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适合棉花品种鉴定的SSR核心引物的筛选和品种鉴别

Screening A Core Set of Informative SSR Primers for Identificating Cotton Cultivars and Lines(G. Hirsutum L.)

作者: 专业:遗传学 导师:沈法富 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Cotton, SSR, Cultivar identification, Core primer

        快速准确地鉴定作物品种对于品种审定、品种保护、假种辨别、产权纠纷均有重要作用,因此对快速、高效、稳定的品种鉴定技术研究具有重要意义,SSR分子标记因其自身具备的优点在品种鉴定中得到越来越广泛的应用。本研究旨在参考前人研究的基础上,筛选出适合棉花品种鉴定的核心引物,形成一套适合棉花品种指纹鉴定技术体系,并利用核心引物对2009年参加山东省棉花区试的67份品系的真实性进行鉴定,同时对参试品种(系)进行遗传多样性分析,主要结果如下:1.研究确立了一套快速适合棉花品种鉴定的SSR检测体系:使用两种不同的提取方法分别提取棉花叶片和种子的DNA,利用TD-PCR反应程序提高了PCR反应的特异性和灵敏性,采用了快速银染的方法,提高了银染的效率。2.从CMD、CottonDB数据库和文献资料公布的引物中选取了2000对引物,对32份通过审定大面积推广的棉花主推品种基因组DNA进行了多态性筛选,共筛选到26对核心引物,其条带清晰,多态性高,稳定性好,占总引物数量的1.3%, 26对核心引物覆盖了棉花的20条染色体。利用数目依次减少的核心引物(26对,11对,8对)的指纹数据,对32份审定品种进行UPGMA聚类分析,11对核心引物和26对核心引物所反映的32份审定品种间DNA水平的差异基本吻合,当引物减少到8对时,每个大类别所包含的品种有大的变化,且品种间遗传相似系数也有较大的差别,所以将11对核心引物确定为首选核心引物。3.提出了核心引物理论上可区分的最大品种数的计算公式:N=G1×…×Gn,出现相同指纹图谱的概率为:P=1/N;11对首选核心引物理论上可区分的最大品种数为419904个,出现相同指纹图谱的概率为:2.38×10-6,理论推测结果表明11对首选核心引物进行棉花品种鉴定是可行的。4. 32份审定品种的DNA指纹数据的聚类分析结果基本反映了品种来源的系谱,有共同亲本的品种其遗传相似系数大,且聚为相同类别, 11对核心引物所反映的32份品种间DNA水平上的差异与品种来源的系谱分析结果基本吻合。5. 11对首选核心引物对匿名取样品种的真实性进行了指纹鉴定,结果表明,该品种DNA指纹图谱与为鑫秋4号棉花品种图谱一致,与匿名取样品种完全相符。6.利用11对首选核心引物,对参加2009年山东省棉花区试的67个品系进行了DNA指纹鉴定,结果表明,省区试4号和鲁棉研37号疑为同一品种,省区试15号和邯棉559疑为同一品种;省区试38号和省区试45号疑为同一品系,省区试11号和省区试46号疑为同一品系。7.利用11对首选核心引物在不同组合品种(系)间产生的指纹数据进行UPGMA聚类分析,结果表明,参试品种(系)整体上遗传基础狭窄,同一育种单位育成的部分品种间遗传距离较近,不同地区和不同育种单位育成的品种间遗传距离相对较大。
    It is very important to identify cultivars rapidly and precisely for plant variety certification, plant varieties protection, fake seeds identification and property right dispute. Thus, it is significantly important to research on the rapid, high-efficient and stable cultivar identification system, SSR molecular makers are more widely used in cultivar identification because of its own advantages. This study aims to select a set of core primer pairs and develop a system for cotton cultivar identification, genetic clustering analysis of the tested materials and authenticity identification of 67 cotton varieties in Shandong provincial regional trial in 2009 were also performed based on DNA fingerprinting database derived from the selected 11 core primer pairs,the main research conclusions are as follows.1. A rapid and stable system applied for the cotton cultivar identification was developed: two different methods of DNA extractiong from cotton leaf and seed are established respectively, touchdown PCR increases specificity and sensitivity in PCR amplification, a sample and rapid method improves the efficiency of silver staining of DNA banding in PAGE gel.2. Genomic DNA from 32 approved and commercial cotton cultivars with larger planting areas were amplified with 2000 primer pairs selected from CMD database, cottonDB database and relevant research papers, 26 core primer pairs with high polymorphism and stability, clear amplified banding were selected, which accounting for 1.3% of primers used in this study and covered 20 cotton chromosomes. Genetic clustering analysis by UPGMA of 32 cotton cultivars was performed based on DNA fingerprinting database derived from the the reducing number of the core primers (26 pairs, 11 pairs, 8 pairs), the results showed that the difference of 32 approved cotton cultivars at DNA level based on 26 and 11 core primer pairs was little, but it was big when the core primer pairs reduced to 8, thus 11 core primer pairs were proposed as preferred primer pairs for identification of cotton cultivars.3.A formula for calculating the maximum number of cotton cultivars that the core primer pairs can identify in theory was advanced: N=G1×...×Gn, G1...Gn, and the probability of the different cultivars having the same DNA fingerprinting was: P=1/N; 11 core primer pairs can identify the maximum number of cotton cultivars in theory is 419904 .The probability of the different cultivars having the same DNA fingerprinting was :2.38×10-6. According to theory, 11 core primer pairs were effective in cotton cultivar identification.4. The results of genetic clustering analysis of 32 cotton cultivars based on DNA fingerprinting database derived from 11 core primer pairs demonstrated the pedigree of them, cultivars with the same parents having a great genetic similarity coefficient, and clustered into the same group of UPGMA dendrogram. Difference of 32 approved cotton cultivars at DNA level was in accordance with the result of pedigree analysis of them.5. The result of authenticity identification of A approved cultivar based on DNA fingerprinting database derived from the 11 core primer pairs showed that A cultivar’s fingerprting was exactly the same as Xinqiu 4 cultivar’s, the fact is that leaves of A cultivar were from Xinqiu 4 cultivar, the result was in accordance with the fact.6. The deduced result of authenticity identification of 67 cotton varieties in Shandong provincial regional trial in 2009 based on DNA fingerprinting database derived from the selected core primer pairs was that the number 2 and number 15 cotton lines in Shandong provincial regional trial was the Lumianyan 37 and Hanmian 559 cotton cultivar respectively, and the number 38 and number 45 cotton lines in Shandong provincial regional trial was the same cotton lines as well as the number 11 and 46.7. Genetic clustering analysis of different combinations of cotton cultivars (lines) used in this study was performed based on DNA fingerprinting database derived from the 11 core primer pairs, the result showed that genetic basis of the tested materials was limited, genetic basis of cultivars from the same breeding unit is limited, and it is relatively large in different areas and different breeding units.
        

