【作者】 赵世浩；

【导师】 周清明；

【作者基本信息】 湖南农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 烟草（Nicotiana tabacum）为茄科（Solanaceae）烟属（Nicotiana）一年或多年生作物,被认为起源南美洲,现已成为一种世界性的重要经济作物,是最早应用于分子生物和基因工程研究的模式植物之一。我国是世界上最大的烟草生产国,但烟草育种面临亲本匮乏、品种遗传背景狭窄、盲目性、重复性大等问题。近年来分子标记等分子生物技术在烟草领域应用中的研究,取得了一定进展。SSR分子标记技术是90年代兴起的一种分子标记,微卫星在染色体上分布均匀,数量也多,具有高度的变异,多态性信息非常丰富等优点被迅速且广泛地运用于生命科学的各领域中。利用现有生物数据库中的烟草EST资源,在了解烟草EST中SSR的信息的基础上,首次建立了EST-SSR标记,同时对其在品种资源分析中的应用进行了探讨。主要研究结果如下:1、明确了烟草EST中SSR的频率及特性。利用生物信息学方法在含有40666条烟草EST序列的数据库中进行微卫星标记筛选,共发现微卫星序列3685个,占整个EST数据库的9.062%。其中双碱基重复序列2614个、三碱基序列1019个,分别占在EST数据库中发现微卫星序列的70.936%和27.652%。烟草EST-SSR中,二核苷酸重复是主要的重复类型,出现最多的重复基元是AG/CT重复。烟草EST中SSR信息的明确为进一步建立和应用EST-SSR标记奠定了基础。2、建立了烟草EST-SSR标记。以烤烟品种K326为材料,研究了烟草EST-SSR分析中PCR反应体系的主要成分对SSR扩增结果的影响。结果表明:在总体积为20μL的PCR反应中,含25ng模板DNA,1.5U Taq DNA聚合酶,MgCl₂终浓度为1.5mmol·L^-1,dNTPs浓度为200μmol·L^-1,引物浓度为0.05μmol·L^-1时效果较好。根据筛选到的微卫星序列设计并合成引物50对,其中30对引物有扩增产物,23对产物条带比较清晰。3、本文应用EST-SSR标记,对烟草属2个种6份材料的遗传多样性进行了分析。从50对SSR引物中共筛选出15个多态性明显、条带清晰、反应稳定的引物,6个样品DNA共扩增出91条谱带,平均每对引物扩增出6.06条谱带,多态性条带比率达51.04%。种间遗传相似系数在0.2681～0.9411之间,平均为0.6046,表现出丰富的遗传多态性。红花种的4个栽培品种之间的遗传相似系数较高,在0.5277～0.9411之间,其中来自美国的G80和革新五号遗传相似系数最高,为0.9411,揭示了烟草栽培种内品种间的遗传基础相对较狭窄。黄花93-1烟草与其它供试材料间的遗传相似系数较低,在0.2681～0.3713之间,揭示出烟草野生种与栽培种不同类型间存在较大的遗传差异性。这些结果说明利用EST-SSR标记对烟草资源评价是有效的。更多还原

【Abstract】 Nicotiana tabacum is annual or perennial crop. It is originated from south America. It is an important world crop. It is the first crop applied in molecule and genie engineering .China is the biggest area for planting tobacco, but there are many problems: such as short of predecessor, etd. Molecular marker technology have gained success .SSR Molecular marker technology is rised from the 90s. Microsatellite is proportioned, bigger quantity. Polymorphism is abundance.This essay searched on tobacco genetic diversity and germplasms theory using SSR. Based on ESTs of Nicotiana tabacum existing in the public database, ESR-SSR marker was developed after mining and evaluating SSRs in the first time. As well as the application in resource evaluation of these EST-SSR markers were discussed and explored.First, the frequency and characteristics of SSR were analyzed and clarified in tobacco ESTs. Approximately 40666 EST sequences were searched for identification of SSRs using the Simple Sequences Repeat Identification Tool（SSRIT）.3685 microsatellite sequences were obtained from 40666 EST sequences, about 9.062 % of the whole database in which 2614（70.936%） dinucleotide repeats and 1019（27.652%） trinucleolide repeats were found. The dinucleotide repeat is the dominant type with AG/CT repeated motif being the most common. These results provide a base for the development and future application of EST-SSR markers in tobacco plant.Second, the EST-SSR marker was developed in tobacco.Through the research using fluecured tobacco vc. K326, the effects of major elements in PCR reaction system on SSR analysis were studied. The results showed that under the conditions of 1.5U Taq DNA polymerase, terminal MgCl₂ concentration of 1.5mmol·L^-1, 200μmol·L^-1 dNTPs, 0.25μmol·L^-1. 50 primer pairs were designed from microsatellite. Among the 50 primer pairs, 30 have amplification products,23 can achieve clear PCR products by electrophoresis.Third, the EST-SSR markers reported in this study were highly polymorphic between （Nicotiana）2. 15 primers those selected from 50 SSR primers. and 91 DNA fragments were amplified from 6 samples. The average number of DNA ban& was 6.06 per primer 51.04% PPB. The genetic similarity among all species was 0.29～0.52, anrange from the four species was 0.5277～0.9411, anrange from the 93-1 was 0.2681～0.3713. The results show that the EST-SSR marker is very effective in evaluation of tobacco germplasms.更多还原