Co-expression network analysis and identification of core genes in the interaction between wheat and Puccinia striiformis f. sp. tritici.

The epidemic of stripe rust, caused by the pathogen Puccinia striiformis f. sp. tritici (Pst), would reduce wheat (Triticum aestivum) yields seriously. Traditional experimental methods are difficult to discover the interaction between wheat and Pst. Multi-omics data analysis provides a new idea for efficiently mining the interactions between host and pathogen. We used 140 wheat-Pst RNA-Seq data to screen for differentially expressed genes (DEGs) between low susceptibility and high susceptibility samples, and carried out Gene Ontology (GO) enrichment analysis. Based on this, we constructed a gene co-expression network, identified the core genes and interacted gene pairs from the conservative modules. Finally, we checked the distribution of Nucleotide-binding and leucine-rich repeat (NLR) genes in the co-expression network and drew the wheat NLR gene co-expression network. In order to provide accessible information for related researchers, we built a web-based visualization platform to display the data. Based on the analysis, we found that resistance-related genes such as TaPR1, TaWRKY18 and HSP70 were highly expressed in the network. They were likely to be involved in the biological processes of Pst infecting wheat. This study can assist scholars in conducting studies on the pathogenesis and help to advance the investigation of wheat-Pst interaction patterns.

Comparative transcriptome analysis reveals nicotine metabolism is a critical component for enhancing stress response intensity of innate immunity system in tobacco.

The pyridine alkaloid nicotine acts as one of best-studied plant resistant traits in tobacco. Previous research has shown that NtERF199 and NtERF189, acting as master regulators within the NIC1 and NIC2 locus, quantitatively contribute to nicotine accumulation levels in N. tabacum. Genome editing-created Nic1(Nterf199) and Nic2 (Nterf189) double mutant provides an ideal platform for precisely dissecting the defensive role of nicotine and the connection between the nicotine biosynthetic pathway with other putative metabolic networks. Taking this advantage, we performed a comparative transcriptomic analysis to reevaluate the potential physiological and metabolic changes in response to nicotine synthesis defect by comparing the nic1nic2 and NIC1NIC2 plants. Our findings revealed that nicotine reduction could systematically diminishes the expression intensities of genes associated with stimulus perception, signal transduction and regulation, as well as secondary metabolic flux. Consequently, this global expression reduction might compromise tobacco adaptions to environmental fitness, herbivore resistances, and plant growth and development. The up-regulation of a novel set of stress-responsive and metabolic pathway genes might signify a newly established metabolic reprogramming to tradeoff the detrimental effect of nicotine loss. These results offer additional compelling evidence regarding nicotine's critical defensive role in nature and highlights the tight link between nicotine biosynthesis and gene expression levels of quantitative resistance-related genes for better environmental adaptation.

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Residues, potential source and ecological risk assessment of polycyclic aromatic hydrocarbons (PAHs) in surface water of the East Liao River, Jilin Province, China.

The environmental risks posed by polycyclic aromatic hydrocarbons (PAHs) and the diversity of their anthropogenic origins make them a global issue. Therefore, it is of utmost significance for protecting the aquatic environment and the growth of neighboring populations to identify their possible origins and ecological risk. Here, we detail the contamination profiles of 15 PAHs found in the East Liao River's surface waters in Jilin Province and use the receptor model Absolute Principal Component Analysis - Multiple Linear Regression (APCS-MLR) and diagnostic ratios method to identify the primary potential sources of pollution. Based on the natural hazard risk formation theory (NHRFT), an ecological risk assessment (ERA) model for PAHs in the East Liao River was developed. The method assesses the ecological risk status of PAHs by integrating the risk quotient (RQ) approach and the DPSIRM (driving force, pressure, state, impact, response, management) conceptual framework. Total concentrations in the surface water body were between 396.42 and 624.06 ng/L, with an average of 436.99 ng/L. The source research revealed that coal, biomass, and traffic emission sources are the most likely PAH contributors to the East Liao River. The ERA found that the majority of the sites' locations of the study were at low risk for PAHs in surface water bodies (30.7 % and 32.2 %, respectively), while only a tiny percentage of sites were at high or very high risk (1.8 % and 13.6 %). The study results provide theoretical support for the East Liao River's ecological, environmental protection, and policy formulation.

