甘蓝型油菜C2连锁群种子含油量QTL簇及品质性状的分析

Analyzing the QTL Cluster of Seed Oil Content and Quality Traits on C2 Linkage Group in Brassica Napus L.

作者: 专业:发育生物学 导师:孟金陵 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Brassica napus L., oil content, molecular marker, QTL, quality trait, Association analysis

        油菜是我国主要油料作物之一,优质菜油饱和脂肪酸含量低,是食用植物油之佳品;由于菜籽油中脂肪酸的碳链长度与柴油相近,也是生产生物柴油的理想原料。因此,提高油菜含油量一直是众多科学家与育种家关注的目标。本实验室以甘蓝型油菜欧洲冬性品种Tapidor和中国半冬性栽培种Ningyou7为亲本构建了TN DH作图群体和遗传图谱,利用该图谱定位了种子含油量QTL,并在C2连锁群上发现了一个含油量QTL簇(命名为qcOCC2)。通过回交、表型筛选和分子标记辅助选择,构建了导入供体亲本Tapidor的qcOCC2片段的高世代回交群体和近等基因系。为了解析qcOCC2的遗传效应及克隆相关基因,必须在C2连锁群尤其是目标区间qcOCC2内加密足够的分子标记。本研究共采用了两种方法加密分子标记:1. Federico等基于甘蓝序列信息一共开发了587对SSR引物。经本实验前期的研究,目标区间qcOCC2与拟南芥第1染色体的E区段(C1E)、第5染色体的A区段(C5A)及E区段(C5E)同源,基于此,我们挑选出63个标记用于本研究;另外,从浙江农科院赵坚义博士获得5个定位于SG DH群体的C2连锁群的ATH及ZAAS标记用于本研究。最终,共8个标记定位于TN DH群体的C2连锁群,其中7个ATH及ZAAS标记定位于目标区间内。2.在实验室已构建Tapidor BAC文库的基础上,对Tapidor BAC末端进行了测序。将BAC末端序列与拟南芥基因组进行比对,挑选与拟南芥C1E、C5A或C5E高度同源(80%)的BAC作为候选BAC。然后将候选BAC的BAC-end序列与白菜A2染色体的BAC序列进行BLAST比对,从中挑选与A2 BAC同源性在80%-95%的Tapidor BAC-end序列进行引物设计,将其命名为BES引物。共设计出41对BES引物用于本研究,最终有6个标记定位于目标区间qcOCC2。随后,我们用定位在TN遗传图谱C2连锁群上的68个分子标记检测142份甘蓝型油菜的品种材料。用关联分析的方法来检测在自然群体中含油量表型及其它品质性状与这些标记之间的关系,并将关联分析的结果与QTL分析的结果进行比较分析,发现两者结果基本吻合。在关联分析中检测到的与含油量以及其它品质性状显著相关联的分子标记,大多数都位于家系连锁定位(FBL)的QTL置信区间内。因此可以得出如下结论:1.含油量表型与C2连锁群上标记之间确实存在一定的关联性;2.在TN群体以及该自然群体中,有相同的位点与含油量表型相关,这也验证了本研究中含油量QTL的真实性。
    Brassica napus is one of the main oil crops in our country. High quality seed oil is a good choice for edibility because of the low content of saturated fatty acids. On the other side, the long carbon chain fatty acids components in seed oil is more like the diesel fuel component, thus seed oil can be a suitable material for biodiesel production. So, increasing the seed oil content of rapeseed is always the main objective of many researchs.A doubled haploid population of B. napus named as TN DH population was constructed with the winter-typed variety Tapidor and the semi-winter variety Ningyou7 in our lab. Seed oil content of the population and the two parent lines in the multi-enviroment tests have been used to detect QTL with the linkage map. Hundreds of QTL related to seed oil content were identified, and a QTL cluster was detected on the C2 linkage group named as qcOCC2. Then, we developed the near isogenic lines (NILs) of the QTL cluster through backcross with the negative effect parent Ningyou 7 under phenotype screening and molecular marker assisted selecting. In order to dissect the qcOCC2 and clone the related genes, we need useful selected markers on C2 linkage group, especially in the confidence interval of qcOCC2.Two kinds of molecular markers were used to densify the linkage group C2 in the research:1. A total of 587 FITO primer flanking SSR markers were developed by Federico et al. according to the sequences of Brassica oleracea. In the previous research, we found that there were three synteny blocks (C5A, C5E and C1E) of Arabidopsis genome identified sequentially along the confident interval of qcOCC2 by in silico mapping analysis. Based on this, we picked out 63 FITO primers for our research. In addition,5 ATH and ZAAS markers which located on C2 linkage group in SG DH population were used in the research. Finally,8 markers are anchored on C2 linkage group in TN DH population, and 7 ATH and ZAAS markers are anchored in the region of qcOCC2. 2. We sequenced the Tapidor BAC-end sequence and aligned the Tapidor BAC-end sequences with the genome sequences of Arabidopsis thaliana to pick out the candidate BACs which were highly homologous (80%) with the C1E, C5A or C5E region of Arabidopsis thaliana. Then, the candidate BAC-end sequences were blasted with the BAC sequences of A2 linkage group in Brassica rapa. The Tapidor BAC-end sequences which were highly homologous (80%-95%) with the BACs of A2 linkage group will be picked out. According to these Tapidor BAC-end sequences, we designed 41 primer pairs, named them as BES marker. Finally,6 BES markers were anchored in the region of qcOCC2.We used the 68 markers anchored on C2 linkage group of TN DH genetic map to analysis 142 rapeseed varieties of Brassica napus, and detected the relevance between the seed quality traits, including seed oil content and mainly fatty acid content, and the tested markers in the natural population with the method of Association Analysis. Then, we find the results of Association Analysis were coincident with the previous results of QTL analysis. The markers which significantly associated with the seed quality traits were almost located in the confidence interval of seed quality traits QTL. So, we make a conclusion:1. There is a certain relevance between seed oil content and the tested markers.2. It has the same alleles which are related with seed oil content in TN population and this natural population. This result affirms the authenticity of seed oil content QTL detection in TN DH population.
        

