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Background: Tobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV. Results: Here, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development. Conclusions: The two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding. [ABSTRACT FROM AUTHOR]

Background: At present, the distinctness, uniformity, and stability (DUS) testing of flue-cured tobacco (Nicotiana tabacum L.) depends on field morphological identification, which is problematic in that it is labor intensive, time-consuming, and susceptible to environmental impacts. In order to improve the efficiency and accuracy of tobacco DUS testing, the development of a molecular marker-based method for genetic diversity identification is urgently needed. Results: In total, 91 simple sequence repeats (SSR) markers with clear and polymorphic amplification bands were obtained with polymorphism information content, Nei index, and Shannon information index values of 0.3603, 0.4040, and 0.7228, respectively. Clustering analysis showed that the 33 study varieties, which are standard varieties for flue-cured tobacco DUS testing, could all be distinguished from one another. Further analysis showed that a minimum of 25 markers were required to identify the genetic diversity of these varieties. Following the principle of two markers per linkage group, 48 pairs of SSR markers were selected. Correlation analysis showed that the genetic relationships revealed by the 48 SSR markers were consistent with those found using the 91 SSR markers. Conclusions: The genetic fingerprints of the 33 standard varieties of flue-cured tobacco were constructed using 48 SSR markers, and an SSR marker-based identification technique for new tobacco varieties was developed. This study provides a reliable technological approach for determining the novelty of new tobacco varieties and offers a solid technical basis for the accreditation and protection of new tobacco varieties. [ABSTRACT FROM AUTHOR]