Construction of a high-density genetic map and dissection of genetic architecture of six agronomic traits in tobacco (Nicotiana tabacum L.).

Tobacco (Nicotiana tabacum L.) is an economic crop and a model organism for studies on plant biology and genetics. A population of 271 recombinant inbred lines (RIL) derived from K326 and Y3, two elite flue-cured tobacco parents, has been constructed to investigate the genetic basis of agronomic traits in tobacco. Six agronomic traits including natural plant height (nPH), natural leaf number (nLN), stem girth (SG), inter-node length (IL), length of the largest leaf (LL) and width of the largest leaf (LW) were measured in seven environments, spanning the period between 2018 and 2021. We firstly developed an integrated SNP-indel-SSR linkage map with 43,301 SNPs, 2,086 indels and 937 SSRs, which contained 7,107 bin markers mapped on 24 LGs and covered 3334.88 cM with an average genetic distance of 0.469cM. Based on this high-density genetic map, a total of 70 novel QTLs were detected for six agronomic traits by a full QTL model using the software QTLNetwork, of which 32 QTLs showed significant additive effects, 18 QTLs showed significant additive-by-environment interaction effects, 17 pairs showed significant additive-by-additive epistatic effects and 13 pairs showed significant epistasis-by-environment interaction effects. In addition to additive effect as a major contributor to genetic variation, both epistasis effects and genotype-by-environment interaction effects played an important role in explaining phenotypic variation for each trait. In particular, qnLN6-1 was detected with considerably large main effect and high heritability ( h a 2 =34.80%). Finally, four genes including Nt16g00284.1, Nt16g00767.1, Nt16g00853.1, Nt16g00877.1 were predicted as pleiotropic candidate genes for five traits.

Transcriptomics and iTRAQ-proteomics analyses provide novel insights into the defense mechanism of black shank disease in tobacco.

Black shank disease caused by Phytophthora nicotianae is one of the most important diseases in tobacco worldwide and can result in a devastating loss in tobacco cultivation. Many efforts have been carried out to identify the chromosome segment from Nicotiana plumbaginifolia containing a resistance locus carrying a gene named Php; however, the Php gene has not been cloned, and knowledge of the potential mechanism of the Php gene in the resistant lines is limited. To further characterize the resistance mechanism of the Php gene, we first used the resistant line "RBST" and the susceptible cultivar "Honghuadajinyuan" (HD) to obtain the near-isogenic line RBS89 containing the Php gene from RBST. RBS89 showed high resistance to black shank disease. Transcriptomic and iTRAQ analyses were applied to explore the potential defense mechanisms in RBS89 plants in comparison with HD plants with or without inoculation. Many differentially expressed genes (DEGs) and proteins were identified, and some pathogenesis-related (PR) proteins were extensively abundant in the RBS89 plants when compared with the HD plants in response to black shank disease. Importantly, overexpression of the PR gene NtPR-1B in HD plants improved the resistance of tobacco plants to black shank disease, indicating that NtPR-1B and Php genes might have similar roles in protecting tobacco from black shank disease. However, the relationship between NtPR-1B and Php genes requires further analysis. Therefore, our study provides valuable information for breeding tobacco cultivars with black shank disease resistance and sheds light on the defense mechanism of black shank disease in tobacco for enhancing Phytophthora resistance in other Solanaceae crops.

