用分子标记技术初步解析甘蓝型油菜种子含油量QTL簇qcO.C2
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摘要
油菜是世界范围内的重要油料作物之一,油菜籽含油量是决定油菜经济价值的主要因素,提高油菜含油量成为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系作图群体和遗传图谱,并利用该图谱定位了种子含油量QTL,其中在C2连锁群上发现了一个含油量QTL簇(命名为qcO.C2)。通过回交、表型筛选和分子标记辅助选择,构建了导入了供体亲本Tapidor的qcO.C2区段的高世代回交群体和近等基因系。鉴于C2连锁群及qcO.C2区段分子标记稀疏(分别为25个和8个),为了解析qcO.C2的遗传效应及克隆相关基因,必须在C2连锁群尤其是目标QTL区间内加密分子标记。
本研究采用了多种分子标记技术加密分子标记:
1.采用SRAP分子标记技术。结合分离群体分组分析法(BSA)和三步筛选原则筛选了58条SRAP引物随机组合发展的672对SRAP引物对,在C2连锁群上加密了11个SRAP分子标记,在TN遗传图谱的其他14个连锁群上增加了37个SRAP标记;同时对定位在C2连锁群上的SRAP标记进行了测序以及序列分析,将其中的2个SRAP标记成功转化为SCAR标记,已用于近等基因系的筛选和构建中。
2.采用AFLP分子标记技术。结合BSA法筛选了200对AFLP引物组合,在C2连锁群上加密了5个分子标记,在TN连锁图的其它13个连锁群增加了34个AFLP标记,随后也对这些定位在C2连锁群上的AFLP标记进行了测序以及序列分析。
3.采用STS分子标记技术。利用C2连锁群上已有DNA序列信息的标记与拟南芥基因组进行了电子比对作图(in silico mapping),根据初步比对结果对白菜BAC序列和Tapidor BAC末端序列(BES)等信息进行分析,从而开发新的STS标记,最终在C2连锁群上加密了8个STS标记,在TN连锁图的其它4个连锁群上增加了6个STS标记。
通过以上几种分子标记技术,在C2连锁群,一共增加了27个新标记,C2连锁群上的平均标记密度从1Marker/5cM增加到1Marker/2.6cM。对TN DH群体12个不同环境下的含油量表型变异再次进行QTL分析,C2连锁群上检测到的QTL数目从13个增加到20个。且经过QTL整合后,QTL的分布较以往更加集中,置信区间也有所缩小。
将qcO.C2区间内的所有新标记的序列信息与拟南芥基因组进行了电子比对作图后,基本明确了此目标区段从上到下分别与拟南芥第5条染色体的A区段(C5A)、第5条染色体的E区段(C5E)和第1条染色体的E区段(C1E)同源。结合QTL整合后的置信区间,初步预测qcO.C2与油脂合成相关的9个候选基因。
Rapeseed {Brassica napus L.) is an important oil crop worldwide, and seed oil content is the main determinant of its value. So, increasing the seed oil content of rapeseed becomes the main objective of many scientists.
There is a segregating population of 202 double haploid lines (designated as TN DH population) and a genetic map in our Lab. This population is derived from a cross between Tapidor, a European winter cultivar of Brassica napus L., and Ningyou7, a Chinese semi-winter cultivar. Tens of QTL related to seed oil content variation with the populaton were identified, and a QTL cluster was detected on the C2 linkage group of the map (designated as qcO.C2). This cluster explained 25% of the phenotypic variation with superior alleles contributed from Tapidor. Based on the resources and information above, we developed near isogenic lines (NILs) for dissecting the QTL cluster with backcrossing, phenotype screening and molecular marker assisted selecting. Because of the limitation of markers on C2 and the QTL region (25 markers and 8 markers, respectively), adding more powerful markers in the QTL region is required for dissecting genetic control of qcO.C2 and gene cloning.
Different methods of developing molecular markers were applied for adding more markers on C2 in this study:
1. A total of 672 pairs of SRAP primer were screened in three-step procedure with Bulked Segregant Analysis (BSA) strategy. As a result, 11 SRAP markers were located on C2, and 37 SRAP markers were located on another 14 linkage groups. Then these newly developed markers on C2 were sequenced and analysed. Two SRAP markers were transformed into SCAR markers successfully, and these SCAR markers have been involved in marker assiated selection for developing NILs.
2. A total of 200 pairs of AFLP primer were screened with the same strategy above. As a result, five AFLP markers were located on C2, and 34 AFLP markers were located on another 13 linkage groups. And then, these new markers on C2 were also done sequence-analysis.
3. Base on the sequence of new markers on C2, in silico mapping with Arabidopsis genome sequence was done. We analysed some sequence from B. rapa genome and Tapidor BAC end according to the result of in silico mapping. Then, eight STS markers were newly developed and located on C2, and six STS markers were located on another four linkage groups.
In summary, 27 new markers were added onto C2 from this study. The marker density of C2 was increased from lMarker/5cM to lMarker/2.6cM. And then, QTL mapping was practiced for seed oil content variation from 12 experiments again. The number of QTL on C2 was increased, from 13 to 20. After meta-analysis, the location of QTL was concentrated than before, and the QTL confidence interval was also refined. There were three synteny blocks (C5A, C5E and C1E) of Arabidopsis genome identified sequentially along the confident interval of qcO. C2 after in silico mapping analysis, and nine candidate genes related to seed oil formation were discussed.
