Tobacco (Nicotiana tabacum L.), particularly flue-cured tobacco, is one of the most economically important nonfood crops and is also an important model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping, and breeding. Simple sequence repeats (SSR) are one of the most widely-used molecular markers, having significant advantages including that they are generally co-dominant, easy to use, abundant in eukaryotic organisms, and produce highly reproducible results. In this study, based on the genome sequence data of flue-cured tobacco (K326), we developed a total of 13,645 mostly novel SSR markers, which were working in a set of eighteen tobacco varieties of four different types. A mapping population of 213 backcross (BC1) individuals, which were derived from an intra-type cross between two flue-cured tobacco varieties, Y3 and K326, was selected for mapping. Based on the newly developed SSR markers as well as published SSR markers, we constructed a genetic map consisting of 626 SSR loci distributed across 24 linkage groups and covering a total length of 1120.45 cM with an average distance of 1.79 cM between adjacent markers, which is the highest density map of flue-cured tobacco till date.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

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