The complete chloroplast genome of Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Vivianiis 1802 is an annual herb, native to Mexico and South America. It is one of the most widely distributed tobacco species. As a wild tobacco, N. plumbaginifolia has provided several economically important disease-resistance genes to cultivated tobacco. We assembled the complete chloroplast genome of N. plumbaginifolia. The chloroplast genome is 155,945 bp in length, which includes a large single copy region (86,621 bp), a small single copy region (18,528 bp) and two separated inverted repeat regions (25,398 bp). A total of 117 unique genes were annotated, consisting of 84 protein-coding genes, 29 tRNA genes and 4 rRNA genes. Based on chloroplast genomes of 17 Nicotiana species, phylogenetic analyses indicated that N. plumbaginifolia was closely related to N. suaveolens and N. amplexicaulis.

Comprehensive Risk Assessment of High Temperature Disaster to Kiwifruit in Shaanxi Province, China.

In recent years, the main kiwifruit producing region, central-south Shaanxi Province, has often suffered from the threat of extreme high temperatures. Assessing the risk of high-temperature disasters in the region is essential for the rational planning of agricultural production and the development of resilience measures. In this study, a database was established to assess the risk of a high-temperature disaster to kiwifruit. Then, four aspects, hazard, vulnerability, exposure and disaster prevention and mitigation capacity, were taken into account and 19 indexes were selected to make an assessment of the risk of a high-temperature disaster. At the same time, 16 indexes were selected for the assessment of the climatic suitability of kiwifruit in terms of light, heat, water, soil and topography, and were used as one of the indexes for exposure assessment. The analytic hierarchy process and the entropy weighting method were combined to solve the weights for each index. The results reveal that: (1) The Guanzhong Plain has a high climatic suitability for kiwifruit, accounting for 15.14% of the study area. (2) The central part of the study area and southern Shaanxi are at high risk, accounting for 22.7% of the study area. The major kiwifruit producing areas in Shaanxi Province (e.g., Baoji) are at a low risk level, which is conducive to the development of the kiwifruit industry. Our study is the first to provide a comprehensive assessment of the risk of a high-temperature disaster to the economic fruit kiwifruit, providing a reference for disaster resilience and mitigation.

Wan-Nian-Qing, a Herbal Composite Prescription, Suppresses the Progression of Liver Cancer in Mice by Regulating Immune Response.

The Wan-Nian-Qing prescription (WNQP), an herbal composite containing Ornithogalum caudatum, has been used in China for several years for cancer treatment. However, the mechanism of its pharmacological action against liver cancer is not clear. This study aimed to investigate the role of WNQP in inhibiting tumor growth in hepatocellular carcinoma model mice and determine its mechanism of action. We established HepG2- and SMMC-7721-xenografted tumor models in nude mice and BALB/c mice. The mice were administered WNQP for 2 weeks. The bodyweight of each mouse was monitored every day, and the tumor size was measured using vernier caliper before each round of WNQP administration. After the last dose, mice were sacrificed. The tumors were removed, lysed, and subjected to proteome profiling, enzyme-linked immunosorbent assay, and western blotting. The liver, spleen, and kidney were collected for histopathological examination. The effects of WNQP against liver cancer were first systematically confirmed in HepG2- and SMMC-7721-xenografted nude mice and BALB/c mice models. WNQP inhibited tumor growth, but failed to affect bodyweight and organ structures (liver and spleen), confirming that it was safe to use in mice. In BALB/c mice, WNQP regulated immune function, inferred from the modulation of immune-related cytokines such as interleukins, interferon, tumor necrosis factors, and chemokines. Further results confirmed that this regulation occurred via the regulatory effects of WNQP on Nrf2 signaling. WNQP can inhibit the growth of HepG2- and SMMC-7721-xenografted tumors in nude mice and BALB/c mice. This effect manifests at least partially through immunomodulation mediated apoptosis.

GIS- and MCD-based suitability assessment for optimized location of solid waste landfills in Dar es Salaam, Tanzania.

