Isolation of a gibberellin-insensitive dwarfing gene, Rht-B1e, and development of an allele-specific PCR marker

Gibberellins (GAs) are important phytohormones in plants. GAs promote plant growth by inducing the degradation of DELLA proteins, which serve as GA signal repressors. The semi-dwarfing genes Rht-B1b and Rht-D1b, derived from the Japanese variety Norin 10, are gain-of-function mutant alleles of the reduced height-1 genes (Rht-B1 and Rht-D1) encoding wheat DELLA proteins. Wheat varieties carrying these Rht alleles are shorter and insensitive to the GA response. At the Rht-B1 loci, an alternative GA-insensitive dwarfing gene, Rht-B1e, was found in the Russian mutant of Bezostaya1, or Krasznodari 1, by breeders, but its molecular mechanism for causing dwarfism remains unknown. In this study, the Rht-B1e allele was isolated using homology-based cloning. Sequence comparison between Rht-B1e and the wild-type Rht-B1a revealed an A-to-T substitution at nucleotide position 181 in Rht-B1e, which introduced a stop codon into the DELLA domain. Alignment of deduced amino acid sequences of Rht-B1e and Rht-B1b showed that the stop codon position in Rht-B1e was earlier than that of Rht-B1b by three amino acid residues, and it was also followed closely by several methionines, which may permit translational re-initiation, as seen in Rht-B1b. Yeast two-hybrid analysis revealed that the predicted Rht-B1e proteins did not interact with the GA receptor GID1 in the presence of GA, suggesting that the stop codon mutation in the DELLA domain is the molecular cause of GA insensitivity and dwarfism conferred by Rht-B1e in wheat. Meanwhile, we developed an allele-specific PCR marker for Rht-B1e, which may facilitate the use of the Rht-B1e dwarfing gene in wheat breeding programs.