为提高抗PVY烟草品种的分子育种效率,本研究针对抗病种质资源半坤村晒烟中隐性抗病基因eIF4E1的SNP位点G149C建立一种简单快速的双向等位基因特异性PCR(Bi-directional PCR amplification of specific alleles,Bi-PASA)检测方法,并对该检测方法的特异性、准确度及实际应用效果进行验证.结果表明,建立的Bi-PASA检测方法能够在一个PCR反应中有效区分eIF4E1基因G149C位点3种基因型:野生型GG、杂合突变型GC、纯合突变型CC.利用Bi-PASA检测方法可以对K326×半坤村晒烟F2分离群体的基因型进行有效鉴别,且鉴定结果与普通的等位基因特异性PCR(allele-specific PCR,AS-PCR)以及直接测序法的鉴定结果一致.综上所述,本研究建立的Bi-PASA检测方法特异性强、准确度高、操作简便,可更好地应用于eIF4E1基因的分子标记辅助育种.

为提高烟草白粉病隐性抗病基因(感病基因)NtMLO1(M1)和NtMLO2(M2)在分子标记辅助选择育种中基因型的选择效率,根据已报道的M1和M2突变基因序列,设计4对单基因特异性引物,组成2个双重PCR反应体系,分别用于同时扩增M1和M2突变型或M1和M2野生型等位基因.结果表明,建立的2个双重PCR体系扩增的特异性片段与单重PCR体系扩增的片段完全吻合,突变基因纯合体只在突变基因多重PCR体系中产生191和306 bp的特异性片段,野生基因纯合体只在野生基因多重PCR体系中产生437和652 bp的特异性片段,杂合体在2个反应体系中均有特异性片段.利用该体系可经过1次或2次PCR扩增准确检测出回交转育过程中回交或自交后代M1和M2的基因型,加快抗病品种育种进程.

[Introduction] Seven smoke constituents, including hydrogen cyanide (HCN), ammonia (NH3), phenol, benzo[alpha] pyrene (B[a]P), carbon monoxide (CO), crotonaldehyde, and 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK), are proposed be the most relevant constituents for smoking-related diseases. [Methods] Different combinations of leaf stalk positions, varieties and locations were used to create variable chemistry of cigarette filler and smoke. Experimental cigarettes were measured for emission level of seven smoke toxicants and content of seventy-three filler components. [Results] The ranges of coefficient of variation (CV) for seven smoke toxicants were 15.435%-43.15%. The emission pattern of NNK and crotonaldehyde were different from that of other five smoke toxicants. Most of the seven smoke toxicants were influenced in following order: stalk position > location > variety. The leaf constitutes closely correlated with seven smoke toxicants were analyzed. [Conclusions] The results showed that seven toxicants were significantly influenced by leaf position and location, and closely correlated with leaf components, such as potassium, malate and alkaloid contents. The results provide useful and comprehensive information on the affecting factors and correlating leaf constituents for the variations of seven smoke toxicants.

[Objective]To screen the new variety of flue-cured tobacco.[Method]With Yunyan 87 as the control,adaptability demonstration of Yunyan 105 in flue-cured tobacco of Shuicheng County was carried out in 2016-2017.[Result]Topping stage of Yunyan 105 was 13-14 d later than that of control.Yunyan 105 showed poor tolerance to maturity,it was easily affected by powdery mildew.Plant height of Yunyan 105 at top-ping and the remained leaves were 25 cm and 4 leaves greater than those of control.Compared with the control,the single leaf weight of Yunyan 105 was 2.65 g greater,stem content was 1.07 percentage points lower,leaf area density was 12.50 g/m2larger.The safety and flammability of Yunyan 105 were superior to those of control,belonging to middle incense type.The quality of Yunyan 105 was middle +,strength was moderate+,and concenration was middle +.[Conclusion]Further adaptability demonstration was suggested to be carried out.