Despite recent advancements in waste management technologies, landfills remain the dominant approach for the final disposal of solid waste (SW) around the world. However, landfills are associated with adverse environmental and health-related impacts. This study is primarily applied to Dar es Salaam, a city in the East African region that has failed to follow appropriate approaches for site selection and that the methods used to select the current sites are manually operated. The study used a geographical information system (GIS) combined with multicriteria decision analysis (MCDA) to objectively determine the optimal landfill sites. A comprehensive list of assessment criteria was examined through a computerized site selection procedure based on GIS to analyze the ground that was surveyed and to remotely obtain data. Analytical hierarchy process (AHP)-one of the best MCDA approaches-was used to assign relative weight and evaluate each criterion. Finally, the study computed site suitability indices (SSIs), which aids in the identification of site suitability levels (SSLs) for the entire region. The final suitability map produced by overlaying the criteria map layers showed that a large part of the study area (60.92%) was completely unsuitable for landfill siting. The remaining areas consisted of land with varying suitability level, which were grouped into four classes: very low (30.92%), low (2.05%), moderate (5.65%), and high (0.46). To this end, only the high suitability class (0.46%) was of acceptable suitability levels (ASLs) (SSI ≥ 2.0). From the numerous identified sites with ASL, only three sites were suggested. Due to concerns regarding the lifespan and environment-related risks associated with these suggested sites, there is a need to further study new environmentally friendly techniques, considering the importance of circular economy agenda of waste to resource/energy for sustainable solid waste management.

Genome-wide association study of leaf chemistry traits in tobacco.

Leaf chemistry traits are some of the key factors influencing tobacco quality, which can be significantly reduced by lower chemical components in cured leaf. To improve tobacco quality through breeding, genetic diversity analysis, population structure analysis, and genome-wide association studies were performed in a panel of 347 tobacco germplasms and the markers associated with five leaf chemistry traits, including total sugar (TS), reducing sugar (RS), total nitrogen (TN), nicotine (NIC), and total potassium (TP) contents were identified. Four groups were classified at a genetic distance of 0.316 by genetic diversity analysis based on coefficient parameter NEI72 using a program NTSYS-pc2.10e, whereas four well-differentiated subpopulations were postulated in the 347 tobacco accessions. A total of 47 target trait-associated SNPs was detected in at least three environments as well as the best linear unbiased predictions (BLUPs) across all environments, among which two, two, four, six, and one highly suggestive associated SNPs were repeatedly detected in all environments and BLUPs for TS, RS, TN, NIC, and TP, respectively. On the basis of the phenotypic effects of the alleles corresponding to suggestive associated SNPs, five tobacco accessions harboring favorable alleles with elite phenotypic performance in leaf chemistry traits were identified. The results could facilitate quality tobacco breeding for higher leaf chemistry trait contents through molecular marker-assisted approaches.

Comprehensive assessment of heavy metals pollution of farmland soil and crops in Jilin Province.

As a major agricultural province in China, it is necessary to study the content of heavy metals in farmland soil and crop in Jilin Province and to evaluate the risks to ecology and human health. This study presented the work completed on 79 soil samples, 10 rice samples, 66 maize samples and 15 soybean samples collected from Jilin Province farmland and evaluated six heavy metals (Zn, Cu, Pb, Cd, Hg and As) concentrations. The results showed that the concentrations of the six heavy metals in farmland soil and crop samples from Jilin Province basically met the soil standards and food health standards of China. The agricultural soil pollution spatial distribution was the most serious in the south of Jilin Province and the lightest in the west. The non-carcinogenic risks faced by children eating crops were higher than those of adults, but the carcinogenic risks were lower than those of adults. Both of the two health risks to adults and children from eating crops were very limited. The results would help determine the heavy metals pollution in farmland soil in Jilin Province efficiently and accurately and helped decision makers to achieve a balance between production and environmental regulation.

Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L.

