摘要
我国按烟叶品质特点、生物学性状和栽培方法,把普通烟草划分为烤烟、晒烟、晾烟、白肋烟、香料烟五个类型。由于目前国际烟草生产上的主要栽培种多以烤烟类型为主,因此有关烟草品种遗传改良研究多集中在烤烟类型内的品种间进行,不同烟草类型间的重要相关有益性状还没有得到充分聚合和有效利用,因此生产上使用的栽培品种其遗传基础相对狭窄,烟叶品质、产量、抗性、适应性等还不能适应烟草生产发展的需求。研究烟草类型间重要性状差异及其遗传特性,探讨寻找烟草类型间的重要相关性状的遗传变异规律,有效利用烟草类型间的有益或优异性状扩大烟草栽培种的遗传变异背景,成为烟草品种改良工作中越来越关注的问题。
研究的目的旨在于:1)分析普通烟草类型间的植物学性状和致香物质差异,为以提高烟叶香气为主攻方向的烟草品质育种,提供改善烟叶香气的有益基因源;2)利用主基因+多基因遗传模型分析方法,研究普通烟草类型间的主要植物学性状的遗传规律,为在新品种选育过程中的性状选择提供理论依据,提高育种工作的预见性、减少选择工作的盲目性;3)利用分子标记技术,在分子水平上筛选普通烟草类型间的分子标记,为烟草类型间杂交育种工作提供稳定、不受环境影响的选择标准,提高性状选择的准确性;4)通过分析普通烟草类型间基因型差异,研究烟草类型间的遗传多样性和亲缘关系,为烟草育种工作中的亲本选择提供理论与实验依据。
研究以普通烟草不同类型间代表品种为供试材料,通过田间种植观察、双列杂交试验、致香物质成分检测与聚类分析、主基因+多基因遗传分离分析、SRAP标记等技术,分别从供试材料的表现型、基因型分析其遗传特点和重要性状差异。主要研究结果如下。
1、利用普通烟草不同类型间代表性品种,测定了烟草类型间的重要植物学性状和烟叶重要致香物质成分,并进行了R因子聚类分析,检测到5种致香物质与不同类型香气的产生有较大关系。供试的12个代表品种的系统聚类分析结果,与我国对普通烟草类型的习惯划分结果基本一致,为我国烟草类型的习惯划分方法提供了实验依据和理论佐证。
2、以烤烟品种与香料烟、白肋烟和名优晾晒烟品种为研究对象,通过杂交构建遗传分析群体,利用数量性状主基因+多基因遗传体系分离分析方法,对普通烟草栽培种内不同品种类型间的株高性状进行了遗传鉴定研究。结果表明:普通烟草类型间的株高性状遗传,符合两对加性-显性-上位性主基因+加性-显性多基因混合遗传模型(E1),各组合的遗传效应都表现出了加性、显性效应。各主基因和多基因遗传率计算结果,烤烟与晒晾烟、烤烟与香料烟组合的主基因遗传率较高,分别为71.60%和88.55%,可作为烟草株高性状早期世代选择的理论依据。同时研究证明,本实验采用的数量性状分离分析方法,适用于研究烟草数量性状的遗传规律,为烟草数量性状的遗传研究奠定了一种有效可行的分析方法。
3、进一步利用数量性状主基因+多基因遗传体系分离分析方法,对烤烟、香料烟及其杂交种一代的节距、叶数、叶长、叶宽等植物学性状的遗传规律进行了研究分析,初步揭示了烤烟与香料烟主要植物学性状的遗传特点,并估计了各性状的遗传力。其中,叶数遗传符合两对显性主基因+加性-显性多基因模型(E6);节距、叶长和叶宽的遗传符合两对加性-显性-上位性主基因+加性-显性多基因混合遗传模型(E1)。研究结果可作为烤烟与香料烟类型间的杂种鉴定利用的实验与理论依据。
4、利用SRAP标记技术,研究和分析了烤烟、晒烟、晾烟、白肋烟、香料烟等不同栽培类型间的基因型差异,初步筛选出了能够在分子水平上反映不同烟草类型差异的特征性标记带:晾烟MD40、Ky17、Burley21、TN90基因型共有的特征带有两对引物组合,分别为SF25*SR2和SF3*SR26;晒烟小牛舌、青梗、小花青和香料烟Sumsun、komotini Basma、Xanthi Basma共有的特征带引物组合为SF20*SR14;NC89、K326、G-28、大白筋和MD40、Ky17、Burley21、TN90共有的特征带引物组合为SF16*SR14,烤烟中大白筋599的特征带引物组合SF22*SR14。
5、利用61对SRAP引物组合,研究了普通烟草不同类型间的亲缘关系,将普通烟草类型间8个不同供试材料划分为3类别。I:烤烟类型(G-28);II:白肋烟类型(MD40、By21、大白筋599);III:香料烟类型(Basma、Sumsun、小牛舌、千层塔)。聚类结果与经验评价结果基本一致。
6、利用正交设计,从DNA浓度、dNTP、Mg2+、引物、Taq DNA聚合酶5个因素4个水平上,获得了最优的ILP标记的PCR反应体系,20ul反应体系包括:模板DNA 20ng,Taq酶1.0U,Mg2+ 2.0mmol/L,dNTP150umol/L,引物0.3umol/L。
According to the quality of tobacco leaf characteristics, biological characteristics and cultivation methods, Nicotiana tabacum is divided into flue-cured tobacco, sun-cured tobacco, air-cured, burley and oriental tobacco in China. Flue-cured tobacco is the most important type in international tobacco production, currently. So the tobacco breeder play most attention in flue-cured tobacco variety development and neglected the useful trait of other types in improving the tobacco variety, causing the genetic background of the cultivars is very narrow and many traits ,such as leaf quality, yield, resistance and adaptability, still can’t meet the demand. Therefore, it is very important to study the differences of traits and their genetic characteristics among different types.
