摘要
烟草青枯病是一种由青枯菌(Ralstonia solanacearum)引起的细菌性病害，是典型的维管束病害，显著的病状是枯萎，一旦发病即可造成全株死亡，严重影响烟草的产量和质量，是一种毁灭性病害。本论文在对安徽省宣州区的寒亭、文昌、黄渡、杨林，以及芜湖县三元镇的青枯病病菌分离鉴定的基础上，研究了安徽省烟草青枯病病菌对烟草的致病力及与相关生物学性状的关系，开展了抗青枯病基因RRS1的烟草同源性筛选研究，利用15个亲本配置的双列杂交组合进行了烟草青枯病抗性种质的主微位点组效应和配合力分析，利用BSA集团分离分析方法对2个烟草F2随机群体（TI448A×Enshu和TI448A×岩烟97）进行了烟草青枯病抗性的QTL定位分析。主要研究结果如下：
1、烟草青枯病病原菌的分离检测及致病力分化研究
采用组织分离法获得青枯病菌株8个。对其中5个进行了较详细研究，分别为：RS1、RS2、RS06、RS07、RS08。从菌株的致病性来看，RS23、RS07、RS71的致病性较强，对烟株叶片进行注射接种，叶片均有不同程度发病，但发病程度没有灌根法严重；供试菌株用注射和灌根对烟株均有致病性。同时针对烟草青枯菌生化型测定分析研究结果显示，分离的菌株除RS3不能利用卫茅醇外，其余都能利用三糖三醇；同时也均能利用硝酸盐，发生还原反应。根据青枯雷尔氏菌生化型划分标准，确定了从皖南烟区分离的菌株为生化型Ⅲ型。分析表明，不同菌株利用糖、醇的能力存在部分差异， RS23利用纤维二糖、卫茅醇的能力均较弱，RS22对卫茅醇利用能力也弱，但对其它糖、醇能完全利用，也能使硝酸盐还原，因此也属于生化型Ⅲ；RS3不能利用卫茅醇，但能利用其它双糖、双醇和还原硝酸盐，这个菌株划为生化型Ⅲ－1。
2、青枯病抗性基因RRS1的烟草同源性研究
研究针对烟草青枯病的田间单棵发病统计，病情等级，大田发病面积，进行重复性，多层次，多样本的人工统计，在拟南芥青枯病抗性基因RRS1的同源性检测试验中共筛选出3个烟草种质（G28，K346，LMAFC34）含有RRS1特异性扩增序列，针对筛选出3个烟草种质(G28、K346、LMAFC34)含有RRS1特异性扩增序列片段进行多重复的特异扩增纯化，增强结果的可靠性和完整性。由于RRS1基因含有有4个内含子，所以在引物设计上需要进一步加密，保证准确的特异性扩增，研究可以选用另外一种分子标记进行辅助和验证检测，能提高试验结果的准确性。
3、烟草青枯病抗性的主微位点组遗传分析
主-微位点组联合分析研究了青枯病病率和病指在始病期后第5天、第15天和第25天的效应值，主位点组效应均达到显著，而微位点组效应未达到显著。其中J1=13056和J1=13055相邻的2个主位点组在第一次和第二次的联合分析中均检测到，且效应值较大；J1=14080和J1=14079相邻的2个主位点组在第一次和第二次的联合分析中同样被检测到，且效应值较大。15个亲本青枯病病率第5天、第15天和第25天的微位点组加性效应中云烟85、DB101和RG11的效应值均最大，该检测结果和这3个品种的田间抗病指标表现一致，与之相对应的广义遗传率均在20%以上，在品种的青枯病抗性改良中可以重点考虑如何利用云烟85、DB101和RG11品种的遗传抗性。遗传纯合显性位点(++)表现为青枯病病指增加(感病)，而纯合隐性位点(––)使青枯病病指减少（抗病），且加性效应值均较大，显性效应值均较小，表明青枯病抗性的遗传规律主要是以加性遗传为主，进而为改良亲本的青枯病抗性能力以创新种质资源提供新策略。
4、烟草青枯病抗性的配合力分析
研究了烟草青枯病抗性各个调查时期各项指标的一般配合力都达到显著差异水平，而特殊配合力表现不一致，其中2010年7月26日调查数据发病率及病情指数都达显著差异，研究列出了该次调查病情指数的特殊配合力分析结果，并提出了几个效应强的组合。该实验结果并不表明整个试验抗性群体的特殊配合力能够这样应用，只能说明如果在一般配合力组合间决选时差异不大或难以确定时，可以选择参考一些特殊配合力分析结果，例如方差分析显著的调查时期或指标，故该研究主要考虑一般配合力，一般情况下可按烟草青枯病抗性强强组合应用于育种，烟草青枯病抗性配合力的差异可能涉及到加性效应、非加性效应以及遗传率等抗性遗传方面的研究分析。
5、烟草青枯病抗性QTL定位研究
研究利用TI448A×Enshu群体及TI448A×岩烟97群体在第3染色体及第5染色体上定位到4个烟草青枯病抗性QTLs （qBWR-3a， qBWR-3b， qBWR-5a和qBWR-5b），在LOD极大似然值大于或等于3的筛选条件下，分别解释青枯病抗性表型变异9.00%，19.70%，17.30%和17.40%。初步推断出测定烟草青枯病抗性的特异引物，以及利用该特异引物测定烟草青枯病抗性的的方法；所述特异性引物为两对，其核苷酸序列如下： PT20275：5′GTTCTATTTGATCGCCCC3′；5′AACAGCACCAACAGCATT3′； PT30229：5′GTTGCTCGTGTGCCTGACTA3′；5′AATGTGGAAACAGGA。利用上述特异性引物对待检测的烟草种质的DNA进行PCR扩增，采用PT20275引物能特异性的扩增出380bp大小的特异带，采用PT30229引物能特异性的扩增出200bp大小的特异带，即可判定其为抗性。本研究首次将青枯病抗性基因初步定位于第三染色体19.45cM区域，在国内外前人的研究中均未有报道，且本研究中3个群体（TI448A×岩烟97、TI448A×Enshu及长脖黄×G28）均验证了此连锁标记，为精细定位工作奠定了基础。