Tobacco (Nicotiana tabacum L.) is an essential commercial crop and an ideal model plant for biological mechanism studies. As an allopolyploid species, tobacco harbors a massive and complex genome, which makes the application of molecular markers complicated and challenging. In our study, we performed whole-genome sequencing of an intraspecific recombinant inbred line (RIL) population, a F1 generation and their parents. With the Nicotiana tabacum (K326 cultivar) genome as reference, a total of 45,081 markers were characterized to construct the genetic map, which spanned a genetic distance of 3486.78 cM. Evaluation of a two-dimensional heat map proved the high quality of the genetic map. We utilized these markers to anchor scaffolds and analyzed the ancestral genome origin of linkage groups (LGs). Furthermore, such a high-density genetic map will be applied for quantitative trait locus (QTL) detection, gene localization, genome-wide association studies (GWAS), and marker-assisted breeding in tobacco.

Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites.

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.

The DPSIR Model for Environmental Risk Assessment of Municipal Solid Waste in Dar es Salaam City, Tanzania.

Environmental risk has become an area of major concern and research, drawing special attention. This study on the environmental risk assessment (ERA) of Dar es Salaam Municipal Solid Waste comes at a time when the Government of Tanzania is becoming increasingly concerned about dealing with high levels of pollution from municipal solid waste (MSW). The paper employed the Driving force-Pressure-State-Impact-Response (DPSIR) model to establish an environmental risk indicator system and the analytical hierarchy process (AHP) to calculate and analyze risk values, based on the actual situation of MSW in the city of Dar es Salaam. It lists several measures that have been taken in response to the current significantly high levels of pollution, which have assisted in maintaining the environmental risk index (ERI) at a medium level (0.4⁻0.6) during the period from 2006⁻2017. However, these measures have not been adequate enough to manage the external pressure. The ERI has been increasing gradually, calling for timely formulation of demand-specific waste management policies to reduce the possibility of reaching the critical point in near future. With the use of the DPSIR model for ERA, this study has become highly valuable, providing empirical justification to reduce environmental risk from MSW, which is one of the main sources of environmental pollution in the urban areas of developing countries.

The Spatial Distributions and Variations of Water Environmental Risk in Yinma River Basin, China.

Water environmental risk is the probability of the occurrence of events caused by human activities or the interaction of human activities and natural processes that will damage a water environment. This study proposed a water environmental risk index (WERI) model to assess the water environmental risk in the Yinma River Basin based on hazards, exposure, vulnerability, and regional management ability indicators in a water environment. The data for each indicator were gathered from 2000, 2005, 2010, and 2015 to assess the spatial and temporal variations in water environmental risk using particle swarm optimization and the analytic hierarchy process (PSO-AHP) method. The results showed that the water environmental risk in the Yinma River Basin decreased from 2000 to 2015. The risk level of the water environment was high in Changchun, while the risk levels in Yitong and Yongji were low. The research methods provide information to support future decision making by the risk managers in the Yinma River Basin, which is in a high-risk water environment. Moreover, water environment managers could reduce the risks by adjusting the indicators that affect water environmental risks.

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection.

BACKGROUND: Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure. METHODS: Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control. RESULTS: De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions. CONCLUSION: Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection.

Transcriptomic profile of tobacco in response to Phytophthora nicotianae infection.

Black shank, caused by Phytophthora nicotianae (P. nicotianae), is a serious disease of cultivated tobacco (Nicotiana tabacum) worldwide. The interactions between tobacco and P. nicotianae are complex and the outcomes of the interactions depend on the tobacco genotype, P. nicotianae strain, and environmental conditions. In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare transcriptional changes in the stems of tobacco upon inoculation with P. nicotianae strain race 0. We used two tobacco varieties: RBST (named from resistance to black shank and tobacco mosaic virus), which was resistant to the P. nicotianae strain race 0, and Honghuadajinyuan (HD), which was susceptible to P. nicotianae race 0. Samples were collected 12 and 72-hour post inoculation (hpi). Analysis of differentially expressed genes (DEGs) and significantly enriched GO terms indicated that several basic defense mechanisms were suppressed in both varieties, which included response to wounding (GO: 0009611), and defense response to fungus (GO: 0050832). We also found some genes that may especially be related to mechanisms of resistance in RBST, such as the one encoding a chitinase. These results will provide a valuable resource for understanding the interactions between P. nicotianae and tobacco plants.

Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco.

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.