The use of bulk segregant analysis to map dwarf mutants
--Tim Helentjaris, Ivone Torres-Jerez and Tom McCreery

In our efforts to study various dwarf mutations in maize, we have found it necessary to establish better linkages between their genomic locations and the maize molecular marker set. We have found it convenient to use bulk segregant analysis as described by Michelmore et al. (PNAS 88:9828-9832, 1991). Usually 100+ F2 seeds were planted either in the greenhouse or field. All of the mutant plants were identified and leaves collected from them as one pool (usually 10+ plants) and multiple normal plants as a second pool. The material from each pool was lyophilized, ground, and DNA was extracted. Genomic DNA for each pool was digested with HindIII, EcoRI, and EcoRV, electrophoresed in an agarose gel, and blotted to a nylon membrane. A collection of RFLP probes were selected for the chromosome arm where each mutant was known to be located. Close linkage of the mutant to a RFLP locus was scored from the hybridization results based upon a polymorphism being evident in the F2 population and that two allele fragments could be detected in the "normal" pool and only one in the mutant pool.

Four mutants have been evaluated so far. br2 was found to be most closely located to a region containing npi272 and bnl5.59 near 1C. The location for ct1 on chromosome 8S falls in a region containing npi110 and umc32b. ct2 is located on chromosome 1S and lies within a region containing umc157 and npi97b. na2 is located in a region on 5S containing umc27a and umc166. 

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