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系作图群体和遗传图谱,并利用该图谱定位了种子含油量QTL,其中在C2连锁群上发现了一个含油量QTL簇(命名为qcO.C2)。通过回交、表型筛选和分子标记辅助选择,构建了导入了供体亲本Tapidor的qcO.C2区段的高世代回交群体和近等基因系。鉴于C2连锁群及qcO.C2区段分子标记稀疏(分别为25个和8个),为了解析qcO.C2的遗传效应及克隆相关基因,必须在C2连锁群尤其是目标QTL区间内加密分子标记。
本研究采用了多种分子标记技术加密分子标记:
1.采用SRAP分子标记技术。结合分离群体分组分析法(BSA)和三步筛选原则筛选了58条SRAP引物随机组合发展的672对SRAP引物对,在C2连锁群上加密了11个SRAP分子标记,在TN遗传图谱的其他14个连锁群上增加了37个SRAP标记;同时对定位在C2连锁群上的SRAP标记进行了测序以及序列分析,将其中的2个SRAP标记成功转化为SCAR标记,已用于近等基因系的筛选和构建中。
2.采用AFLP分子标记技术。结合BSA法筛选了200对AFLP引物组合,在C2连锁群上加密了5个分子标记,在TN连锁图的其它13个连锁群增加了34个AFLP标记,随后也对这些定位在C2连锁群上的AFLP标记进行了测序以及序列分析。
3.采用STS分子标记技术。利用C2连锁群上已有DNA序列信息的标记与拟南芥基因组进行了电子比对作图(in silico mapping),根据初步比对结果对白菜BAC序列和Tapidor BAC末端序列(BES)等信息进行分析,从而开发新的STS标记,最终在C2连锁群上加密了8个STS标记,在TN连锁图的其它4个连锁群上增加了6个STS标记。
通过以上几种分子标记技术,在C2连锁群,一共增加了27个新标记,C2连锁群上的平均标记密度从1Marker/5cM增加到1Marker/2.6cM。对TN DH群体12个不同环境下的含油量表型变异再次进行QTL分析,C2连锁群上检测到的QTL数目从13个增加到20个。且经过QTL整合后,QTL的分布较以往更加集中,置信区间也有所缩小。
将qcO.C2区间内的所有新标记的序列信息与拟南芥基因组进行了电子比对作图后,基本明确了此目标区段从上到下分别与拟南芥第5条染色体的A区段(C5A)、第5条染色体的E区段(C5E)和第1条染色体的E区段(C1E)同源。结合QTL整合后的置信区间,初步预测qcO.C2与油脂合成相关的9个候选基因。
Rapeseed {Brassica napus L.) is an important oil crop worldwide, and seed oil content is the main determinant of its value. So, increasing the seed oil content of rapeseed becomes the main objective of many scientists.
There is a segregating population of 202 double haploid lines (designated as TN DH population) and a genetic map in our Lab. This population is derived from a cross between Tapidor, a European winter cultivar of Brassica napus L., and Ningyou7, a Chinese semi-winter cultivar. Tens of QTL related to seed oil content variation with the populaton were identified, and a QTL cluster was detected on the C2 linkage group of the map (designated as qcO.C2). This cluster explained 25% of the phenotypic variation with superior alleles contributed from Tapidor. Based on the resources and information above, we developed near isogenic lines (NILs) for dissecting the QTL cluster with backcrossing, phenotype screening and molecular marker assisted selecting. Because of the limitation of markers on C2 and the QTL region (25 markers and 8 markers, respectively), adding more powerful markers in the QTL region is required for dissecting genetic control of qcO.C2 and gene cloning.
Different methods of developing molecular markers were applied for adding more markers on C2 in this study:
1. A total of 672 pairs of SRAP primer were screened in three-step procedure with Bulked Segregant Analysis (BSA) strategy. As a result, 11 SRAP markers were located on C2, and 37 SRAP markers were located on another 14 linkage groups. Then these newly developed markers on C2 were sequenced and analysed. Two SRAP markers were transformed into SCAR markers successfully, and these SCAR markers have been involved in marker assiated selection for developing NILs.
2. A total of 200 pairs of AFLP primer were screened with the same strategy above. As a result, five AFLP markers were located on C2, and 34 AFLP markers were located on another 13 linkage groups. And then, these new markers on C2 were also done sequence-analysis.
3. Base on the sequence of new markers on C2, in silico mapping with Arabidopsis genome sequence was done. We analysed some sequence from B. rapa genome and Tapidor BAC end according to the result of in silico mapping. Then, eight STS markers were newly developed and located on C2, and six STS markers were located on another four linkage groups.
In summary, 27 new markers were added onto C2 from this study. The marker density of C2 was increased from lMarker/5cM to lMarker/2.6cM. And then, QTL mapping was practiced for seed oil content variation from 12 experiments again. The number of QTL on C2 was increased, from 13 to 20. After meta-analysis, the location of QTL was concentrated than before, and the QTL confidence interval was also refined. There were three synteny blocks (C5A, C5E and C1E) of Arabidopsis genome identified sequentially along the confident interval of qcO. C2 after in silico mapping analysis, and nine candidate genes related to seed oil formation were discussed.
引文
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