Nicotiana tabacum is an allotetraploid hybrid of Nicotiana sylvestris and Nicotiana tomentosiformis and a model organism in genetics. However, features of subgenome evolution, expression coordination, genetic diversity and complex traits regulation of N. tabacum remain unresolved. Here we present chromosome-scale assemblies for all three species, and genotype and phenotypic data for 5,196 N. tabacum germplasms. Chromosome rearrangements and epigenetic modifications are associated with genome evolution and expression coordination following polyploidization. Two subgenomes and genes biased toward one subgenome contributed unevenly to complex trait variation. Using 178 marker-trait associations, a reference genotype-to-phenotype map was built for 39 morphological, developmental and disease resistance traits, and a novel gene regulating leaf width was validated. Signatures of positive and polygenic selection during the process of selective breeding were detected. Our study provides insights into genome evolution, complex traits regulation in allotetraploid N. tabacum and the use of GeneBank-scale resources for advancing genetic and genomic research.

AbstractNicotiana tabacumis a model organism in plant molecular and pathogenic research and has significant potential in the production of biofuels and active pharmaceutical compounds in synthetic biology. Because of the large allotetraploid genome of tobacco, its genomic features, genetic diversity and genetic regulation of many complex traits remain unknown. In this study, we present a nearly complete chromosome-scale assembly ofN. tabacumand provide evidence that homoeologous exchange between subgenomes and epigenetic remodelling are likely mechanisms of genome stabilization and subgenome coordination following polyploidization. By leveraging GenBank-scale sequencing and phenotyping data from 5196 lines, geography at the continent scale, rather than types assigned on the basis of curing crop practices, was found to be the most important correlate of genetic structure. Using 178 marker□trait associations detected in genome-wide association analysis, a reference genotype-to-phenotype map was built for 39 morphological, developmental, and disease-resistance traits. A novel gene, auxin response factor 9 (Arf9), associated with wider leaves after being knocked out, was fine-mapped to a single nucleotide polymorphism (SNP). This point mutation alters the translated amino acid from Ala203to Pro203, likely preventing homodimer formation during DNA binding. Our analysis also revealed signatures of positive and polygenic selection for multiple traits during the process of selective breeding. Overall, this study demonstrated the power of leveraging GenBank genomics to gain insights into the genomic features, genetic diversity, and regulation of complex traits inN. tabacum, laying a foundation for future research on plant functional genomics, crop breeding, and the production of biopharmaceuticals and biofuels.

Agronomic traits such as plant height (PH), leaf number (LN), leaf length (LL), and leaf width (LW), which are closely related to yield and quality, are important in tobacco (Nicotiana tabacum L.). To identify quantitative trait loci (QTLs) associated with agronomic traits in tobacco, 209 recombinant inbred lines (RILs) and 537 multiparent advanced generation intercross (MAGIC) lines were developed. The biparental RIL and MAGIC lines were genotyped using a 430 K single-nucleotide polymorphism (SNP) chip assay, and their agronomic traits were repeatedly evaluated under different conditions. A total of 43 QTLs associated with agronomic traits were identified through a combination of linkage mapping (LM) and association mapping (AM) methods. Among these 43 QTLs, three major QTLs, namely qPH13-3, qPH17-1, and qLW20-1, were repeatedly identified by the use of various genetically diverse populations across different environments. The candidate genes for these major QTLs were subsequently predicted. Validation and utilization of the major QTL qLW20-1 for the improvement of LW in tobacco were investigated. These results could be applied to molecular marker-assisted selection (MAS) for breeding important agronomic traits in tobacco.

Ralstonia solanacearum species complex (RSSC) is a diverse group of plant pathogens that attack a wide range of hosts and cause devastating losses worldwide. In this study, we conducted a comprehensive analysis of 131 RSSC strains to detect their genetic diversity, pathogenicity, and evolution dynamics. Average nucleotide identity analysis was performed to explore the genomic relatedness among these strains, and finally obtained an open pangenome with 32,961 gene families. To better understand the diverse evolution and pathogenicity, we also conducted a series of analyses of virulence factors (VFs) and horizontal gene transfer (HGT) in the pangenome and at the single genome level. The distribution of VFs and mobile genetic elements (MGEs) showed significant differences among different groups and strains, which were consistent with the new nomenclatures of the RSSC with three distinct species. Further functional analysis showed that most HGT events conferred from Burkholderiales and played a great role in shaping the genomic plasticity and genetic diversity of RSSC genomes. Our work provides insights into the genetic polymorphism, evolution dynamics, and pathogenetic variety of RSSC and provides strong supports for the new taxonomic classification, as well as abundant resources for studying host specificity and pathogen emergence.

