Project description:Acorn squash (Cucurbita pepo) is an iconic fall vegetable in the United States, known for its unique fruit shape and also prized for its culinary properties. Little is known about the metabolism that underlies the development of fruit quality attributes such as color, sweetness, texture and nutritional qualities in acorn squash, or any other winter squash grown worldwide. To provide insight into winter squash fruit and seed development and add to the genomic resources in the Cucurbita genus, RNA sequencing was used to generate an acorn squash fruit and seed transcriptome from the cultivar Sweet REBA at critical points throughout fruit development. 141 838 600 high-quality paired-end Illumina reads were assembled into 55 949 unigenes. 85% of unigenes with predicted open reading frames had homology with previously identified genes and over 62% could be functionally annotated. Comparison with the watermelon and cucumber genomes provided confirmation that the unigenes are full-length and comprehensive, covering an average of 90% of the coding sequence of their homologs and 72% of the cucumber and watermelon exomes. Key candidate genes associated with carotenoid and carbohydrate metabolism were identified toward a resource for winter squash fruit quality trait dissection. This transcriptome represents a major advance in C. pepo genomics, providing significant new sequence information and revealing the repertoire of genes expressed throughout winter squash fruit and seed development. Future studies on the genetic basis of fruit quality and future breeding efforts will be enhanced by tools and insights developed from this resource.
Project description:The phloem sieve elements (SEs), enucleate cells, contain RNAs, which are imported from surrounding tissues and cells, mostly companion cells tightly associated with SEs, and transported via the phloem over the whole plant body. The RNA phloem transport is essential for plant individual development and responses to environmental cues. Recently, we identified primary miRNA (pri-miRNA) sequences in de novo assembled transcriptome of Cucurbita maxima phloem sap and reported 11 most abundant pri-miRNAs . Here, we provide the output of this analysis in complete detail. For the full set of pri-miRNAs identified in the C. maxima phloem sap transcriptome, data on relative abundance are provided along with annotated sequence data.
Project description:MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the response of pumpkin to salt stress, and provided valuable information on novel miRNAs and their target genes in Cucurbita.
Project description:Nectaries are the glands responsible for nectar secretion. To understand the genetic programming underlying nectar production, male and female squash(Cucurbita pepo) floral nectaries at four different time points (pre-secretion #1, pre-secretion #2, secretory, and post-secretory) in biological triplicate were collected, with RNA being isolated and subjected to Illumina RNA-seq analysis. Overall design: Cucurbita pepo (Crookneck Yellow Squash) plants were grown on Sun Gro LC8 soil under a 16 hr day/8 hr night cycle, photosynthetic photon flux of 250 μmol m-2 s-1 at leaf level, and a temperature of 21°C. Four types of RNA samples were separately prepared from the nectaries of both male and female squash flowers, including: ‘pre-secretory #1’ (24 hours prior to anthesis/nectar secretion), ‘pre-secretory #2’ (12 hours prior to anthesis/nectar secretion), ‘secretory’ (full anthesis, 3 hours after dawn), and ‘post-secretory’ (12 hours after the ‘secretory’ stage). All nectary tissues were manually dissected by hand with the RNA being immediately extracted by mechanical disruption with a microcentrifuge pestle and using an RNAqueous® RNA isolation kit (Ambion, Austin, TX) with Plant RNA Isolation Aid (Ambion, Austin, TX). Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality for all samples prior to submission to the University of Minnesota Genomics Center for mRNA isolation, barcoded library creation and Illumina HiSeq 2500 sequencing. Twenty-three TruSeq RNA v2 libraries were created (triplicate samples for male and female nectaries at four timepoints each, except for only duplicate samples of female ‘pre-secretory #2’ nectaries) and sequenced via 50 bp, paired-end runs on the HiSeq 2500 using Rapid chemistry. All libraries were pooled and sequenced across two full lanes. This generated over 240 M reads for each lane and the average quality scores were above Q30.
