Identification of QTL associated with resistance to Phytophthora fruit rot in cucumber (Cucumis sativus L.).

Phytophthora fruit rot (PFR) caused by the soilborne oomycete pathogen, Phytophthora capsici, can cause severe yield loss in cucumber. With no resistant variety available, genetic resources are needed to develop resistant varieties. The goal of this work was to identify quantitative trait loci (QTL) associated with resistance to PFR using multiple genomic approaches and populations. Two types of resistances have been identified: age-related resistance (ARR) and young fruit resistance. ARR occurs at 12-16 days post pollination (dpp), coinciding with the end of exponential fruit growth. A major QTL for ARR was discovered on chromosome 3 and a candidate gene identified based on comparative transcriptomic analysis. Young fruit resistance, which is observed during the state of rapid fruit growth prior to commercial harvest, is a quantitative trait for which multiple QTL were identified. The largest effect QTL, qPFR5.1, located on chromosome 5 was fine mapped to a 1-Mb region. Genome-wide association studies (GWAS) and extreme-phenotype genome-wide association study (XP-GWAS) for young fruit resistance were also performed on a cucumber core collection representing > 96% of the genetic diversity of the USDA cucumber germplasm. Several SNPs overlapped with the QTL identified from QTL-seq analysis on biparental populations. In addition, novel SNPs associated with the resistance were identified from the germplasm. The resistant alleles were found mostly in accessions from India and South Asia, the center of diversity for cucumber. The results from this work can be applied to future disease resistance studies and marker-assisted selection in breeding programs.

The contributions from the progenitor genomes of the mesopolyploid Brassiceae are evolutionarily distinct but functionally compatible.

The members of the tribe Brassiceae share a whole-genome triplication (WGT), and one proposed model for its formation is a two-step pair of hybridizations producing hexaploid descendants. However, evidence for this model is incomplete, and the evolutionary and functional constraints that drove evolution after the hexaploidy are even less understood. Here, we report a new genome sequence of Crambe hispanica, a species sister to most sequenced Brassiceae. Using this new genome and three others that share the hexaploidy, we traced the history of gene loss after the WGT using the Polyploidy Orthology Inference Tool (POInT). We confirm the two-step formation model and infer that there was a significant temporal gap between those two allopolyploidizations, with about a third of the gene losses from the first two subgenomes occurring before the arrival of the third. We also, for the 90,000 individual genes in our study, make parental subgenome assignments, inferring, with measured uncertainty, from which of the progenitor genomes of the allohexaploidy each gene derives. We further show that each subgenome has a statistically distinguishable rate of homoeolog losses. There is little indication of functional distinction between the three subgenomes: the individual subgenomes show no patterns of functional enrichment, no excess of shared protein-protein or metabolic interactions between their members, and no biases in their likelihood of having experienced a recent selective sweep. We propose a "mix and match" model of allopolyploidy, in which subgenome origin drives homoeolog loss propensities but where genes from different subgenomes function together without difficulty.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator, is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene (cytb). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

Genetic and genomic resources to study natural variation in Brassica rapa.

The globally important crop Brassica rapa, a close relative of Arabidopsis, is an excellent system for modeling our current knowledge of plant growth on a morphologically diverse crop. The long history of B. rapa domestication across Asia and Europe provides a unique collection of locally adapted varieties that span large climatic regions with various abiotic and biotic stress-tolerance traits. This diverse gene pool provides a rich source of targets with the potential for manipulation toward the enhancement of productivity of crops both within and outside the Brassicaceae. To expand the genetic resources available to study natural variation in B. rapa, we constructed an Advanced Intercross Recombinant Inbred Line (AI-RIL) population using B. rapa subsp. trilocularis (Yellow Sarson) R500 and the B. rapa subsp. parachinensis (Cai Xin) variety L58. Our current understanding of genomic structure variation across crops suggests that a single reference genome is insufficient for capturing the genetic diversity within a species. To complement this AI-RIL population and current and future B. rapa genomic resources, we generated a de novo genome assembly of the B. rapa subsp. trilocularis (Yellow Sarson) variety R500, the maternal parent of the AI-RIL population. The genetic map for the R500 x L58 population generated using this de novo genome was used to map Quantitative Trait Loci (QTL) for seed coat color and revealed the improved mapping resolution afforded by this new assembly.