适合棉花品种鉴定的SSR核心引物的筛选和品种鉴别

摘要8-10
Abstract10-11
1 前言12-26
    1.1 形态学鉴定在品种鉴定中的应用12-13
        1.1.1 种子形态学鉴定12
        1.1.2 幼苗鉴定12
        1.1.3 田间小区种植鉴定12-13
    1.2 蛋白质指纹技术在品种鉴定中的应用13-15
        1.2.1 同功酶指纹技术13-14
        1.2.2 贮藏蛋白指纹技术14-15
    1.3 DNA 指纹技术在品种鉴定中的应用15-25
        1.3.1 RFLP 及其在品种鉴定中的应用16-17
        1.3.2 RAPD 及其在品种鉴定中的应用17-18
        1.3.3 AFLP 及其在品种鉴定中的应用18-19
        1.3.4 SSR 及其在品种鉴定中的应用19-24
            1.3.4.1 SSR 在马铃薯品种鉴定中的应用20-21
            1.3.4.2 SSR 在玉米品种鉴定中的应用21-22
            1.3.4.3 SSR 在水稻品种鉴定中的应用22-23
            1.3.4.4 SSR 在大麦、小麦品种鉴定中的应用23-24
            1.3.4.5 SSR 在棉花品种鉴定中的应用24
        1.3.5 常用分子标记在品种鉴定应用中的优缺点24-25
    1.4 本研究的目的和意义25-26
2 材料和方法26-34
    2.1 材料26-28
    2.2 方法28-32
        2.2.1 DNA 提取28-29
            2.2.1.1 CTAB 法提取叶片gDNA28
            2.2.1.2 SDS 法提取种子gDNA28-29
        2.2.2 PCR 扩增29
        2.2.3 PCR 扩增产物的检测29-32
            2.2.3.1 聚丙烯酰胺凝胶电泳试剂的配制29-30
            2.2.3.2 胶板的制备30-31
            2.2.3.3 变性聚丙烯酰胺凝胶电泳31
            2.2.3.4 银染显带31-32
        2.2.4 核心引物的筛选32
    2.3 数据记录和统计分析32-33
        2.3.1 数据记录32-33
        2.3.2 数据统计分析33
    2.4 核心引物有效性33-34
3 结果与分析34-48
    3.1 两种方法所提取的DNA 适合SSR 扩增34
    3.2 TD-PCR 能够提高PCR 反应的特异性和灵敏度34-35
    3.3 快速银染方法提高了显带效率35-36
    3.4 核心引物的筛选36-39
    3.5 首选核心引物有效性的理论推测39-40
    3.6 首选核心引物有效性40-42
        3.6.1 匿名取样品种真实性鉴定40
        3.6.2 系谱分析40-42
    3.7 山东省2009 年区试品系真实性鉴定42-45
    3.8 参试品种(系)的遗传多样性分析45-48
4 讨论48-52
    4.1 棉花品种鉴定SSR 分子标记检测体系48
    4.2 核心引物的多态性48-49
    4.3 核心引物理论上可以区分最大品种数49-50
    4.4 首选核心引物的有效性50
    4.5 核心引物的确立50
    4.6 参试品种(系)的遗传多样性50-52
5 结论52-53
参考文献53-61
致谢61-62
攻读学位期间发表的论文情况62
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