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Genetic linkage maps are essential for studies of genetics, genomic structure, and genomic evolution, and for mapping quantitative trait loci (QTL). Identification of molecular markers and construction of genetic linkage maps in tobacco (Nicotiana tabacum L.), a classical model plant and important economic crop, have remained limited. In the present study we identified a large number of single nucleotide polymorphism (SNP) markers and constructed a high-density SNP genetic map for tobacco using restriction site-associated DNA sequencing. In 1216.30 Gb of clean sequence obtained using the Illumina HiSeq 2000 sequencing platform, 99,647,735 SNPs were identified that differed between 203 sequenced plant genomes and the tobacco reference genome. Finally, 13,273 SNP markers were mapped on 24 high-density tobacco genetic linkage groups. The entire linkage map spanned 3421.80 cM, with a mean distance of 0.26 cM between adjacent markers. Compared with genetic linkage maps published previously, this version represents a considerable improvement in the number and density of markers. Seven QTL for resistance to cucumber mosaic virus (CMV) in tobacco were mapped to groups 5 and 8. This high-density genetic map is a promising tool for elucidation of the genetic bases of QTL and for molecular breeding in tobacco. Keywords: Single nucleotide polymorphism, Genetic linkage map, Tobacco, Cucumber mosaic virus, Quantitative trait loci

Cucumber mosaic virus (CMV) is one of the most severe viral diseases transmitted by aphids infecting Solanum crops in China, causing great losses of crop yields and income in rural communities. The tobacco cultivars NC82 and Taiyan 8 are closely related but differ in resistance to CMV. NC82 is susceptible to infection and Taiyan 8 is resistant, but the mechanisms underlying this difference in resistance are not clear. In this study, we conducted RNA sequencing to analyze changes in gene expression induced in the leaves of Taiyan 8 and NC82 upon systemic infection with CMV, compared with gene expression in the leaves of mock-inoculated plants. Leaves were sampled at one, three, eight, and 15 days after infection. In total, 3443 and 747 differentially expressed genes were identified in Taiyan 8 and NC82, respectively. Gene ontology and pathway enrichment analyses revealed that the different responses to CMV infection between cultivars were based on microtubule-based processes, pentose and glucuronate interconversions, plant–pathogen interaction, and hormone signal transduction pathways. Genes encoding pathogenesis-related proteins, disease-resistance proteins, lipoxygenase, cellulose synthase, an auxin response factor, and an ethylene receptor showed different expression patterns. The differences in gene expression following CMV infection likely contributed to the different resistance levels of these two tobacco cultivars. The comprehensive transcriptome dataset described here, which includes candidate response genes, will serve as a resource for further studies of the molecular mechanisms associated with tobacco defense responses against CMV. Keywords: Cucumber mosaic virus, Tobacco, Resistance, Transcriptome, Differential gene expression

Cucumber mosaic virus (CMV) is among the most important plant virus infections, inducing a variety of disease symptoms. However, the molecular mechanisms underlying plant responses to CMV infection remain poorly understood. In this study, we performed RNA sequencing analysis of tolerant (Taiyan8) and susceptible (NC82) tobacco cultivars on CMV-infected plants, using mock-inoculated plants as a control. The propagation of CMV in inoculated leaves did not show obvious difference between two cultivars, whereas virus accumulation in systemic leaves of Taiyan8 was smaller than those of NC82 at the same time point. We observed 765 and 1,011 differentially expressed genes (DEGs) in Taiyan8 and NC82, respectively, in CMV-inoculated leaves. DEGs related to reactive oxygen species, salicylic acid signal transduction, and plant–pathogen interaction were upregulated or downregulated in Taiyan8, which indicates that defense response pathways to CMV were activated in the tolerant cultivar. In addition, we identified several DEGs related to disease defense and stress resistance showing opposing expression patterns in the two cultivars. Our comparative transcriptome analysis will improve our understanding of the mechanisms of CMV tolerance in plants, and will be of great importance in the molecular breeding of CMV- tolerant genotypes.