Developmentally regulated activation of defense allows for rapid inhibition of infection in age-related resistance to Phytophthora capsici in cucumber fruit.

BACKGROUND: Age-related resistance (ARR) is a developmentally regulated phenomenon conferring resistance to pathogens or pests. Although ARR has been observed in several host-pathogen systems, the underlying mechanisms are largely uncharacterized. In cucumber, rapidly growing fruit are highly susceptible to Phytophthora capsici but become resistant as they complete exponential growth. We previously demonstrated that ARR is associated with the fruit peel and identified gene expression and metabolomic changes potentially functioning as preformed defenses. RESULTS: Here, we compare the response to infection in fruit at resistant and susceptible ages using microscopy, quantitative bioassays, and weighted gene co-expression analyses. We observed strong transcriptional changes unique to resistant aged fruit 2-4 h post inoculation (hpi). Microscopy and bioassays confirmed this early response, with evidence of pathogen death and infection failure as early as 4 hpi and cessation of pathogen growth by 8-10 hpi. Expression analyses identified candidate genes involved in conferring the rapid response including those encoding transcription factors, hormone signaling pathways, and defenses such as reactive oxygen species metabolism and phenylpropanoid biosynthesis. CONCLUSION: The early pathogen death and rapid defense response in resistant-aged fruit provide insight into potential mechanisms for ARR, implicating both pre-formed biochemical defenses and developmentally regulated capacity for pathogen recognition as key factors shaping age-related resistance.

QTL and Transcriptomic Analyses Implicate Cuticle Transcription Factor SHINE as a Source of Natural Variation for Epidermal Traits in Cucumber Fruit.

The fruit surface is a unique tissue with multiple roles influencing fruit development, post-harvest storage and quality, and consumer acceptability. Serving as the first line of protection against herbivores, pathogens, and abiotic stress, the surface can vary markedly among species, cultivars within species, and developmental stage. In this study we explore developmental changes and natural variation of cucumber (Cucumis sativus L.) fruit surface properties using two cucumber lines which vary greatly for these traits and for which draft genomes and a single nucleotide polymorphism (SNP) array are available: Chinese fresh market type, Chinese Long '9930' (CL9930), and pickling type, 'Gy14'. Thin-section samples were prepared from the mid-region of fruit harvested at 0, 4, 8, 12, 16, 20, 24 and 30 days post pollination (dpp), stained with Sudan IV and evaluated for cuticle thickness, depth of wax intercalation between epidermal cells, epidermal cell size and shape, and number and size of lipid droplets. 'Gy14' is characterized by columnar shaped epidermal cells, a 2-3 fold thicker cuticular layer than CL9930, increased cuticular intercalations between cells and a larger number and larger sized lipid droplets. In both lines maximal deposition of cuticle and increase in epidermal size coincided with exponential fruit growth and was largely completed by approximately 16 dpp. Phenotyping and quantitative trait locus mapping (QTL) of fruit sampled from an F7:F8 Gy14 × CL9930 recombinant inbred line (RIL) population identified QTL regions on chromosomes 1, 4 and 5. Strong QTL for epidermal cell height, cuticle thickness, intercalation depth, and diameter of lipid droplets co-localized on chromosome 1. SSR markers on chromosome 1 were used to screen for recombinants in an extended RIL population to refine the QTL region. Further fine mapping by KASP assay combined with gene expression profiling suggested a small number of candidate genes. Tissue specificity, developmental analysis of expression, allelic diversity and gene function implicate the regulatory factor CsSHINE1/WIN1 as a source of natural variation for cucumber fruit epidermal traits.

Author Correction: Origin and evolution of the octoploid strawberry genome.

In the version of this article originally published, author Joshua R. Puzey was incorrectly listed as having affiliation 7 (School of Plant Sciences, University of Arizona, Tucson, AZ, USA); affiliation 6 (Department of Biology, College of William and Mary, Williamsburg, VA, USA) is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.

Origin and evolution of the octoploid strawberry genome.