The tobacco bacterial wilt is a kind of fatal bacterial disease caused by Ralstoniasolanacearum. It is a typical vascular disease of which obvious symptom is to fade. Oncethis disease happens, the whole plant die. And the yield and quality affectedseriously. This thesis studied the relationship between the pathogenicity of tobaccoRalstonia solanacearum and the related biological characters based on the isolation andidentification of the germ at towns Hanting; Wenchang; Huangdu;Yanglin, Xuanchengdistrict and at town Sanyuan, county Wuhu in province Anhui. The study of screening onRRS1gene of bacterial wilt resistance in Tobacco for homolog was conducted.15diallelcross combination as parent disposition was used to analyze the effects of main and minorloci groups and combining ability of the resistance germplasm of tobacco bacterial wilt.QTL Analysis of the resistance of tobacco bacterial wilt from two F2random populations(TI448A×Enshu and TI448A×Yanyan97) was conducted by the method of BSA groupseparation analysis. The results showed as follows:
1. Study on the separation-detection of Pathogen of tobacco bacterial wilt andpathogenicity differentiation
Tissue isolation was used so eight strains with tobacco bacterial wilt was obtained.And five of them have been analyzed in details, they are RS1; RS2; RS06; RS07and RS08.The pathogenicity of RS23; RS07and RS71was found to be stronger. While both of thetwo ways are pathogenic, injection to leaves will lead to tobacco bacterial wilt at differentdegree; however they are less serious than root-drenching method. At the same time theresults of the determination and analysis of the biochemical variant of tobacco ralstoniasolanacearum indicate that apart from RS3among the isolated strains which cannot usedulcitol, the others can absorb trisaccharide and triol. And nitrates take part in theirreduction reaction. The division standard of the biochemical variant of ralstoniasolanacearum ensure that the strain separated from south Anhui is type Ⅲ. The analysisshows that the ability of different strains to utilize sugar and alcohol are differential. RS23to cellobiose and dulcitol are weak. RS22to dulcitol is weak. But they can use other sugarand alcohol entirely and also the nitrates, so they must be type Ⅲ. As RS3can utilizedisaccharide and alcohol but dulcitol, RS3belong to type Ⅲ－1.
