Since last summer, we have been actively isolating a set of publicly available RFLP clones for maize. We were graciously provided a genomic library by Tim Helentjaris containing over 700 unscreened clones. Each of these clones is being screened against Southern blots of EcoRI, EcoRV, and HindIII digests of genomic DNA from the inbreds Tx303 and CO159 and the F1, Tx303/CO159. So far we have identified over 75 clones that detect polymorphic loci between these two inbreds. We have mapped approximately half of these in an F2 involving these same two inbreds to produce a "rough" RFLP map. In addition to our own clones, we have also probed the F2 blots with a select group of NPI clones and other cloned loci in order to establish linkages with already existing loci. Tim Helentjaris has also provided linkage data of their loci in order for us to use our data with theirs to produce a combined RFLP map. The current map with both NPI and UMC loci is presented with the working maps toward the end of this newsletter. Any of the clones we have isolated are available upon request.
On the technical side, we have resorted to isolating the insert from each clone prior to hybridizations. The presence of several "contaminating bands" in one of the inbreds and several of the F2 samples made interpretation of the blots difficult. These bands appear to be present in the original leaf material used for DNA isolation and hybridized to the plasmid (puC 8) alone. There appears to be a dichotomy among RFLP researchers for those who have problems and those who do not. We felt that even the possibility of additional bands could lead to future problems and have decided to take the extra time to isolate inserts. Basically our procedure involves digesting a miniplasmid prep with the appropriate enzyme, electrophoresis in 1% DNA grade agarose, and excising the insert band. Labelling of the isolated insert is achieved through oligolabelling of an aliquot of the diluted gel slice directly. So far, all of the clones from the genomic library have been labelled successfully. Copies of our procedures are available.
Dave Hoisington and Jack Gardiner
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