Dissection of genetic architecture of nine hazardous component traits of mainstream smoke in tobacco (Nicotiana tabacum L.).

Tobacco (Nicotiana tabacum L.) use is the leading cause of preventable death, due to deleterious chemical components and smoke from tobacco products, and therefore reducing harmful chemical components in tobacco is one of the crucial tobacco breeding targets. However, due to complexity of tobacco smoke and unavailability of high-density genetic maps, the genetic architecture of representative hazardous smoke has not been fully dissected. The present study aimed to explore the genetic architecture of nine hazardous component traits of mainstream smoke through QTL mapping using 271 recombinant inbred lines (RILs) derived from K326 and Y3 in multiple environments. The analysis of genotype and genotype by environment interaction (GE) revealed substantially greater heritability over 95% contributed mostly by GE interaction effects. We also observed strong genetic correlations among most studied hazardous smoke traits, with the highest correlation coefficient of 0.84 between carbon monoxide and crotonaldehyde. Based on a published high-density genetic map, a total of 19 novel QTLs were detected for eight traits using a full QTL model, of which 17 QTLs showed significant additive effects, six showed significant additive-by-environment interaction effects, and one pair showed significant epistasis-by-environment interaction effect. Bioinformatics analysis of sequence in QTL region predicted six genes as candidates for four traits, of which Nt21g04598.1, Nt21g04600.1, and Nt21g04601.1 had pleiotropic effects on PHE and TAR.

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.

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.

Highly sensitive time-resolved fluoroimmunoassay for the quantitative onsite detection of Alternaria longipes in tobacco.

AIMS: Alternaria longipes is a causal agent of brown spot of tobacco, which remains a serious threat to tobacco production. Herein, we established a detection method for A. longipes in tobacco samples based on the principle of time-resolved fluoroimmunoassay, in order to fulfil the requirement of rapid, sensitive and accurate detection in situ. METHODS AND RESULTS: A monoclonal antibody against A. longipes was generated, and its purity and titration were assessed using western blot and ELISA. The size of europium (III) nanospheres was measured to confirm successful antibody conjugation. The method described here can detect A. longipes protein lysates as low as 0.78 ng ml-1 , with recovery rates ranging from 85.96% to 99.67% in spiked tobacco. The specificity was also confirmed using a panel of microorganisms. CONCLUSIONS: The fluorescent strips allow rapid and sensitive onsite detection of A. longipes in tobacco samples, with high accuracy, specificity, and repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel detection method provides convenience of using crude samples without complex procedures, and therefore allows rapid onsite detection by end users and quick responses towards A. longipes, which is critical for disease control and elimination of phytopathogens.

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.

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.

Characterization and evolution of gene clusters for terpenoid phytoalexin biosynthesis in tobacco.

MAIN CONCLUSION: The study performed genome-wide identification, characterization and evolution analysis of gene clusters for phytoalexin terpenoid biosynthesis in tobacco, and specifically illustrated ones for capsidiol, an efficient defensive specialized metabolite. Terpenoid phytoalexins play an important role in plant self-defense against pest and pathogen attack. Terpenoid biosynthesis involves terpene synthase and cytochrome P450, which always locate and function as cluster(s). In this study, we performed genome-wide investigation of metabolic gene clusters involved in terpenoid production in tobacco (Nicotiana tabacum). Due to the complexity of the tobacco genome, we modified a published prediction pipeline to reduce the influence of the large number of repeats and to improve the annotation of tobacco genes with respect to their metabolic functions. We identified 1181 metabolic gene clusters with 34 of them potentially being involved in terpenoid biosynthesis. Through integration with transcriptome and metabolic pathway annotation analyses, 3 of the 34 terpenoid biosynthesis-related gene clusters were determined to be high-confidence ones, with 2 involved in biosynthesis of capsidiol, a terpenoid recognized as 1 of the effective resistance compounds in the Nicotiana species. The capsidiol-related gene cluster was conserved in N. sylvestris, N. tomentosiformis and N. attenuate. Our findings demonstrate that phytoalexins in tobacco can arise from operon-like gene clusters, a genomic pattern characterized as being beneficial for rapid stress response, gene co-regulation, co-function and co-heredity.

NaPDR1 and NaPDR1-like are essential for the resistance of Nicotiana attenuata against fungal pathogen Alternaria alternata.

Pleiotropic drug resistance (PDR) transporters are widely distributed membrane proteins catalyzing the export or import of a diverse array of molecules, and are involved in many plant responses to biotic and abiotic stresses. However, it is unclear whether PDRs are involved in Nicotiana attenuata resistance to the necrotic fungal pathogen Alternaria alternata. In this study, transcriptional levels of both NaPDR1 and NaPDR1-like were highly induced in N. attenuata leaves after A. alternata inoculation. Interestingly, silencing NaPDR1 or NaPDR1-like individually had little effect on N. attenuata resistance to A. alternata; however, when both genes were co-silenced plants became highly susceptible to the fungus, which was associated with elevated JA and ethylene responses. Neither NaPDR1 nor NaPDR1-like was significantly elicited by exogenous treatment with methyl jasmonate (MeJA), whereas both were highly induced by ethylene. The elicitation levels of both genes by A. alternata were significantly reduced in plants with impaired JA or ethylene signaling pathways. Thus, we conclude that both NaPDR1 and NaPDR1-like function redundantly to confer resistance against A. alternata in N. attenuata, and the elicitation of the transcripts of both genes by the fungus is partially dependent on ethylene and jasmonate signaling.

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.

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

BACKGROUND: Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive. FINDINGS: We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome. CONCLUSIONS: These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.