Marivi Colle

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.

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

Abstract 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.

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.

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

AbstractMain conclusionMorphological, 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.

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.

Genomic Analysis of Cucurbit Fruit Growth

Fruit development in cucurbit species follows the canonical progression of ovary development, fruit set, expansive fruit growth, and maturation and ripening. This commonality, however, belies tremendous morphological diversity. Variation in timing, amount, and orientation of cell division and cell expansion pre- and post-anthesis, as well as factors influencing carpel number, floral sex, photosynthetic capacity and trichome development all drive extreme variability in fruit size and shape. New genomic approaches utilizing recently assembled draft genomes for the four major cucurbit crop species (Cucumis sativus, Cucumis melo, Citrullus lanatus, Cucurbita spp), next generation high throughput sequencing, molecular mapping methods, transcriptomic analyses, gene cloning, and transgenic approaches are all contributing to an increased understanding of the key processes underlying cucurbit fruit development. Extensive quantitative trait locus (QTL) analyses have identified numerous QTL for features such as ovary length, width, and shape, and fruit length, width, shape, flesh thickness and cavity diameter. Most recently, multi-pronged approaches combining mapping, sequence, and transcriptional analyses have allowed for identification specific candidate genes influencing cucurbit fruit morphology.

Cucumber (Cucumis sativus) breeding line with young fruit resistance to infection by Phytophthora capsici

Cucumber (Cucumis sativus) production in the eastern and midwestern United States is subject to severe losses due to fruit rot caused by the soilborne oomycete pathogen, Phytophthora capsici (Granke et al., 2012; Sonogo and Ji, 2012).P. capsici preferentially infects cucumber fruits, especially young fruit, while leaves and vines remain healthy (Ando et al., 2009, 2015; Gevens et al., 2006). Disease is manifested by extensive mycelial growth, sporulation, fruit rot, and tissue collapse. There are currently no commercial cucumber cultivars with resistance to this disease. Our prior screening of young fruit from 1076 accessions from the U.S. cucumber PI collection identified three PIs with potential resistance: PI 109483 and PI 178884 from Turkey and PI 214049 from India (Colle et al., 2014). Although each accession had multiple plants with mean fruit scores in the resistant category, there were mixtures of resistant and susceptible individuals within the PI seed sample. Heterogeneity and heterozygosity among samples have been frequently observed for PI accessions, including for disease resistance in cucurbits (e.g., Davis et al., 2007; Donahoo et al., 2013; Wechter et al., 2011), making it necessary to stabilize resistance before further breeding efforts. PI 214049 from India was consistently very late or failed to produce female flowers and fruits in the field inMichigan conditions and so was not pursued further. Of the two PIs from Turkey, progeny of PI 109483 appeared to have stronger and more consistent resistance in subsequent generations as expressed by either no lesion development or lesions largely limited to the site of inoculation. Here we report the development of a PI 109483derived S6 progeny line with young fruit resistant to infection by P. capsici as a possible source of resistance for use in breeding programs.

Genomic Resources for the Woodland Strawberry (Fragaria vesca)

Fragaria vesca is one of the putative diploid progenitors and donor of subgenome A in the octoploid genome of the economically important cultivated strawberry (Fragaria × ananassa). With its small genome size, short generation time, and well-established transformation system, F. vesca is an ideal model species for Fragaria and other rosaceous crops. The F. vesca genome was first sequenced in 2011 and since then, with the availability of other molecular genetics and genomic resources, the assembly and annotation of the draft genome were further improved. The F. vesca draft genome has been an invaluable resource to the strawberry research community, with numerous studies using it as reference genome to identify candidate genes related to agronomically important traits. It has led to the generation of a 90 K array, metabolic pathways database, and various other new genetic resources. The strawberry genome also has been used in comparative genomics studies that elucidated the relationship among members of the Rosaceae family and identified genes and genomic signals across several rosaceous species. Altogether, the discoveries made with this genome will provide potential ways to improve cultivated strawberry and expand on our understanding of the Rosaceae family.

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

Abstract 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.