Cucumber Fruit Transcriptome Analysis by 454 Sequencing

Cucumber mRNA samples from fruit tissue at 8 days post pollination (dpp), were subjected to 454-sequencing. These analyses yielded 187,406 clean reads, of which 88 % could be assembled into 13,879 contigs, 52 – 2,824 nt in length. The number of sequences per contig, which is reflective of transcript abundance, ranged from 2 – 5,167. BLAST analysis of the most highly represented transcripts (71 contig groups showing > 0.15 % frequency in the transcript pool) against the nr protein sequence NCBI database, indicated high representation of genes associated with protein synthesis, flowers, fruits or seeds of other species, latex related proteins, lipid biosynthesis, cell expansion, defense, phloem transport, and photosynthesis. Approximately 10 % of the highly abundant transcripts did not have a match in the NCBI in the nr protein database, suggesting that these genes could be unique to cucumber fruit. The 126 most highly expressed contigs (0.9 % of total contigs) accounted for 36.8 % of total reads.  The most frequently expressed contig (> 3 %), was previously reported to be highly expressed in cucumber fruits, but is not present in the melon or watermelon fruit ESTs in the NCBI database. Putative homologs of this gene frequently appear in root tissue of other species. The majority of transcripts occurring at a frequency of 0.2 % or greater were also expressed in melon or watermelon fruit, and there was more overlap of these ESTs with cucumber flower ESTs than leaf ESTs.  Several genes showing differential expression at 4 dpp vs. 20 dpp, based on a test run 454 analysis of samples from 0, 4, 8, 12, 16, 20, 26, and 32 dpp fruit, were selected for qRT-PCR analysis.  Results of 454 and qRT-PCR for selected genes were comparable, indicating that the 454 transcriptome sequencing can be used for analyzing relative gene expression during fruit development.