烤烟新品种中烟207是以优质高香气为主攻目标,兼顾抗病性和产量,采用云烟85的雄性不育同型系(MS云烟85)为母本,以香气浓郁且耐气候性斑点病的烤烟新品系0953为父本,经杂交选育而成的雄性不育一代杂交种.该品种株型筒形,叶形长椭圆,田间生长势较强;综合经济性状略优于K326,略低于云烟87.烤后原烟以金黄色为主,结构、身份和油分较好;化学成分较协调;感官香气质、香气量、浓度和余味优于K326和云烟87;综合质量与K326相当,优于云烟87.中抗黑胫病和根结线虫病,中感青枯病和赤星病,感TMV、CMV和PVY,气候性斑点病的抗耐性优于K326和云烟87.综合评价,中烟207是品质、经济、抗病性状较能兼顾的烤烟新品种,适宜在东南、四川及陕西烟区种植.

Cigar tobacco is an important economic crop that is widely grown around the world. In recent years, varietal identification has become a frequent problem in germplasm preservation collections, which causes considerable inconvenience and uncertainty in the cataloging and preservation of cigar germplasm resources, in the selection of parental lines for breeding, and in the promotion and use of high quality varieties. Therefore, the use of DNA fingerprints to achieve rapid and accurate identification of varieties can play an important role in germplasm identification and property rights disputes. In this study, we used genotyping-by-sequencing (GBS) on 113 cigar tobacco accessions to develop SNP markers. After filtering, 580,942 high-quality SNPs were obtained. We used the 580,942 SNPs to perform principal component analysis (PCA), population structure analysis, and neighbor joining (NJ) cluster analysis on the 113 cigar tobacco accessions. The results showed that the accessions were not completely classified based on their geographical origins, and the genetic backgrounds of these cigar resources are complex and diverse. We further selected from these high-quality SNPs to obtained 163 SNP sites, 133 of which were successfully converted into KASP markers. Finally, 47 core KASP markers and 24 candidate core markers were developed. Using the core markers, we performed variety identification and fingerprinting in 216 cigar germplasm accessions. The results of SNP fingerprinting, 2D barcoding, and genetic analysis of cigar tobacco germplasm in this study provide a scientific basis for screening and identifying high-quality cigar tobacco germplasm, mining important genes, and broadening the basis of cigar tobacco genetics and subsequent breeding work at the molecular level.

为进一步挖掘四川、山东产区烟叶致香物质含量特色,本研究基于51份烤烟种质,在四川、山东两产区开展了烟叶8类致香物质含量检测分析比较工作.结果显示,整体上看,四川多酚类、叶绿素降解物绝对和相对含量高于山东产区,苯丙氨酸降解物、高级脂肪酸、多元酸绝对和相对含量低于山东产区;从不同基因型上看,四川92.2％以上种质多酚类、叶绿素降解物的绝对和相对含量高于山东产区,76.5％以上种质苯丙氨酸降解物、高级脂肪酸和多元酸的绝对和相对含量低于山东产区,进一步印证了两产区致香物质含量在整体上表现出的特点;51份种质中,四川产区有76.5％烤烟种质预测具有清香型风格倾向,在山东产区有90.2％烤烟种质预测具有中间香型风格倾向,多酚类和叶绿素降解物含量较山东产区高可能是四川清香型风格烟叶的重要物质基础;苯丙氨酸降解物、高级脂肪酸和多元酸含量较四川产区高可能是山东中间香型风格烟叶的重要物质基础.基因型和生态条件对香型指数均有较大影响,生态条件影响更大.

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.

为将“第三次全国农作物种质资源普查与收集行动”重庆项目组收集到的63份烟草种质资源入国家种质资源库进行编目和保存,对其进行田间鉴定和拍照记录.形态特征和主要农艺性状的遗传多样性分析表明,供试种质的数量性状和质量性状遗传多样性丰富,质量性状以叶形的多样性指数最高,数量性状中叶长、叶宽和茎围的多样性指数较高.聚类分析将新收集烟草种质资源分为4个类群,其中第Ⅰ类群以黄花烟为主,第Ⅲ类群的植株最高、叶片最大,可用于高产育种.主要病毒病的抗性鉴定发现,有11份种质TMV和CMV的抗性均较强,其中2份种质对TMV免疫且高抗CMV,可用于抗病育种.另外,通过田间表型和SSR分子标记对拟编目资源进行鉴定整理,剔除了重复的种质资源.因此,烟草种质资源的普查收集工作对于丰富国家烟草种质资源库和新品种选育具有重大意义.