Cultivated strawberry emerged from the hybridization of two wild octoploid species, both descendants from the merger of four diploid progenitor species into a single nucleus more than 1 million years ago. Here we report a near-complete chromosome-scale assembly for cultivated octoploid strawberry (Fragaria × ananassa) and uncovered the origin and evolutionary processes that shaped this complex allopolyploid. We identified the extant relatives of each diploid progenitor species and provide support for the North American origin of octoploid strawberry. We examined the dynamics among the four subgenomes in octoploid strawberry and uncovered the presence of a single dominant subgenome with significantly greater gene content, gene expression abundance, and biased exchanges between homoeologous chromosomes, as compared with the other subgenomes. Pathway analysis showed that certain metabolomic and disease-resistance traits are largely controlled by the dominant subgenome. These findings and the reference genome should serve as a powerful platform for future evolutionary studies and enable molecular breeding in strawberry.

Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry.

BACKGROUND: Highbush blueberry (Vaccinium corymbosum) has long been consumed for its unique flavor and composition of health-promoting phytonutrients. However, breeding efforts to improve fruit quality in blueberry have been greatly hampered by the lack of adequate genomic resources and a limited understanding of the underlying genetics encoding key traits. The genome of highbush blueberry has been particularly challenging to assemble due, in large part, to its polyploid nature and genome size. FINDINGS: Here, we present a chromosome-scale and haplotype-phased genome assembly of the cultivar "Draper," which has the highest antioxidant levels among a diversity panel of 71 cultivars and 13 wild Vaccinium species. We leveraged this genome, combined with gene expression and metabolite data measured across fruit development, to identify candidate genes involved in the biosynthesis of important phytonutrients among other metabolites associated with superior fruit quality. Genome-wide analyses revealed that both polyploidy and tandem gene duplications modified various pathways involved in the biosynthesis of key phytonutrients. Furthermore, gene expression analyses hint at the presence of a spatial-temporal specific dominantly expressed subgenome including during fruit development. CONCLUSIONS: These findings and the reference genome will serve as a valuable resource to guide future genome-enabled breeding of important agronomic traits in highbush blueberry.

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome.

Background: The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies. Findings: Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ∼200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis. Conclusions: The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

Background: Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Findings: Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Conclusions: Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions.

Transcriptomic and metabolomic analyses of cucumber fruit peels reveal a developmental increase in terpenoid glycosides associated with age-related resistance to Phytophthora capsici.

The oomycete, Phytophthora capsici, infects cucumber (Cucumis sativus L.) fruit. An age-related resistance (ARR) to this pathogen was previously observed in fruit of cultivar 'Vlaspik' and shown to be associated with the peel. Young fruits are highly susceptible, but develop resistance at ~10-12 days post pollination (dpp). Peels from resistant (16 dpp) versus susceptible (8 dpp) age fruit are enriched with genes associated with defense, and methanolic extracts from resistant age peels inhibit pathogen growth. Here we compared developing fruits from 'Vlaspik' with those of 'Gy14', a line that does not exhibit ARR. Transcriptomic analysis of peels of the two lines at 8 and 16 dpp identified 80 genes that were developmentally upregulated in resistant 'Vlaspik' 16 dpp versus 8 dpp, but not in susceptible 'Gy14' at 16 dpp. A large number of these genes are annotated to be associated with defense and/or specialized metabolism, including four putative resistance (R) genes, and numerous genes involved in flavonoid and terpenoid synthesis and decoration. Untargeted metabolomic analysis was performed on extracts from 8 and 16 dpp 'Vlaspik' and 'Gy14' fruit peels using Ultra-Performance Liquid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry. Multivariate analysis of the metabolomes identified 113 ions uniquely abundant in resistant 'Vlaspik' 16 dpp peel extracts. The most abundant compounds in this group had relative mass defects consistent with terpenoid glycosides. Two of the three most abundant ions were annotated as glycosylated nor-terpenoid esters. Together, these analyses reveal potential mechanisms by which ARR to P. capsici may be conferred.

Variation in cucumber (Cucumis sativus L.) fruit size and shape results from multiple components acting pre-anthesis and post-pollination.

