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

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 similar to 7.9 million base pairs (Mb), representing a similar to 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 similar to 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.Peer reviewe

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (\u3ci\u3eFragaria vesca\u3c/i\u3e) 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 (\u3e99.8%) 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

Functional enrichment of genes preferentially expressed in peels at 16 days post pollination (dpp).

<p>^a Functional distribution, normalized frequency, and bootstrap standard deviation (SD) of contigs with putative Arabidopsis homologs was determined using the categories classification from the Classification SuperViewer from Bio-Array Resource for Arabidopsis Functional Genomics for Gene Ontology [<a href="http://compbio.dfci.harvard.edu/tgi/982" target="_blank">http://compbio.dfci.harvard.edu/tgi/982</a> software96/].</p><p>Transcripts were selected based on criteria of top 5% for expression in peel and top 5% for increase in 16 dpp peel vs. 8 dpp peel. The resulting 105 transcripts had >72% of reads in peel samples and >8-fold increase above 8 dpp peel samples.</p

Expression analysis of putative defense related genes.

<p>(A) qRT-PCR verification of potential pathogen defense-related genes with elevated expression in 16 days post pollination (dpp) peels. (B) Relative expression of SYP121/SNAP33 co-expressed genes in 8 and 16 dpp pericarp and peel samples as assessed by 454 pyrosequencing. Genes shown are in the order listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142133#pone.0142133.t004" target="_blank">Table 4</a>. (C) Expression of <i>CsFM01</i> as assessed by 454 pyrosequencing (left) and qRT-PCR analysis (right). 8 = 8 dpp pericarp; 8p = 8 dpp peel; 16 = 16 dpp pericarp; 16p = 16 dpp peel.</p

Co-expression analysis of putative cucumber homologs of MLO2/SYP121/SNAP33- co-expressed genes from Arabidopsis.

<p>Putative homologs of MLO2/SYP121/SNAP33- co-expressed genes from Arabidopsis (as identified by Humphrey et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142133#pone.0142133.ref031" target="_blank">31</a>] were tested for co-expression with cucumber SYP121 and SNAP33 genes in cucumber pericarp (0, 4, 8, 12, and 16 dpp) and peel (8 and 16 dpp) samples.</p

Chemical and physical properties of cucumber fruit peel at 8 and 16 days post pollination (dpp).

<p>(A, B) Effect of aqueous and methanolic extracts from cucumber fruit peel on growth of <i>Phytophthora capsici</i><i>in vitro</i>. Media in individual wells were treated with 10 μl methanolic or aqueous extracts of cucumber fruit peel or solvent controls prepared as described in methods and inoculated with 20 μl of zoospores at 10⁵ zoospores ml^-1. (A) Visual growth rating of isolate OP97 in response to extracts from 8 dpp and 16 dpp fruit peels, 3 days post inoculation. Rating was based on a 1–5 scale as illustrated in the sidebar. (B) Fluorescence emission from isolate NY0664-1RFP in response to aqueous and methanolic extracts. Each value is the mean of 4–5 replicate samples ± S.E. Bars marked with different letters are significantly different (LSD, P<0.05). Each experiment was performed twice with equivalent results. (C) Surface of cucumber fruit at 8 and 16 dpp. Magnification = 1200x; white bar = 10 microns.</p

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

<div><p>Very young cucumber (<i>Cucumis sativus</i>) fruit are highly susceptible to infection by the oomycete pathogen, <i>Phytophthora capsici</i>. 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 <i>P</i>. <i>capsici</i>. Exocarp from 16 dpp fruit had thicker cuticles, and methanolic extracts of peel tissue inhibited growth of <i>P</i>. <i>capsici</i><i>in vitro</i>, 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.</p></div

<i>Phytophthora capsici</i> disease development on cucumber fruit and peels.

<p>(A) Pathogen growth on whole, unwounded cucumber fruit. Fruit were harvested at 8 or 15 days post pollination (dpp) and inoculated with zoospore suspensions of <i>P</i>. <i>capsisi</i>. The photograph was taken at 5 days post-inoculation. The 8 dpp fruit is covered with mycelial growth. (B) Disease development on peel sections and underlying fruit, or directly inoculated whole fruit as described in methods. x/y indicates age of overlying peel section/age of underlying intact fruit. Symptom score: 1 = no symptoms or localized necrosis; 2 = water soaking; 3 = sporulation. Each value is the mean of at least 9 fruit ±se. Bars marked with different letters indicate significant different, ANOVA, LSD, P<0.05.</p