Genetic demonstration of the involvement of WUSCHEL-related homeobox (WOX) genes in narrow-petal and narrow-leaf mutations in traditional Japanese azalea cultivars

Abstract

The spider-type azalea cultivars, Rhododendron kaempferi ‘Kin-kujyaku’ and Rhododendron macrosepalum ‘Seigaiha’, are characterized by extremely narrow leaves and choripetalous flowers with narrow petals. Previous morphological analysis of these cultivars revealed that narrowed organs would be a common mutation in the growth of lateral organs in the transverse plane. In the present study, we focused on the analysis of WUSCHEL-related homeobox (WOX) genes, WOX1 and WOX3, which regulate the laminar width and development of lateral organs. Sequence analysis of the two genes confirmed a fatal mutation in Rk/RmWOX3/SPIDER of ‘Kin-kujyaku’ and ‘Seigaiha’. To demonstrate the role of SPIDER in the spider-type phenotype in azalea, we performed genotyping using DNA markers developed for multiplex PCR that can distinguish between the wild-type and the spider-type allele based on the partial mutated region of RkWOX3/SPIDER in ‘Kin-kujyaku’. The investigation of BC1 population derived from ‘Kin-kujyaku’ confirmed complete co-segregation between spider-type mutation and Rkwox3/spider band pattern of DNA markers. Therefore, we concluded that SPIDER is responsible for the spider-type mutation in azalea.