associated with tissue-specific powdery mildew resistance. In total, six stable QTN were found to be associated with foliar resistance, with one QTN on chromosome 7D associated with a 61% increase in resistance. In contrast to the foliage results, there were no QTN associated with fruit disease resistance and there was a high level of resistance observed on strawberry fruit, with no genetic correlation observed between fruit and foliar symptoms, indicating a tissue-specific response. Beyond the identification of genetic loci, we also demonstrate that genomic selection can lead to rapid gains in foliar resistance across genotypes, with the potential to capture >50% of the genetic foliage resistance present in the population. To date, breeding of robust powdery mildew resistance in strawberry has been impeded by the quantitative nature of natural resistance and a lack of knowledge relating to the genetic control of the trait. These results address this shortfall, through providing the community with a wealth of information that could be utilized for genomic informed breeding, implementation of which could deliver a natural resistance strategy for combatting powdery mildew.

Dose-Dependent Genetic Resistance to Azole Fungicides Found in the Apple Scab Pathogen.

The evolution of azole resistance in fungal pathogens presents a major challenge in both crop production and human health. Apple orchards across the world are faced with the emergence of azole fungicide resistance in the apple scab pathogen Venturia inaequalis. Target site point mutations observed in this fungus to date cannot fully explain the reduction in sensitivity to azole fungicides. Here, polygenic resistance to tebuconazole was studied across a population of V. inaequalis. Genotyping by sequencing allowed Quantitative Trait Loci (QTLs) mapping to identify the genetic components controlling this fungicide resistance. Dose-dependent genetic resistance was identified, with distinct genetic components contributing to fungicide resistance at different exposure levels. A QTL within linkage group seven explained 65% of the variation in the effective dose required to reduce growth by 50% (ED50). This locus was also involved in resistance at lower fungicide doses (ED10). A second QTL in linkage group one was associated with dose-dependent resistance, explaining 34% of variation at low fungicide doses (ED10), but did not contribute to resistance at higher doses (ED50 and ED90). Within QTL regions, non-synonymous mutations were observed in several ATP-Binding Cassette and Major Facilitator SuperFamily transporter genes. These findings provide insight into the mechanisms of fungicide resistance that have evolved in horticultural pathogens. Identification of resistance gene candidates supports the development of molecular diagnostics to inform management practices.

A Genomic Resource for the Strawberry Powdery Mildew Pathogen Podosphaera aphanis.

Powdery mildew is one of the most economically destructive diseases in protected strawberry production. Here we present the first genome assembly for Podosphaera aphanis, the causal agent of powdery mildew on strawberry. This obligate-biotrophic fungal pathogen was sampled from a naturally occurring outbreak on Fragaria × ananassa 'Malling Centenary' plants grown under cover in the United Kingdom. Assembled reads resolved a 55.6 Mb genome, composed of 12,357 contigs whose annotation led to prediction of 17,239 genes encoding 17,328 proteins. The genome is highly-complete, with 97.5% of conserved single-copy Ascomycete genes shown to be present. This annotated P. aphanis genome provides a molecular resource for further investigation into host-pathogen interactions in the strawberry powdery mildew pathosystem.

Towards smart and sustainable development of modern berry cultivars in Europe.

Fresh berries are a popular and important component of the human diet. The demand for high-quality berries and sustainable production methods is increasing globally, challenging breeders to develop modern berry cultivars that fulfill all desired characteristics. Since 1994, research projects have characterized genetic resources, developed modern tools for high-throughput screening, and published data in publicly available repositories. However, the key findings of different disciplines are rarely linked together, and only a limited range of traits and genotypes has been investigated. The Horizon2020 project BreedingValue will address these challenges by studying a broader panel of strawberry, raspberry and blueberry genotypes in detail, in order to recover the lost genetic diversity that has limited the aroma and flavor intensity of recent cultivars. We will combine metabolic analysis with sensory panel tests and surveys to identify the key components of taste, flavor and aroma in berries across Europe, leading to a high-resolution map of quality requirements for future berry cultivars. Traits linked to berry yields and the effect of environmental stress will be investigated using modern image analysis methods and modeling. We will also use genetic analysis to determine the genetic basis of complex traits for the development and optimization of modern breeding technologies, such as molecular marker arrays, genomic selection and genome-wide association studies. Finally, the results, raw data and metadata will be made publicly available on the open platform Germinate in order to meet FAIR data principles and provide the basis for sustainable research in the future.