Phytophthora nicotianae is highly pathogenic to Solanaceous crops and is a major problem in tobacco production. The tobacco cultivar Beihart1000-1 (BH) is resistant, whereas the Xiaohuangjin 1025 (XHJ) cultivar is susceptible to infection. Here, BH and XHJ were used as models to identify resistant and susceptible genes using RNA sequencing (RNA-seq). Roots were sampled at 0, 6, 12, 24, and 60 h post infection. In total, 23,753 and 25,187 differentially expressed genes (DEGs) were identified in BH and XHJ, respectively. By mapping upregulated DEGs to the KEGG database, changes of the rich factor of “plant pathogen interaction pathway” were corresponded to the infection process. Of all the DEGs in this pathway, 38 were specifically regulated in BH. These genes included 11 disease-resistance proteins, 3 pathogenesis-related proteins, 4 RLP/RLKs, 2 CNGCs, 7 calcium-dependent protein kinases, 4 calcium-binding proteins, 1 mitogen-activated protein kinase kinase, 1 protein EDS1L, 2 WRKY transcription factors, 1 mannosyltransferase, and 1 calmodulin-like protein. By combining the analysis of reported susceptible (S) gene homologs and DEGs in XHJ, 9 S gene homologs were identified, which included 1 calmodulin-binding transcription activator, 1 cyclic nucleotide-gated ion channel, 1 protein trichome birefringence-like protein, 1 plant UBX domain-containing protein, 1 ADP-ribosylation factor GTPase-activating protein, 2 callose synthases, and 2 cellulose synthase A catalytic subunits. qRT-PCR was used to validate the RNA-seq data. The comprehensive transcriptome dataset described here, including candidate resistant and susceptible genes, will provide a valuable resource for breeding tobacco plants resistant to P. nicotianae infections.

Cigar line Beinhart 1000-1 has effective durable resistance to black shank (BS) and is considered one of the most resistant sources in tobacco (Nicotiana tabacum L.). To investigate the inheritance and identification of stable quantitative trait loci (QTL) for BS response, F2, BC1F2 individuals and BC1F2:3 lines were produced from a cross between Beinhart 1000-1 and Xiaohuangjin 1025. Two major quantitative trait loci (M-QTL) named qBS7 and qBS17 were repeatedly detected under different conditions. QTL qBS7 was mapped to the region between PT30174 and PT60621 and explained 17.40%–25.60% of the phenotypic variance under different conditions. The other QTL qBS17 in interval PT61564–PT61538 of linkage group 17 was detected in a BC1F2 population in the field and in BC1F2:3 in both the field and at the seedling stage, explaining 6.90% to 11.60% of the phenotypic variance. The results improve our understanding of the inheritance of resistance to BS and provide information that can be used in marker-assisted breeding. Keywords: Phytophthora nicotianae, Quantitative trait locus, Plant disease resistance, Simple sequence repeats

Brown spot (BS) is a destructive foliar disease in tobacco (Nicotiana tabacum L.) and is caused by Alternaria alternata. BS poses a serious threat to tobacco production worldwide. To develop molecular markers that are tightly linked to BS resistance for marker-assisted selection (MAS), F2, F2:3 and BC3F2:3 populations were developed from a cross between a source of BS resistance Jingyehuang (JYH) and a BS susceptible flue-cured variety NC82. One hundred eighty-one F2 individuals, 180 F2:3 lines and 256 BC3F2:3 lines were evaluated for field resistance under different environments and quantitative trait loci (QTL) were identified by linkage mapping. A major QTL was mapped on chromosome15; this QTL explained 8.6–18.0% of the phenotypic variation under different conditions. Furthermore, 219 accessions were evaluated for their responses to BS at two sites, and association mapping (AM) was used to verify the chromosomal region harboring the major QTL. The AM results showed that six significant marker-trait associations were detected at two sites. Among these markers, the marker Indel53 within the specific chromosomal region exhibited the most significant association with resistance to BS and explained 20.0 and 21.5% of the phenotypic variation at the two sites, respectively. An approximately 2-Mb physical interval at the locus of marker Indel53 contained 31 predicted genes; quantitative real-time PCR results suggested that two of these genes (Nitab 4.5_0000264g0050.1 and Nitab 4.5_0000264g0130.1) were probable candidate genes for resistance to BS. In summary, our results suggested that the novel major QTL from tobacco variety JYH for resistance to BS provided partial effective resistance against A. alternata and was useful for MAS of resistance to BS in tobacco breeding.

Tobacco mosaic virus (TMV) caused serious loss in yield and quality of tobacco every year. It is a long-term goal to improve the tobacco resistance against TMV by tobacco breeding. N gene was the firstly reported TMV-resistant gene, which showed resistance against all Tobamoviruses except the Ob stain and belonged to the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance (R) genes. At present, N gene had already been widely used in tobacco conventional breeding, but there is rare available molecular maker used in marker-assisted selection of TMV resistance. In this study, we designed a pair of primers that specific amplify N gene fragment based on the sequence of N gene intron III, named N-marker. Then, we identified TMV resistance by two selecting methods, PCR with N-marker and inoculated with the TMV-C strain. Results from the two method showed that (1) 13 varieties among 67 tobacco varieties displayed hypersensitive reaction when inoculated with the TMV-C strain, also contained N gene fragments screened by PCR with N-marker; (2) 105 strains of 200 BC1 strains showed resistance against TMV when inoculated with TMV-C strain, meanwhile, 103 of the 105 strains contained N gene fragment verified by PCR with N-marker. Therefore, the N-marker is reliable for high throughput screening of germplasm resources and tobacco breeding materials in selection of N-mediated TMV resistance. Our study not only developed a molecular marker for tobacco breeding, but also identified new germplasm resources that are resistant to TMV.