There are four purpose in this study. The first purpose analyzes the differences of the botany traits and the flavor matters to provide the genetic source of aroma for quality breeding. The second uses the mixed major-gene plus polygene inheritance model to study the inheritance of the main botany traits to reduce the difficulty and errors in selection. The third uses the molecular marker technology to select the belt of different culture spawns and provide a stable selection criteria for the tobacco breeding. The fourth analyze the genotypic differences to study the diversity and the relationship among different tobacco to provide the theoretical basis for breeding.
Therefore, this study analyzes the phenotypic differences and the genetic differences among the representative varieties of the different types of tobacco, by observing the traits, detecting aroma substances and using diallel crossing test, cluster analysisthe, mixed major-gene plus polygene inheritance model, SRAP technology. The results are as follows:
1) The differences of traits and aroma of different types of tobacco are analyzed and clustered into five groups by R factor cluster method based on 12 varieties; meanwhile, the 12 varieties are systematically clustered based on 32 traits. The result is similar to the practice.
2) The mixed major-gene plus polygene inheritance model is used to analyze the inheritance of plant height in Nicotiana tabacum L. cross between flue-cured tobacco and burley tobacco, oriental tobacco, sun-cured tobacco. The results showed that the inheritance of plant height is accorded with the E1 model, and there were additive gene effect and dominant gene effect in the three cross combinations; the heritability of major genes for G-28×Qiancengta and G-28×Basma, were 71.60% and 88.55% respectively. The higher heritability will provide the theoretical basis for choosing the plant height at earlier generation. Meanwhile, this result shows that the mixed major-gene plus polygene inheritance model could be used in the inheritance study of quantitative characters of tobacco.
3) The mixed major-gene plus polygene inheritance model is also used to analyze the inheritance of the main botanical traits, including pitch, leaf number, leaf length and leaf width etc., cross between flue-cured tobacco (G-28)and oriental tobacco (Basma). The results showed that the inheritance of pitch, leaf length and width are all accorded with the E1 model, the leaf number is accorded with the E6 model. The result would be used in tobacco breeding.
4) SRAP technology is used to study the genetic differences among different types of tobacco, including flue-cured tobacco, oriental tobacco, maryland tobacco, burley tobacco, and air/sun cured tobacco, five pairs of primer combinations which can amplify characteristic bands has been screened. Among the five combinations, two of them (SF25*SR2 and SF3*SR26) can distinguish air-cured tobacco including maryland tobacco and Burley tobacco from the other cultivated forms; SF20*SR14 can distinguish sun cured tobacco including oriental tobacco and sun-cured tobacco; SF16*SR14 can distinguish flue-cured tobacco and air-cured tobacco, and SF22*SR14 can distinguish DBJ599 with specisl aroma and the other flue-cured tobacco.
5) 8 varieties , which represent the different types of tobacco, are divided into 3 groups. I: G-28, II:DBJ599、MD40、By21, III: Basma、Sumsun、XNS、QCT. The cluster analysis result es consistant with experience evatuation.
6) Anorthogonal design was used to optimize a ILP-PCR system with 5 factors (DNA, Mg2+, dNTP, primer and Taq polymerase) at 4 levels.The result showed the optimized ILP-PCR system for Nicotiana tabacum L. was: 2uL10×PCRbuffer, 20ng template DNA, Mg2+ 2.0mmol/L, dNTP 150umol/L, primer 0.3umol/L, Taq DNA polymerase 1 U in a total of 20uL reactionsolution. Each factor had different effecton on the results of PCR. Taq DNA polymerase had the greatest effect, while DNA concentration had the least. The effection of Taq DNA polymerase, primer concentration and dNTP concentration all reached significant level.
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