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the 2,860 Bafilomycin A1 SSR containing unige nes, 1,679 had sufficient flanking sequences for primer design. The complete list of SSRs and their corre sponding primer pair information Bafilomycin A1 were provided in Addi tional file 3. Since the ESTs generated under the present study using the 454 technology are from two different cultivars, we expect SNPs to be present in our EST collection. We identified a total of 114 SNPs between WI1983G and WI1983H, among which 42 were transitions, 16 were transversions, and 56 were indels, The frequency of SNP occurrence in our EST collection is rel atively low, which is not unexpected since the sequences were derived from two near isogenic lines. In OAC1 summary, the SSRs and SNPs identified in this study provided a valuable resource for future studies on genetic linkage mapping and the analysis of interesting traits in cucumber.
Conclusion In this study, we describe the generation of more than 350,000 cucumber cDNA sequences from flower buds of two near isogenic lines with different floral sex types, a gynoecious line and a hermaphroditic Erythropoietin line, using the rapid and cost effective massive parallel pyrosequencing technology. Currently in public domains, only 8,000 ESTs are available for cucumber and 150,000 for all the cucurbit species. The ESTs generated in the present study represent a significant addition to the existing genomics and functional genomics resources of cucurbit species. These ESTs have been used to facilitate the annotation of cucumber genome and to identify alternatively spliced genes.
In addition, these ESTs can also be served as a valuable source to derive SSR and SNP markers, which can help to further identify genes linked to inter esting traits. A biochemical pathway database containing more than 300 predicted metabolite pathways was derived from these EST sequences. Digital expression analysis Fer-1 by comparing transcriptomes of two sex type flowers provided some novel insights into the molecular mechanisms of cucumber sex determination, as well as a rich list of candidate genes for further functional analysis. To facilitate public usages of this EST resource, all the EST sequences, annotations, their alignments to the cucumber genome, and the derived pathway database have been made available in a searchable manner through the Cucurbit Genomics Database, Methods Plant material Seeds of gynoecious and hermaphrodite nearly isogenic cucumber lines were kindly provided by Dr J.
E. Staub, WI1983G originated from a cross between inbred WI5821 and WI5822, An andromonoecious Bafilomycin A1 near isogenic line WI1983A was developed using a hermaphrodite line as the donor parent. Five direct backcrosses to WI1983G were made followed by three subsequent generations of self pollina Fer-1 tion. The hermaphrodite WI1983H line was selected from a cross between WI1983G and WI1983A, Seeds were germinated and grown in trays containing a soil mixture, Plants were adequately watered and grown at day night temperatures of 24 18 C with a 16 h photoperiod. Bafilomycin A1 Flower buds of approximately 5 mm in diameter, which represents a crit ical stage of cucumber sex determination, were col lected from both lines and immediately frozen in liquid nitrogen.
Frozen flower buds were stored at 80 C till use. cDNA preparation and sequencing Total RNA was extracted from cucumber flower buds using the TRIzol Reagent, mRNA was purified from the total RNA using the Oligotex mRNA Midi Kit, Double strand cDNA was then synthesized using the SMART cDNA Fer-1 Library Con struction kit following the manufac turers protocol. The PCR products of cDNA were purified using the QIAquick PCR Purification Kit and checked for quality using the Agi lent 2100 Bioanalyzer. Approximately 10 ug cDNA from each of the two flower samples were used for sequencing on a GS FLX platform. A half plate sequencing run was performed for each sample at the Virginia Bioinformatics Institute Core Laboratory Facility following manufac turers protocols. All the sequences can be downloaded and queried at the Cucub