Positional cloning and molecular identification of EHY5. A, Map-based cloning. The genetic locus of ehy5 mutation was first mapped between markers ER and T20P8 on chromosome 2. Fine mapping using genetic markers designed from BAC clones. The direction of the BAC clone is indicated by the arrow. The numbers above and below the arrow indicate the marker number and the corresponding recombinants for the respective marker. Sequence of the genomic DNA fragment from wild-type (WS) and ehy5 mutant plants and comparison with wild-type (Col) genomic DNA sequence indicate C to T mutation. B, DNA polymorphism between ehy5 and wild-type (WS) plants. The C to T mutation in ehy5 genomic DNA adds a DdeI recognition site. The DNA fragments flanking the DdeI site were amplified from the wild-type and ehy5 plants, digested with DdeI, and separated on native PAGE. C, Genetic complementation of ehy5. Phenotypes of 6-day-old wild-type (WS), ehy5 and ehy5* (HY1 complemented) are shown. D, RT- PCR results show the expression of HY1 in wild-type (WS), ehy5 and ehy5*. Actin bands show the loading control. The RT-PCR experiment was repeated thrice and a representative result is shown.