Main conclusion NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco. Abtract The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase ( ALS ) genes in tobacco through homology-based gene prediction using Arabidopsis ALS . Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.

为了提高野生烟种质资源的开发利用效率,本研究利用表型性状和SSR分子标记技术对30份野生烟种质资源进行遗传多样性分析.结果 表明,30份种质资源表型性状具有丰富的变异类型,遗传多样性指数(H')的变幅为0.831～1.955,其中节距的多样性指数最高,叶宽和叶长次之.16对SSR引物共检测到98个等位基因,平均为6.125,Shannon信息指数(Ⅰ)、期望杂合度(He)、观测杂合度(Ho、多态性信息含量(PIC)的均值分别为1.5425、0.7596、0.2404、0.8668.聚类分析和群体结构分析显示供试材料可分为4个类群,分类结果大致相同,各类群聚集的地域性规律较明显.利用这16对引物构建了30个野生烟种质的指纹图谱,为野生烟种质资源鉴定体系的研究奠定理论基础.抗病性鉴定发现,N.exigua Wheeler等7份种质表现为抗黑胫病;N.nesophila Johnston高抗青枯病,N.debneyi Domin和N.stocktonii Brandegee抗青枯病;N.alata Link&Otto等5份种质对烟草花叶病免疫;N.alata Link&Otto等9份种质抗黄瓜花叶病;N.debneyi Domin等15份种质抗马铃薯Y病.本研究可为野生烟种质资源的鉴定评价、种质创新和优异基因发掘奠定基础.

冷杉醇是一些香料烟和部分雪茄烟中主要的赖百当类物质,对烟草的香气风格起着重要作用,然而烤烟中基本不含有.开发分子标记是将这种香气特征转入烤烟的有效途径.本研究基于等位基因特异性PCR(AS-PCR)技术,设计了一系列引物,并最终筛选到引物对1F/R1与其互补引物对8F/R1具有良好的多态性,这两对引物在6个亲本和F1群体也得到了验证.利用两个引物对47份烤烟品种和98份'中烟100'的高香气突变体进行基因分型,同时利用GC-MS检测表型,最终获得材料'大白筋599'、'革新三号'、'NC2326'和'TBT6'突变株含有冷杉醇.结果表明,这两对标记可用于烟草育种分子标记辅助选择,含冷杉醇的烤烟种质资源可作为烟草育种的重要亲本材料.

In order to clarify the usability of SSR molecular markers closely linked to sugar esters in the actual breeding of tobacco, and to accelerate the genetic improvement of flue-cured tobacco sugar esters, in this study, using polyacrylamide gel electrophoresis and GC-MS detection techniques, 5 tobacco germplasms with different genetic backgrounds and a genetic population with 192 recombinant inbred lines were used as experimental materials for screening and testing of 5 SSR molecular markers. The results showed that, among the 5 molecular markers, the polymorphisms of 2 molecular markers PT20315 and PT30354 were stable in different genetic backgrounds, and the correspondence between their polymorphic bands and the phenotypic difference of sugar esters were better. In the recombinant inbred line population, the consistency rate between the genotypes identified by the 2 molecular markers and phenotypes of sugar esters reached 93.75% and 90.58% respectively. The 2 molecular markers could be used in the molecular-assisted breeding of high-sugar ester tobacco. Currently, using of the 2 molecular markers in assisted selection has directionally improved new lineage materials such as high sugar ester K326 etc.

Tobacco bacterial wilt is a bacterial disease caused by Ralstonia solanacearum, and is one of the main diseases that harm China's tobacco production. Analysis of the resistance genetics of tobacco bacterial wilt has important significance in guiding disease resistance breeding. In this study, a multi-generation combined analysis method of the main gene + multi-gene mixed genetic model was used to construct two different hybrid combination groups for the analysis of population genetic effects with multiple resistance/susceptible materials as parents. The results showed that bacterial wilt resistance of tobacco appeared to be a quantitative trait and the inheritance of Yanyan 97 fit to a mixed genetic model of two major genes with additive-dominance-epistatic effects plus poly-genes with additive-dominance-epistatic effects. The inheritance of Fandi No.3-C fit to a mixed genetic model of one major gene with additive-dominance effects plus poly-genes with additive-dominance-epistatic effects. The results also indicated that tobacco bacterial wilt resistance is dominated by additive effects, which is supplemented by dominance effects, and is conducive to allele aggregation breeding and early generation selection.

