Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae)

Abstract

BackgroundCucurbita 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.ResultsWe 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.ConclusionWe 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.

Genetic diversity of Spanish Cucurbita pepo landraces: an unexploited resource for summer squash breeding

“…Most of the SSRs are within genes with a putative known function and were mainly located in ORFs (open reading frames), with only afew of them in UTRs (untranslated regions). Most of the ESTs-SSRs selected were in unigenes that showed homology to annotated protein sequences (Blanca et al 2011). Some were involved in catabolism, transport, regulation of transcription, stress and defense response (Table 4).…”

“…Pumpkin, 1 Styrian Pumpkin, 1 Croockneck and 1 Scallop) and 1 C. moschata accession (Blanca et al, 2011). …”

“…Some were involved in catabolism, transport, regulation of transcription, stress and defense response (Table 4). Primers used for each SSRs locus are listed in Blanca et al (2011).…”

“…We used a set of 27 ESTs-SSRs selected among those in silico identified by Blanca et al (2011) after screening for SSR motifs the 49,610 unigenes of the first C. pepo transcriptome (assembled using ESTs from the cultivars ZU-MU16 (Zucchini Group) and UPV-196 (Scallop Group)) ( Table 4). EST sequences containing perfect repetitions of more than seven units were selected.…”

“…Gong et al (2008) developed the first collection of genomic SSRs (gSSR), about 400, using an enriched genomic library from an oil-seed Pumpkin cultivar. Recent advances in next-generation sequencing technologies (Metzker, 2010) has allowed us to generate the first Cucurbita transcriptome (Blanca et al 2011), with 49,610 Cucurbita unigenes de novo assembled from 512,751 high quality ESTs, sequenced using Roche GS/454. These unigenes were screened for SSRs motifs, leading to the discovery of a collection of 1,882 unigenes with SSR motifs (EST-SSR), what could be considered as the first genomic resource in this genus.…”