[Objective]The agronomic traits and chemical compositions of tobacco germplasm resources resistant to brown spot disease were analyzed in order to provide reference for the parent selection of tobacco breeding and full utiliza-tion of the germplasm resources.[Method]The experiment was conducted in field,the field growth period,agronomic traits,economic characters and chemical compositions of 24 tobacco germplasm resources that were resistant to brown spot disease were analyzed,and principal component analysis and hierarchical cluster analysis were also conducted.[Re-sult]The results showed that the growth period of all 24 tobacco germplasm resources showed great differences. The germ-plasm resources with suitable field growth period were Jingyehuang,Zhongyan 86,Yunyan 110,Yunyan 100 and Xiaofeng No. 8. Besides,Yunyan 100,Yunyan 110,8813 and Xiaofeng No. 8 were the germplasm resources with sound comprehensive agronomic traits. Yunyan 100,Yunyan 110,Xiaofeng No. 8,YA-2,Huishui-1,Huishui-2 and Zijiang No. 1 were the germplasm resources with fine main economic characters. Yunyan 100,Yunyan 110,Oxford 26,Danyu No.2 and Huishui-1 were the germplasm resources with coordinated chemical composition in middle tobacco leaves. YA-2,Huishui-1,Zijiang No.1,Yunyan 100,9042,Huishui-2,Coker spedlgreed,YQ-1,Fuyan No.6 and Yunyan 110 were the germplasm resources obtaining high comprehensive score in principal component analysis.[Conclusion]There are great differences among various tobacco germplasm resources that are resistant to brown spot disease. Ten germplasm re-sources,including YA-2,Huishui-1,Zijiang No.1,Yunyan 100,9042,Huishui-2,Cokers pedlgreed,YQ-1,Fuyan No.6 and Yunyan 110,obtain high comprehensive score,so they can be used as parent materials for the breeding of new toba-cco varieties that are resistant to brown spot disease.

2016-2017年通过对云烟99在水城烤烟生产上的应用价值分析,与对照云烟87相比,云烟99结果表现:同年各大田生育期与对照相当;易感白粉病,产值量偏低;叶面积大于对照品种,单叶重大于对照3.49克;其含梗率和叶面密度均低于对照0.88个百分点和3.94g/m2;总糖含量和钾含量大于对照达2.13和0.49个百分点;其烟碱、还原糖、总氮和氯含量均低于对照0.97、0.91、0.07和0.40个百分点;云烟99属"中间"香型,劲头"适中+",浓度"中等+",质量档次为"中等+".

在贵州兴义、 凤冈和贵定3个生态环境种植烤烟品种红花大金元、K326,研究不同生态环境下烤烟品种碳氮代谢关键酶活性及其与致香物质的关系.结果表明,在烟叶生长发育过程中,硝酸还原酶活性持续降低,而蔗糖转化酶表现为先上升后下降的变化趋势,不同生态环境、 品种间的碳氮代谢关键酶活性差异明显.生态环境、 品种及其互作均对致香物质、 感官评吸质量产生影响,其中生态环境、 因素的作用效应较大.相关分析结果显示,烤烟致香物质总量受到移栽后35、50,65d与80 d的NR/INV的影响,且除65 d外,其影响效应均达到极显著水平.NR/INV与烟叶感官评吸质量各指标无明显直线相关关系,但各感官评吸质量指标及评吸质量总分均与某一时期或者几个时期的NR/INV的相关性分别达到差异显著或者极显著水平.

气候要素是否影响地表物理侵蚀速率目前还存在诸多争议.以往对此方面的研究均集中在高降雨区域,而对于低降雨条件下气候因子的控制机制研究目前还很有限.青海湖流域对气候变化十分敏感.本文通过对青海湖流域最大两条河流布哈河和沙柳河每日的温度、降雨、径流和河流悬浮物浓度进行为期一年的持续监测,探讨干旱/半干旱区气候要素对地表物理侵蚀速率的影响.研究结果显示:在气候要素中,降雨以及其产生的径流是青海湖流域物理侵蚀速率最主要的控制因素,而温度的影响较弱.最重要的是,降雨强度决定着最终的侵蚀速率.布哈河和沙柳河流域一次瞬时高强度降雨能产生全年侵蚀通量的30％以上.此外,相同流量条件下,季风前期的侵蚀速率要高于季风中、后期,反映了季风前期低温条件下的冰冻作用,以及春季降尘产生的大量细颗粒物质,增加了雨季来临时侵蚀物质的输出.这些认识对于理解长时间尺度上青海湖沉积物钻孔中沉积速率和古气候要素的关系具有重要意义.