The new flue-cured tobacco variety Zhongchuan208 (CF228) is a male sterile hybrid bred by crossbreeding with MS-Zhongyan103 as the female parent and T136 as the male parent, aiming at high quality, suitable yield and TMV resistance. In the field, Zhongchuan208 grows neatly and uniformly with strong vigor, with tower shape plant type and oval leaf shape. Comprehensive economic traits are significantly better than K326, slightly better than Yunyan87, and the appearance quality is better than K326, slightly better or equivalent to Yunyan87. The overall coordination of chemical components in flue-cured tobacco leaves is good with slightly higher potassium content. It is characterized by regional style, strong industrial usability, clear fragrance style and outstanding sweet fragrance in southwest tobacco areas. It has immunity to TMV, moderate susceptibility to black shank disease, root knot nematode disease and PVY, and is susceptible to bacterial wilt disease, brown spot disease and CMV. In summary, Zhongchuan208 is a new flue-cured tobacco variety with good quality, economic characters and disease resistance, which has strong adaptability and is suitable for planting in southwest and central China tobacco areas.

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,BC1 F2 individuals and BC1 F2:3 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 BC1 F2 population in the field and in BC1 F2:3 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.

To catalog and preserve 77 tobacco germplasm resources collected from Hubei Province into the National Bank of Tobacco Germplasm Resources,field identification and evaluation of these tobacco germplasm resources were carried out.Data on period of duration,morphological characteristics,main agronomic traits and resistance to major virus diseases,have been obtained,and the photos of plant type,leaf,inflorescence,corolla and capsule also have been taken individually.Result showed that the accessions B035,B075 and B076 have resistance or mid-resistance for more than two virus diseases,while B025 is completely resistant to TMV.Field phenotypic characterization and SSR marker analysis were applied to identify the cataloging germplasm,in order to remove the repeated collection germplasm resources.The obtained results of this study will lay a good foundation for enriching the genetic diversity of National Bank of Tobacco Germplasm Resources and assisting the tobacco breeding.

The objective of this study was to explore the key aroma component and molecular basis responsible for the specific aroma traits of flue-cured tobacco Dabaijin599, which will lay the foundation for developing flue-cured tobacco varieties with specific aroma traits and industrial utilization of their leaves. Leaf superficial exudates of different tobacco varieties (lines) were detected and smoking evaluation of the corresponding tobacco leaves was conducted to determine the main aroma components contributed to the specific aroma traits of Dabaijin599. Sequence polymorphism analysis of the key genes regulating the aroma component predicted was conducted to explore the molecular mechanism of specific aroma traits of Dabaijin599. The results showed that leaf superficial exudates of flue-cured tobacco Dabaijin599 and Oriental tobacco Samsun contain labdanoid diterpene abienol, while other flue-cured tobacco variety G28 etc. have no labdanoid diterpene abienol. Among the backcross or hybridization population of G28 with Dabaijin599, the offsprings contained abienol present special aroma characteristics like Dabaijin599, however, the ones without abienol had no special aroma characteristics. The 292th base of the key gene NtCPS2 controlling the synthesis of abienol was the wild type G in Dabaijin599 and the mutation type T in common flue-cured tobacco cultivars. Abienol was the key aroma component contributed to specific aroma of flue-cured tobacco variety Dabaijin599. SNP polymorphism in the 292th base of the gene NtCPS2, among Dabaijin599 and other varieties, results in the aroma characteristic difference between Dabaijin599 and the common flue-cured tobacco varieties. Enriching the type and content of aroma components by genetic improvement is an effective way to breed flue-cured tobacco varieties with special aroma.

Two resistant × susceptible hybrid combinations were made between the line resistant to black shank (Beinhart1000-1) and two susceptible materials, SNN (Samsun NN) and XHJ (Xiaohuangjin1025). All the three parents and the two crosses were performed black shank artificial inoculation and resistance evaluation at mature stage. The genetic characteristics of Beinhart1000-1 resistance were studied using the major gene plus poly gene model. The results showed that: The black shank resistance genetic model of the cross Beinhart1000-1 × SNN was identified to fit model E2 and the heritability of major genes and poly-genes were estimated to be 99.14% and 0.48%, respectively. For cross Beinhart1000-1 × XHJ1025, it fitted B1 model very well, and the heritability of major genes was estimated to be 99.52%. Because of the higher heritability of the major genes, the selection of black shank resistant plants could be done in early generations. These results could provide information on MAS for improvement of resistance to black shank disease in tobacco.