III. Designing ToRuGs
To identify root-expressed genes in LeGI, we queried the TiGR tomato gene database and identified approximately 4,300 genes (as tentative clusters: TCs). We named this set the Tomato Root Unigene Set (ToRuGs).
Using a Perl script, the TCs defining each of 4,300 root-expressed genes were searched to identify the clone that contained the longest member of each TC (Fig. 4). The script’s initial output was a list of clones from 20 tomato libraries distributed across ~400 library plates.
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To reduce the number of library plates, we identified those which contained only 1-3 clones of interest, and deployed an iterative process to find other members of the TC. First, the TC for each clone was queried to identify the 2nd longest clone, and its plate location examined. If present in one of the ~200 multiple-clone plates destined for ToRuGs, it was selected; if not, the TC was re-examined for the next-longest clone, and so on. This yielded a set of 202 primary plates.
These clones, along with clones from the giant cell library, are being re-arrayed into 12, 384-well plates to assemble ToRuGs.