甘蓝型油菜C2连锁群种子含油量QTL簇及品质性状的分析

摘要7-9
Abstract9-10
缩略词表11-12
1 文献综述12-26
    1.1 油菜含油量性状的研究进展12-14
    1.2 油菜品质性状QTL的研究进展14-17
        1.2.1 含油量QTL的研究进展14-15
        1.2.2 其它品质性状QTL的研究进展15-17
    1.3 特异引物的PCR分子标记17-19
        1.3.1 基于重复序列的分子标记(SSR标记,FITO标记)18
        1.3.2 基于同源基因序列的分子标记(STS标记)18-19
        1.3.3 分子标记在基因组测序中的意义19
    1.4 植物比较基因组学的研究19-23
        1.4.1 芸薹属A、B、C基因组间的关系20-21
        1.4.2 芸薹属植物与拟南芥的比较基因组学研究21-22
        1.4.3 比较基因组学的应用22-23
    1.5 关联分析23-25
        1.5.1 植物关联分析的应用以及研究进展23-25
    1.6 本课题的研究基础、由来、目的以及研究意义25-26
2 材料与方法26-31
    2.1 实验材料26
        2.1.1 Tapidor、Ningyou7及TN DH群体26
        2.1.2 自然群体26
    2.2 表型数据检测方法26-27
        2.2.1 油菜种子收获方法26
        2.2.2 表型数据检测方法26-27
    2.3 DNA提取27
    2.4 特异分子标记的开发27-29
        2.4.1 FITO和ZAAS标记27-28
            2.4.1.1 FITO标记的来源27
            2.4.1.2 ZAAS标记的来源27-28
        2.4.2 Tapidor BAC-end序列分析及BES标记28-29
            2.4.2.1 Tapidor BAC-end序列的分析与候选BAC的确认28
            2.4.2.2 BES标记的开发28-29
    2.5 PCR反应体系和反应程序29
    2.6 基因型检测方法29-30
    2.7 遗传图谱的构建方法30
    2.8 QTL分析及QTL的整合分析30
    2.9 C2连锁群品质性状相关位点的关联分析30-31
        2.9.1 实验材料30
        2.9.2 分子标记的全基因组扫描30-31
        2.9.3 群体结构分析31
        2.9.4 关联分析31
3 研究结果31-60
    3.1 FITO与ZAAS标记分析31-34
        3.1.1 FITO与ZAAS标记的多态性筛选31-32
        3.1.2 FITO与ZAAS标记在遗传图谱上的定位32-34
    3.2 Tapidor BAC-end序列分析与定位34-36
        3.2.1 电子杂交(in silico mapping)筛选Tapidor BAC-end34-35
        3.2.2 BES标记的筛选及在遗传图谱上的定位35-36
    3.3 TN群体中C2连锁群上含油量QTL的扫描结果与分析36-40
    3.4 TN DH群体C2连锁群上其他品质性状QTL的初步定位40-46
    3.5 C2连锁群含油量及其他品质性状相关位点关联分析的结果46-60
        3.5.1 分子标记全基因组扫描46-47
        3.5.2 群体结构的评估47-49
        3.5.3 关联分析的结果49-60
            3.5.3.1 种子含油量关联分析的结果49-50
            3.5.3.2 其他种子品质性状关联分析的结果50-56
            3.5.3.3 关联分析结果小结56-60
4 分析与讨论60-64
    4.1 电子杂交与BES标记的定位在后续研究中的作用和意义60-61
    4.2 关联分析中群体结构分析的必要性61-62
    4.3 关联分析对QTL分析的补充与佐证62-64
参考文献64-71
致谢71-72
附录72-79
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