RNA-Seq analysis of Orobanche resistance in Nicotiana tabacum: development of molecular markers for breeding recessive tolerance from ‘Wika’ tobacco variety

Orobanche spp. (broomrape) is an obligate root parasite that can attack a wide spectrum of plants, including tobacco. It has been responsible for economic losses in Europe since 2002 and its incidence in many tobacco-growing countries is increasing. Preventive and curative methods exist, including the use of agrochemicals, however efficacy is limited and pest dissemination remains important due to a high rate of multiplication of the parasite and very small long lasting seeds. The tobacco variety ‘Wika’ induces lower or delayed germination of Orobanche seeds. This seems to be conditioned by a single recessive gene (Cailleteau et al. in: CORESTA Congress, Paris, 2006). Artificial testing in Petri dishes was developed to evaluate the ability of tobacco plantlets to stimulate seed germination. Different lines derived from ‘Wika’, with susceptible control lines, were tested and studied by RNA-Seq. Candidate markers including SNPs or genes differentially expressed between susceptible and resistant lines were identified. An F2 population segregating for ‘Wika’ recessive tolerance was then used for marker validation and mapping. All candidates were situated on chromosome 14 of the tobacco genetic map. The Nicotiana variety collection from Imperial Brands was also tested for these markers, highlighting or confirming other potential tolerant lines. KASP™ genotyping or markers for conventional gel electrophoresis are now available to drive the transfer of ‘Wika’ recessive tolerance into elite lines. RNA-Seq technology combined with sound experimental testing has again proven its high efficiency to identify useful markers for tobacco breeding.