Project description:The complete genome sequence of a Papaya ringspot virus (PRSV) isolate from South Korea (SK) infecting squash (Cucurbita pepo) was obtained using paired-end RNA sequencing. A BLASTn search of the PRSV SK isolate full-genome sequence showed nucleotide sequence identity ranging from 81% to 83% with previously reported PRSV isolates (GenBank accession numbers KX655874 and EF017707).
Project description:BACKGROUND: Cucurbita pepo belongs to the Cucurbitaceae family. The "Zucchini" types rank among the highest-valued vegetables worldwide, and other C. pepo and related Cucurbita spp., are food staples and rich sources of fat and vitamins. A broad range of genomic tools are today available for other cucurbits that have become models for the study of different metabolic processes. However, these tools are still lacking in the Cucurbita genus, thus limiting gene discovery and the process of breeding. RESULTS: We report the generation of a total of 512,751 C. pepo EST sequences, using 454 GS FLX Titanium technology. ESTs were obtained from normalized cDNA libraries (root, leaves, and flower tissue) prepared using two varieties with contrasting phenotypes for plant, flowering and fruit traits, representing the two C. pepo subspecies: subsp. pepo cv. Zucchini and subsp. ovifera cv Scallop. De novo assembling was performed to generate a collection of 49,610 Cucurbita unigenes (average length of 626 bp) that represent the first transcriptome of the species. Over 60% of the unigenes were functionally annotated and assigned to one or more Gene Ontology terms. The distributions of Cucurbita unigenes followed similar tendencies than that reported for Arabidopsis or melon, suggesting that the dataset may represent the whole Cucurbita transcriptome. About 34% unigenes were detected to have known orthologs of Arabidopsis or melon, including genes potentially involved in disease resistance, flowering and fruit quality. Furthermore, a set of 1,882 unigenes with SSR motifs and 9,043 high confidence SNPs between Zucchini and Scallop were identified, of which 3,538 SNPs met criteria for use with high throughput genotyping platforms, and 144 could be detected as CAPS. A set of markers were validated, being 80% of them polymorphic in a set of variable C. pepo and C. moschata accessions. CONCLUSION: We present the first broad survey of gene sequences and allelic variation in C. pepo, where limited prior genomic information existed. The transcriptome provides an invaluable new tool for biological research. The developed molecular markers are the basis for future genetic linkage and quantitative trait loci analysis, and will be essential to speed up the process of breeding new and better adapted squash varieties.
Project description:The genus Cucurbita (squashes, pumpkins, gourds) contains numerous domesticated lineages with ancient New World origins. It was broadly distributed in the past but has declined to the point that several of the crops' progenitor species are scarce or unknown in the wild. We hypothesize that Holocene ecological shifts and megafaunal extinctions severely impacted wild Cucurbita, whereas their domestic counterparts adapted to changing conditions via symbiosis with human cultivators. First, we used high-throughput sequencing to analyze complete plastid genomes of 91 total Cucurbita samples, comprising ancient (n = 19), modern wild (n = 30), and modern domestic (n = 42) taxa. This analysis demonstrates independent domestication in eastern North America, evidence of a previously unknown pathway to domestication in northeastern Mexico, and broad archaeological distributions of taxa currently unknown in the wild. Further, sequence similarity between distant wild populations suggests recent fragmentation. Collectively, these results point to wild-type declines coinciding with widespread domestication. Second, we hypothesize that the disappearance of large herbivores struck a critical ecological blow against wild Cucurbita, and we take initial steps to consider this hypothesis through cross-mammal analyses of bitter taste receptor gene repertoires. Directly, megafauna consumed Cucurbita fruits and dispersed their seeds; wild Cucurbita were likely left without mutualistic dispersal partners in the Holocene because they are unpalatable to smaller surviving mammals with more bitter taste receptor genes. Indirectly, megafauna maintained mosaic-like landscapes ideal for Cucurbita, and vegetative changes following the megafaunal extinctions likely crowded out their disturbed-ground niche. Thus, anthropogenic landscapes provided favorable growth habitats and willing dispersal partners in the wake of ecological upheaval.