Genomic Informed Breeding Strategies for Strawberry Yield and Fruit Quality Traits.

Over the last two centuries, breeders have drastically modified the fruit quality of strawberries through artificial selection. However, there remains significant variation in quality across germplasm with scope for further improvements to be made. We reported extensive phenotyping of fruit quality and yield traits in a multi-parental strawberry population to allow genomic prediction and quantitative trait nucleotide (QTN) identification, thereby enabling the description of genetic architecture to inform the efficacy of implementing advanced breeding strategies. A negative relationship (r = -0.21) between total soluble sugar content and class one yield was identified, indicating a trade-off between these two essential traits. This result highlighted an established dilemma for strawberry breeders and a need to uncouple the relationship, particularly under June-bearing, protected production systems comparable to this study. A large effect of quantitative trait nucleotide was associated with perceived acidity and pH whereas multiple loci were associated with firmness. Therefore, we recommended the implementation of both marker assisted selection (MAS) and genomic prediction to capture the observed variation respectively. Furthermore, we identified a large effect locus associated with a 10% increase in the number of class one fruit and a further 10 QTN which, when combined, are associated with a 27% increase in the number of marketable strawberries. Ultimately, our results suggested that the best method to improve strawberry yield is through selecting parental lines based upon the number of marketable fruits produced per plant. Not only were strawberry number metrics less influenced by environmental fluctuations, but they had a larger additive genetic component when compared with mass traits. As such, selecting using "number" traits should lead to faster genetic gain.

Fitness Cost Associated With Enhanced EPSPS Gene Copy Number and Glyphosate Resistance in an Amaranthus tuberculatus Population.

The evolution of resistance to pesticides in agricultural systems provides an opportunity to study the fitness costs and benefits of novel adaptive traits. Here, we studied a population of Amaranthus tuberculatus (common waterhemp), which has evolved resistance to glyphosate. The growth and fitness of seed families with contrasting levels of glyphosate resistance was assessed in the absence of glyphosate to determine their ability to compete for resources under intra- and interspecific competition. We identified a positive correlation between the level of glyphosate resistance and gene copy number for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) glyphosate target, thus identifying gene amplification as the mechanism of resistance within the population. Resistant A. tuberculatus plants were found to have a lower competitive response when compared to the susceptible phenotypes with 2.76 glyphosate resistant plants being required to have an equal competitive effect as a single susceptible plant. A growth trade-off was associated with the gene amplification mechanism under intra-phenotypic competition where 20 extra gene copies were associated with a 26.5 % reduction in dry biomass. Interestingly, this growth trade-off was mitigated when assessed under interspecific competition from maize.

Defining strawberry shape uniformity using 3D imaging and genetic mapping.

Strawberry shape uniformity is a complex trait, influenced by multiple genetic and environmental components. To complicate matters further, the phenotypic assessment of strawberry uniformity is confounded by the difficulty of quantifying geometric parameters 'by eye' and variation between assessors. An in-depth genetic analysis of strawberry uniformity has not been undertaken to date, due to the lack of accurate and objective data. Nonetheless, uniformity remains one of the most important fruit quality selection criteria for the development of a new variety. In this study, a 3D-imaging approach was developed to characterise berry shape uniformity. We show that circularity of the maximum circumference had the closest predictive relationship with the manual uniformity score. Combining five or six automated metrics provided the best predictive model, indicating that human assessment of uniformity is highly complex. Furthermore, visual assessment of strawberry fruit quality in a multi-parental QTL mapping population has allowed the identification of genetic components controlling uniformity. A "regular shape" QTL was identified and found to be associated with three uniformity metrics. The QTL was present across a wide array of germplasm, indicating a potential candidate for marker-assisted breeding, while the potential to implement genomic selection is explored. A greater understanding of berry uniformity has been achieved through the study of the relative impact of automated metrics on human perceived uniformity. Furthermore, the comprehensive definition of strawberry shape uniformity using 3D imaging tools has allowed precision phenotyping, which has improved the accuracy of trait quantification and unlocked the ability to accurately select for uniform berries.