2. Study on RRS1gene of bacterial wilt resistance in tobacco for homolog
According to the repeated, multi-level and diversiform statistics on the infected singleplant which infected tobacco bacterial wilt, disease intensity and the pathogenetic area. The specific amplified sequence RRS1have been detected in three tobacco germplasm (G28;K326and LMAFC34) in the experiment of “detection on RRS1gene of bacterial wiltresistance from Arabidopsis for homolog”. The amplification and purification of theexpected sequence in the three germplasm proved to be credible and integrated. The primerdesign must be more seriously encrypted to make the test reliable as there are four intronsin gene RRS1. The study can be assisted and verified detection with another molecularmarker, and it can also enhance the accuracy of the experiment.
3. Genetic analysis on main and minor loci groups of bacterial wilt resistance intobacco
The united main and minor loci groups analysis studied disease rate and disease indexand the effect value on day5,15,25after disease starting period, it indicates that the effectof main loci groups is significant while the minor is not. Two main loci group J1=13056,J1=13055among them have been detected in the first and second united analysis. So haveJ1=14080, J1=14079and the adjacent two main loci group. The effect value is high two.The additive effect of minor loci groups of Yunyan85, DB101and RG11are the highestamong the fifteen parent-bacterial wilt disease index of day5,15and25. The fieldperformance accord with the test result and at the same time the broad heritability is higherthan20%. The genetic resistance of Yunyan85, DB101and RG11can be considered toutilize in variety improvement of bacterial wilt resistance. The dominant homozygous genelocus(++) of resistance index of bacterial wilt manifested as index increasing but therecessive gene is decreasing. And the additive value of recessive gene is higher while thedominant one is low. It shows that the inheritance of bacterial wilt resistance is mainlyadditive genetic and can afford new strategy for modifying parent’s ability of bacterial wiltresistance.
4. The analysis about general combining ability of bacterial wilt resistance intobacco
The study on bacterial wilt resistance in tobacco showed that general combiningability of every index in every investigation period reached significant level. However thespecific combining ability proved variable. In the survey data about the disease rate andindex, the data of26th, July,2010showed significant. The analyzed result of specificcombining ability was listed out and some combination with strong effect value pointed out.This experiment did not mean that specific combining ability of the whole resistance groupcan be all used like this, but can choose some analyzed result of some specific combiningability as a reference when the difference of general combining ability among combinations is not big or not sure, for example the significant investigation or index inanalysis of variance. So the study is most on general combining ability. Generally the ruleof strong to strong combination can be applied into breeding in bacterial wilt resistance intobacco. And the difference of the combining ability of the resistance may involve additiveeffect, non-additive effect, heritability and other genetic heredity in resistance research.
5. QTL mapping research in bacterial wilt resistance in tobacco
Utilizing the groups Ti448A×Enshu and groups TI448A×Yanyan97four QTLS ofbacterial wilt resistance in tobacco were found on chromosome three and five. With thatand at the selection condition of maximum likelihood method of LOD higher than orequals3, the phenotypic variation of bacterial wilt resistance which are at9.00%,19.70%,17.30%and17.40%were clear. Specific primer of bacterial wilt resistance in tobaccowas preliminarily detected and the way to detecting bacterial wilt resistance in tobaccowith the specific primer is clear. The character is that: the sequence of the pair of specificprimer is: PT20275:5′GTTCTATTTGATCGCCCC3′;5′AACAGCACCAACAGCATT3′;PT30229:5′GTTGCTCGTGTGCCTGACTA3′;5′AATGTGGAAACAGGA. With the upspecific primer to amplify the DNA of tobacco germplasm by PCR, when the primer isPT20275a specific of380bp while PT30229will get a specific of200bp, and this canprove to be resistant. It firstly located the bacterial wilt resistance in tobacco at19.45cmarea on chromosome three. This study never reported before both at home and abroad. Andthe three groups TI448A×Yanyan97, TI448A×Enshu and Changbohuang×G28all provethe linkage marker designed in detail and they lay the foundation of the encryption work.
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