The functional homologs WS1A and WS1B, identified by map-based cloning, control the burley character by affecting chloroplast development in tobacco, contributing to gene isolation and genetic improvement in polyploid crops. Burley represents a special type of tobacco (Nicotiana tabacum L.) cultivar that is characterized by a white stem with a high degree of chlorophyll deficiency. Although important progress in the research of burley tobacco has been made, the molecular mechanisms underlying this character remain unclear. Here, on the basis of our previous genetic analyses and preliminary mapping results, we isolated the White Stem 1A (WS1A) and WS1B genes using a map-based cloning approach. WS1A and WS1B are functional homologs with completely identical biological functions and highly similar expression patterns that control the burley character in tobacco. WS1A and WS1B are derived from Nicotiana sylvestris and Nicotiana tomentosiformis, the diploid ancestors of Nicotiana tabacum, respectively. The two genes encode zinc metalloproteases of the M50 family that are highly homologous to the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) protein of Arabidopsis and the Lutescent 2 (L2) protein of tomato. Transmission electron microscopic examinations indicated that WS1A and WS1B are involved in the development of chloroplasts by controlling the formation of thylakoid membranes, very similar to that observed for EGY1 and L2. The genotyping of historical tobacco varieties revealed that a two-step mutation process occurred in WS1A and WS1B during the evolution of burley tobacco. We also discussed the strategy for gene map-based cloning in polyploid plants with complex genomes. This study will facilitate the identification of agronomically important genes in tobacco and other polyploid crops and provide insights into crop improvement via molecular approaches.
Keywords: Burley tobacco; Chloroplast; Duplicated genes; Map-based cloning; Polyploid; White stem.