Genetic and phenotypic associations between root architecture, arbuscular mycorrhizal fungi colonisation and low phosphate tolerance in strawberry (Fragaria × ananassa).

BACKGROUND: Phosphate is an essential plant macronutrient required to achieve maximum crop yield. Roots are able to uptake soil phosphate from the immediate root area, thus creating a nutrient depletion zone. Many plants are able to exploit phosphate from beyond this root nutrient depletion zone through symbiotic association with Arbuscular Mycorrhizal Fungi (AMF). Here we characterise the relationship between root architecture, AMF association and low phosphate tolerance in strawberries. The contrasting root architecture in the parental strawberry cultivars 'Redgauntlet' and 'Hapil' was studied through a mapping population of 168 progeny. Low phosphate tolerance and AMF association was quantified for each genotype to allow assessment of the phenotypic and genotypic relationships between traits. RESULTS: A "phosphate scavenging" root phenotype where individuals exhibit a high proportion of surface lateral roots was associated with a reduction in root system size across genotypes. A genetic correlation between "root system size" traits was observed with a network of pleiotropic QTL found to represent five "root system size" traits. By contrast, average root diameter and the distribution of roots appeared to be under two discrete methods of genetic control. A total of 18 QTL were associated with plant traits, 4 of which were associated with solidity that explained 46% of the observed variation. Investigations into the relationship between AMF association and root architecture found that a higher root density was associated with greater AMF colonisation across genotypes. However, no phenotypic correlation or genotypic association was found between low phosphate tolerance and the propensity for AMF association, nor root architectural traits when plants are grown under optimal nutrient conditions. CONCLUSIONS: Understanding the genetic relationships underpinning phosphate capture can inform the breeding of strawberry varieties with better nutrient use efficiency. Solid root systems were associated with greater AMF colonisation. However, low P-tolerance was not phenotypically or genotypically associated with root architecture traits in strawberry plants. Furthermore, a trade-off was observed between root system size and root architecture type, highlighting the energetic costs associated with a "phosphate scavenging" root architecture.

Identifying Verticillium dahliae Resistance in Strawberry Through Disease Screening of Multiple Populations and Image Based Phenotyping.

Verticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in multiple strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar "Redgauntlet" and subclade II-2 specific resistance in "Fenella" and "Chandler." A large-scale low-cost phenotyping platform was developed utilizing automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores. A co-dominant resistant QTL; FaRVd3D, present in both "Redgauntlet" and "Hapil" cultivars exhibited a major effect of 18.3% when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the "Emily" cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the F1 mapping population. Markers identified in populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from "Redgauntlet" and FaRVd6D from "Chandler" were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment.

Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes.

The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

Identification of powdery mildew resistance QTL in strawberry (Fragaria × ananassa).

Key Message Powdery mildew resistance in two strawberry mapping populations is controlled by both stable and transient novel QTL of moderate effect. Some low transferability of QTL across wider germplasm was observed. The obligate biotrophic fungus Podosphaera aphanis is the causative agent of powdery mildew on cultivated strawberry (Fragaria × ananassa). Genotypes from two bi-parental mapping populations 'Emily' × 'Fenella' and 'Redgauntlet' × 'Hapil' were phenotyped for powdery mildew disease severity in a series of field trials. Here, we report multiple QTL associated with resistance to powdery mildew, identified in ten phenotyping events conducted across different years and locations. Six QTL show a level of stable resistance across multiple phenotyping events; however, many other QTL were represented in a single phenotyping event and therefore must be considered transient. Subsequent screening of identified QTL across a validation set determined whether identified QTL remained closely linked to the associated resistance gene in the wider germplasm. Furthermore, a preliminary association analysis identified a novel conserved locus for further investigation. Our data suggest that resistance is highly complex and that multiple, primarily additive, sources of quantitative resistance to powdery mildew exist across strawberry germplasm. Utilisation of the reported markers in marker-assisted breeding or genomic selection would lead to improved powdery mildew-resistant strawberry cultivars, particularly where the studied parents, progeny and close pedigree material are included in breeding germplasm.

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.