为研究烟草低温早花的分子机理,采用RT-PCR结合RACE法,从烟草品种NC82中克隆到2个Nup96基因c DNA全长序列,分别命名为Nt Nup96a和Nt Nup96b,Gen Bank登陆号分别为KU558693和KU558694。序列分析表明2个基因均编码1037个氨基酸残基的蛋白,氨基酸序列一致性为97%,与拟南芥Nup96蛋白（NP 1 78183）的序列一致性为60%;2个基因编码蛋白具有典型的植物Nup96结构特性,包含1个Nup96结构域和1个自酶解结构域。荧光定量PCR分析表明:在正常的生长条件下,Nt Nup96a和Nt Nup96b在烟草各组织中均有表达,在叶片中的表达量较弱,且随着植株生长其表达量显著下降;在12℃处理10天后,在烟草叶片中2基因的表达都出现了明显的下调,这一结果提示Nup96可能是烟草开花时间调控的抑制因子,其受低温诱导下调表达可能与NC82低温敏感,易早花特性相关。

In order to identify and check the adaptability of new flue-cured tobacco variety of Guiyan 2 to soil, cultivation and curing conditions and its utility value in production in Qianxinan Prefecture ecological areas, a comparative trail with the leading cultivar Yunyan 97 as the control (CK) was made according to the regula-tions of local characteristic and high-quality tobacco production. The experimental results showed that Guiyan 2 had moderate growth duration after transplant, strong field growing power, good uniformity and good agronomic traits; moreover, its yield, production value and rate of first-class tobacco were respectively higher than these of CK by 349.2 kg/hm2, 4746.3 yuan/hm2 and 6%, while its average price and grade index were respectively lower than these of CK by 1.13 yuan/kg and 0.06;Guiyan 2 also had the advantages of appropriate intrinsic chemical component con-tent and proportion, as wel as better curing characteristics, and it got general y better evaluation from tobacco growers, indicating that Guiyan 2 had a better poten-tial to generalize.

为研究中间香型主要烟区化学成分特征和区域特点，对中间香型主要烟区的7个试点11个品种的主要化学成分进行检测分析和聚类。结果表明：中间香型各烟区烟叶主要化学成分除氯含量偏低（为0．2％）和钾氯比偏高（为12．64）外，其余化学成分均符合优质烟的要求。不同产区、不同品种间的烟叶主要化学成分均存在显著性差异。将7个烟区系统聚类为3类，第一类主要化学成分表现为烟碱含量较高，总糖和还原糖的含量较低，糖碱比低，烟区包括陕西旬阳、重庆武隆和贵州遵义；第二类主要化学成分表现为总氮含量、蛋白质含量、氮碱比和两糖比较低，钾含量、钾氯比较高，其余成分含量中等，主要为山东蒙阴烟区；第三类主要化学成分表现为烟碱含量较低，总糖含量、还原糖含量、糖碱比和氮碱比较高，烟区主要为贵州贵定、湖北咸丰和湖南桑植。

The dynamic of key enzyme activities involved in carbon and nitrogen metabolism in different flue-cured tobacco cultivars were studied with K326 and Honghuadajinyuan in field condition, and their correlations with cartenoids was also investigated. The results showed that the activities of nitrate reductase, glutamine synthetase, invertase and amylase all increased at the earlier stage and decreased at later stage in the growth and development period of tobacco. The activities of nitrate reductase, glutamine synthetase, and amylase reached maximum at 60 d after transplanting, but the peak activity value of invertase was at 75 d after transplanting. The contents of carotenoids components (β-carotene, lutein, neoxanthin, violaxthin) decreased gradually with the proceeding of tobacco growth and development process. In the whole growth period, Honghuadajinyuan had higher enzyme activity and carotenoids content than K326. There was significant negative correlation between the activity of invertase and the contents of carotenoids components.

为明确烟叶DNA甲基化的特征与化学成分的关系,利用烤烟DNA甲基化DArT芯片,分析了K326、红花大金元(红大)和遵烟6号在贵定和兴义两个试点,3个部位烟叶在不同发育时期的DNA甲基化特征和化学成分含量.结果显示,烤烟DNA甲基化水平变异较高,最高为64％,最低为35.4％,平均为47.61％,变幅为28.6％.品种间的甲基化模式差异最大,其次是生长部位.DNA甲基化水平与12种物质代谢关键酶活性和76种致香成分的相关分析发现,DNA甲基化与这些酶活性和化学成分呈极显著相关(p＜0.01),在3个品种中同步呈现极显著相关(p＜0.0001)的酶及化学成分有34个,不同品种中也呈现出受特异性调控的化学物质.

