The origin and distribution of ‘Kokubu’-type splice-site mutations of the MLO genes in tobacco varieties

The Japanese domestic tobacco (Nicotiana tabacum L.) cultivar ‘Kokubu’ shows high powdery mildew resistance that is controlled by splice-site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. We investigated the existence of the same NtMLO1/2 splice mutations in the genomes of various tobacco varieties cultivated in Japan and other countries. In total, 14 Japanese domestic cultivars, which were mainly distributed in Kagoshima, had splice-site mutations in both NtMLO1 and NtMLO2. In addition, tobacco cultivars containing only the NtMLO1 splice-site mutation were found in various tobacco production areas in Japan, but no cultivars with only the NtMLO2 splice-site mutation were detected. Moreover, the NtMLO1 splice-site mutation was detected in native Asian, Oriental and cigar tobacco varieties. Consequently, we speculate that these powdery mildew-resistant tobacco cultivars were generated relative recently in the Kagoshima area when a spontaneous mutation occurred at the NtMLO2 splice site in a cultivar already containing the NtMLO1 splice-site mutation and that the NtMLO1 splice-site mutation occurred during the early period of tobacco seed dissemination from the Americas to Asia and Japan.