MAIN CONCLUSION: Morphological, QTL, and gene expression analyses indicate variation in cucumber fruit size and shape results from orientation, timing, and extent of cell division and expansion, and suggest candidate gene factors. Variation in cucumber (Cucumis sativus L.) fruit size and shape is highly quantitative, implicating interplay of multiple components. Recent studies have identified numerous fruit size and shape quantitative trait loci (QTL); however, underlying factors remain to be determined. We examined ovary and fruit development of two sequenced cucumber genotypes with extreme differences in fruit size and shape, Chinese Long '9930' (CL9930), and pickling type 'Gy14'. Differences were observed in several independent factors that can influence size and shape: ovule number, rate and period of cell division in longitudinal and cross section in ovaries and fruit, timing and rate of fruit expansion in length and diameter, and cell shape. Level and timing of expression of select fruit growth stage marker genes and candidate fruit size gene homologs associated with cucumber fruit size and shape QTL were examined from 5-day pre-anthesis to 20-day post-pollination. Our results indicate that variation in fruit size and shape results from differences in cell number and shape in longitudinal and cross section, driven in turn by differences in orientation, timing, and duration of cell division and expansion, both pre- and post-anthesis, and suggest candidate genes contributing to determination of cucumber fruit size and shape.

Exocarp Properties and Transcriptomic Analysis of Cucumber (Cucumis sativus) Fruit Expressing Age-Related Resistance to Phytophthora capsici.

Very young cucumber (Cucumis sativus) fruit are highly susceptible to infection by the oomycete pathogen, Phytophthora capsici. As the fruit complete exponential growth, at approximately 10-12 days post pollination (dpp), they transition to resistance. The development of age-related resistance (ARR) is increasingly recognized as an important defense against pathogens, however, underlying mechanisms are largely unknown. Peel sections from cucumber fruit harvested at 8 dpp (susceptible) and 16 dpp (resistant) showed equivalent responses to inoculation as did whole fruit, indicating that the fruit surface plays an important role in defense against P. capsici. Exocarp from 16 dpp fruit had thicker cuticles, and methanolic extracts of peel tissue inhibited growth of P. capsici in vitro, suggesting physical or chemical components to the ARR. Transcripts specifically expressed in the peel vs. pericarp showed functional differentiation. Transcripts predominantly expressed in the peel were consistent with fruit surface associated functions including photosynthesis, cuticle production, response to the environment, and defense. Peel-specific transcripts that exhibited increased expression in 16 dpp fruit relative to 8 dpp fruit, were highly enriched (P<0.0001) for response to stress, signal transduction, and extracellular and transport functions. Specific transcripts included genes associated with potential physical barriers (i.e., cuticle), chemical defenses (flavonoid biosynthesis), oxidative stress, penetration defense, and molecular pattern (MAMP)-triggered or effector-triggered (R-gene mediated) pathways. The developmentally regulated changes in gene expression between peels from susceptible- and resistant- age fruits suggest programming for increased defense as the organ reaches full size.

QTL mapping in multiple populations and development stages reveals dynamic quantitative trait loci for fruit size in cucumbers of different market classes.

KEY MESSAGE: QTL analysis in multi-development stages with different QTL models identified 12 consensus QTLs underlying fruit elongation and radial growth presenting a dynamic view of genetic control of cucumber fruit development. Fruit size is an important quality trait in cucumber (Cucumis sativus L.) of different market classes. However, the genetic and molecular basis of fruit size variations in cucumber is not well understood. In this study, we conducted QTL mapping of fruit size in cucumber using F2, F2-derived F3 families and recombinant inbred lines (RILs) from a cross between two inbred lines Gy14 (North American picking cucumber) and 9930 (North China fresh market cucumber). Phenotypic data of fruit length and diameter were collected at three development stages (anthesis, immature and mature fruits) in six environments over 4 years. QTL analysis was performed with three QTL models including composite interval mapping (CIM), Bayesian interval mapping (BIM), and multiple QTL mapping (MQM). Twenty-nine consistent and distinct QTLs were detected for nine traits from multiple mapping populations and QTL models. Synthesis of information from available fruit size QTLs allowed establishment of 12 consensus QTLs underlying fruit elongation and radial growth, which presented a dynamic view of genetic control of cucumber fruit development. Results from this study highlighted the benefits of QTL analysis with multiple QTL models and different mapping populations in improving the power of QTL detection. Discussion was presented in the context of domestication and diversifying selection of fruit length and diameter, marker-assisted selection of fruit size, as well as identification of candidate genes for fruit size QTLs in cucumber.

Ultrahigh-density linkage map for cultivated cucumber (Cucumis sativus L.) using a single-nucleotide polymorphism genotyping array.

Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a '9930' × 'Gy14' recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.