Data analysis
Cloned sequences were aligned using the CLUSTALW algorithm (Omiga 2.0, Oxford Molecular) to assess sequence quality and identify multiple captures of the same sequence. Each unique clone (167 out of 288) was compared against the GenBank database using the BLAST-X and BLAST-N algorithms at the site maintained by the US National Center for Biotechnology Information [21]. Matches showing a probability score of < 1.0 × 10-3 were treated as significant matches. Of the 167 unique clones, nine were dropped from the analyses as a result of evidence of multiple bands or inconsistent PCR amplification. Of the remaining clones, 64 showed a match to one or more sequences in GenBank and, of these, 63 could be placed into one of the gene function groups defined in [10] and the Gene Ontology database [22]. All gene fragment sequences used are available, along with their BLAST search results, at the Beenome Project website [23], and in the NCBI dbEST database [24].
Scanned images of exposed films were scored using the software program Zero Dscan (Scanalytics), giving a densitometry score for each cell in the membrane's 384-cell matrix. Spreadsheet macros (Microsoft Excel) were then used to generate an average intensity value for each of 158 unique clones that consistently showed a single band in the PCR amplifications. These values were normalized as a proportion of the strongest signal on each membrane, then were further normalized and centered on unity across genes and samples [18]. Centroid clustering was then carried out and visualized using the programs Cluster 1.0 and TreeView 1.0, respectively [25].