The 8, 12, and 16 dpp librar ies contained nearly twice as many contigs without iden tified homologs in Arabidopsis as was observed for the 0 and 4 dpp libraries. Of the 91 very highly abundant tran scripts without known homologs outside of cucurbits, only three were not observed in the 8, 12 or 16 dpp sam ples. In contrast, 17 of the cucurbit specific transcripts did not appear in 0 or 4 dpp samples. To validate usefulness of the 454 sequence data for ana lysis of transcript abundance, a set of fourteen genes repre senting different levels of EST representationcontig across the different fruit ages were selected for quantitative real time PCR analysis. These included genes such as cyclin dependent kinase B2.2 with high transcript levels early in development or expansin A5 with higher transcript levels at 816 dpp.
Comparison of transcript level at a given age relative to baseline expression at 0dpp showed good correspondence between values obtained by 454 sequencing and qRT PCR. There was also good correspondence between the qRT PCR results obtained from two different growth experiments in the selleck chemical p38 inhibitor greenhouse, indicating biological reproducibility of patterns of gene expression across fruit ages, and validity of the use of frequency of EST representation in the 454 library as a measure of level of gene expression. Principal component analysis was performed on transcript levels among the libraries from the five fruit ages. The first two components, which accounted for nearly 90% of the variation, separated the fruit ages into three groups, 0 and 4 dpp, 8 dpp, and 12 and 16 dpp.
Examination of fruit growth rate indicated that these age groups correspond with cell divisionpre ex ponential growth, peak exponential expansion, and late post exponential expansion stages of growth, respectively. Comparison of the transcripts present a knockout post in each of the age groups showed that the great majority were detected in all three age groups. The fewest unique transcripts were present in the 8 dpp sample, consistent with a developmental gradient of transcription moving from 04 to 8 to 1216 dpp. Both the PCA and Venn Dia gram show the least commonality between the 04 and 1216 dpp age groups. The most highly represented contigs in each age group exhibited markedly differ ent profiles of putative gene function. Among those in common to all three groups were housekeeping genes in cluding numerous ribosomal protein genes, and several tubulins, actins, and redox related genes, as well as several with unknown function or no identifiable homolog in Arabidopsis. Examination of the transcripts that were very highly represented in only one age group, showed that 04 dpp was the only one to include histone genes.