Plant germplasm is an important gene resource for plant breeding and genetic improvement of traits . Core collection , as a representative subset of germplasm , provides an effective entry to investigate genetic diversity and screen potential breeding material . Most agronomic traits of tobacco often exhibit strong genetic variation which can be decomposed into genotypic and genotype × environment interaction variations . A tobacco collection of 805 accessions were evaluated in two locations , Fuquan and Jinsha City of Guizhou Province , China . The genetic variations of 12 agronomic traits were analyzed and the genotypic values of each accession were predicted by a mixed linear model approach . Significant genotypic variations were detected for 11 traits , as well as genotype × environment interaction for 10 traits . Based on the predicted genotypic values , the combination strategies of sampling and clustering methods in the procedure of stepwise clustering for constructing core collection were screened in terms of magnitude of genetic variation captured by sampled subsets . Finally , the subset ( S1C3_10) , sampled by a centroid method combined with preferred sampling at 10% proportion , was determined to be a core collection of the tobacco germplasm . The representative and validation of the core collection was examined visually by the accession distribution pattern in the plot established by first two principal components , as well as by the correlation coefficients in terms of magnitude and significance between the core and the initial collections . The results showed that the initial collection was well represented by the core collection , and some potential breeding materials for high yield breeding program of tobacco , such as Y 177 and Y178 , were included in the core collection . In sum , the constructed core collection will facilitate tobacco breeder to access to whole resources and promote the utilization of genetic diversity in future breeding program of tobacco in China .

To assess the genetic diversity of flue-cured tobacco germplasm,developing the elite gene resources,we selected 258 flue-cured tobacco varieties from China and abroad to investigate 13 agronomic traits in two locations with different environments.The genetic diversity and population structure were analyzed with 597 SRAP markers produced by 16 primer combinations.Different association models were tested and the most effective model was chosen for the population to make the association analysis between agronomic traits and SRAP markers.The results revealed that abundant phenotypic and genetic diversity existed in the flue-cured tobacco germplasm accessions.Flue-cured tobaccos from China showed less diversity than those from other countries.The accessions could be divided into seven subpopulations and the classification result was significantly correlation with geographic origins and hereditary relationship.Population structure within exotic varieties was multiplex.MIM_Q+K and MIM_ PCA+K were effective models for association analysis between agronomic traits and SRAP markers.A total of 18 markers were found to be significantly associated(-log P2,P0.01) with six agronomic traits.Among them,markers associated with plant height(me1/em9-16,me1/em9-36,me4/em9-1),leaf number(me1/em2-14,me7/em5-34),bottom leaf width(me6/em2-6) and lumbar leaf length(me2/em6-15) were stably detected in different environments.This study provides evidence for identification of elite flue-cured tobacco germplasm,prediction of candidate gene and marker-assisted selection.

In order to assess the high yield ,stability and adaptability of flue-cured tobacco varieties objectively , and provide scientific proofs for the cognizance and spread of varieties .In this paper ,GGE biplot was introduced into flue-cured tobacco regional trial analysis .GGE biplot method and AMMI model were used to analyze the yields and production values from the regional trials for flue-cured tobacco in 2011 .Thirteen lines of flue-cured tobacco were tested in eight sites .The results showed that both the methods could objectively evaluate the high yield and stabili -ty,but GGE biplot reflected the most suitable regions of variaties more accurately .YQ-1 had the highest yield and the best production value .GY1007 ,A18 and 2010 A4 had special suitability .GY1007 and A18 had expansion value in Dejiang,while 2010A4 had expansion value in Daozhen .

The relationship between main chemical components of flue-cured tobacco and visibility of the intermediate aroma style was investigated through analysis of the 151 tobacco samples from the main area of the intermediate aroma style. The results showed that the intermediate flavor style showed a higher degree when the nicotine content was 3.00%, the ratio of potassium to chlorine ranged from 12 to 16, the ratio of total sugar to nicotine ranged from 6 to 8, the ratio of total nitrogen to nicotine ranged from 0.6 to 0.8 and the ratio of reducing sugar to total sugar was 0.7. In addition, a regression analysis indicated that the intermediate aroma style value was significantly positive correlated with the nicotine and potassium content, and negatively correlated with the ratio of reducing sugar to total sugar.

Using newly developed methods and software, association mapping was conducted for chromium content and total sugar in tobacco leaf, based on four-omics datasets. Our objective was to collect data on genotype and phenotype for 60 leaf samples at four developmental stages, from three plant architectural positions and for three cultivars that were grown in two locations. Association mapping was conducted to detect genetic variants at quantitative trait SNP(QTS) loci, quantitative trait transcript(QTT) differences,quantitative trait protein(QTP) variability, and quantitative trait metabolite(QTM) changes,which can be summarized as QTX locus variation. The total heritabilities of the four-omics loci for both traits tested were 23.60% for epistasis and 15.26% for treatment interaction.Epistasis and environment × treatment interaction had important impacts on complex traits at all-omics levels. For decreasing chromium content and increasing total sugar in tobacco leaf, six methylated loci can be directly used for marker-assisted selection, and expression of ten QTTs, seven QTPs and six QTMs can be modified by selection or cultivation.