杜仲遗传连锁图谱构建及重要性状的分子标记
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摘要
杜仲为我国所特有的杜仲科植物,开发利用前景十分广阔,但由于其遗传基础研究比较薄弱,从而制约着育种工作的有效开展。现代分子标记技术的发展,为杜仲早期选择工作的开展提供了有力的工具。构建分子标记遗传连锁图谱,并对其重要性状进行QTLs定位,不但为将来杜仲重要性状的分子标记辅助选择奠定了基础,而且对杜仲重要性状相关基因的图位克隆及分子遗传改良具有十分重要的意义。
本文利用AFLP标记技术和拟测交作图策略,以人工杂交获得的F1作图群体为材料,构建了杜仲AFLP遗传连锁图谱,并利用作图群体的表型资料对F1杂种重要数量性状的QTLs进行分析,找出控制杜仲叶片性状、生长性状及与性别决定相关的分子标记。本研究取得以下主要研究结果:
(1)通过对影响杜仲AFLP反应各因素的分析,建立了一套适合杜仲的AFLP反应体系。结果表明,最适合提取杜仲基因组DNA的方法是改良的CTAB法,酶切模板DNA用量为500ng、反应时间6h,连接反应时间6h,预扩增产物稀释30倍后用于选择性扩增。
(2)利用12个杜仲优良品种(无性系)作为杂交亲本,按析因交配设计,共组成了35个杂交组合进行人工杂交。采集种子后对杂交种子进行实生繁殖,共得到了7个较大的分离群体。结合表型变异情况及亲本间的AFLP多态性分析,确定在表型及DNA水平均差异较大的小叶×秦仲1号F1群体作为构建杜仲遗传连锁图谱的分离群体。
(3)利用F1作图群体和拟侧交作图策略,构建了杜仲AFLP遗传连锁图谱。亲本Q1遗传连锁图包含12个连锁群,共含有108个标记,连锁位点覆盖基因组总长929.57 cM,连锁图上相邻标记的平均间距为8.61 cM。亲本LF遗传连锁图含有14个连锁群,共含有127个标记位点,连锁位点覆盖LF基因组总长约1116.08cM,连锁图上相邻标记间的平均距离为8.78 cM。
(4)利用构建的杜仲AFLP遗传连锁图和LF×Q1的F1群体的表型数据,对杜仲的生长性状和叶片性状进行了QTL分析。共检测到控制8个性状的29个QTL位点,其中位于Q1连锁群上的有14个,位于LF连锁群上的有15个。
(5)通过对64对AFLP引物组合的筛选及验证,得到了两个杜仲雄性相关分子标记,分别命名为Eum E3M8-350和Eum E8M4-279。经测序和SCAR引物设计,将Eum E3M8-350的350 bp的雄性特有AFLP片段转化成为247 bp的SCAR雄性特有标记。经反复验证,确定该SCAR标记可用于杜仲性别的苗期鉴定。
Eucommia ulmoides Oliv., the single extant species of the genus Eucommia (Eucommiaceae), is endemic to China. The prospects of exploitation and utilization are very extensive, but the breeding programs of Eucommia ulmoides Oliv. is constraining by its narrow genetic background. A powerful tool for early selection of Eucommia ulmoides is provided by development of modern molecular markers. The construction of molecular genetic linkage map and analysis of quantitative traits loci not only provide information for the early selection of important traits but also produce the important significance on map-based cloning and molecular breeding.
Based on the F1 permanent mapping population, useing AFLP marker combined the Pseudo-test-cross strategy, the parent-specific genetic linkage maps in Eucommia ulmoides Oliv. were constructed, some important traits were mapped based on the analysis of phenotype date. And two male-specific AFLP markers were identified. The major results and conclusions are described as follows.
(1) An AFLP reaction system had been established, and it provided technical supporting for the construction of genetic linkage map in Eucommia ulmoides Oliv. With the analysis of main factors including extraction of genomic DNA method, reaction system of digestion and ligation, pre-amplification and selective amplification processes. The result shows extraction of genomic DNA with improved CTAB method, 500ng template DNA should be used in the digestion system with the reaction for 6 h, the time of ligation should be 6 h, 30 times of dilution for the products of pre-amplification for selective amplification. Repeated results show that it is feasible to use the reaction system constructed by the study to conduct the AFLP analysis of Eucommia ulmoides Oliv.
(2) Using 12 selected varieties (clones) as the hybrid parents, a total of 35 crossing combinations were hybridized according to the analysis of mating design. By the way of sowing and culture of seedlings, seven F1 familes have been obtained. Based on the variation analysis of field phenotypic characteristics and detecting the levels of polymorphisms between parents using AFLP, LF×Q1 has been selected as the permanent mapping population for construction the first genetic linkage map in Eucommia ulmoides Oliv..
(3) The first AFLP genetic linkage map in Eucommia ulmoides Oliv. was constructed. For the paternal tree of Q1, 108 markers were ordered in 12 linkage groups, covering the total distance of 929.57 cM of Q1 genome, with an average distance of 8.61cM between adjacent markers. For the maternal tree of LF, 14 linkage groups comprising 127 markers were identified, covering the total distance of 1116.08 cM of LF genome, with an average distance of 8.78cM between adjacent markers.
(4) Based on the parent-specific genetic linkage maps and analysis the data of phenotypic characteristics in the permanent F1mapping population, the Quantitative Trait Loci (QTL) linked to 8 phenotypic traits was analyzed. 29 QTLs was located in the genetic linkage map, there are 14 QTLs located on the Q1 linkage group, and the other 15 QTLs located on the LF linkage group.
(5) Two male specific AFLP markers for Eucommia ulmoides Oliv. which named Eum E3M8-350 and Eum E8M4-279 were identified according to screen 64 primer combinations. The SCAR primers were designed from the sequences of these two determined AFLP fragments. The 350 bp AFLP marker Eum E3M8-350 was converted into a 247 bp male-specific SCAR marker, suggested this SCAR marker can be utilized for early sexual identification in Eucommia ulmoides Oliv. accurately.
本文利用AFLP标记技术和拟测交作图策略,以人工杂交获得的F1作图群体为材料,构建了杜仲AFLP遗传连锁图谱,并利用作图群体的表型资料对F1杂种重要数量性状的QTLs进行分析,找出控制杜仲叶片性状、生长性状及与性别决定相关的分子标记。本研究取得以下主要研究结果:
(1)通过对影响杜仲AFLP反应各因素的分析,建立了一套适合杜仲的AFLP反应体系。结果表明,最适合提取杜仲基因组DNA的方法是改良的CTAB法,酶切模板DNA用量为500ng、反应时间6h,连接反应时间6h,预扩增产物稀释30倍后用于选择性扩增。
(2)利用12个杜仲优良品种(无性系)作为杂交亲本,按析因交配设计,共组成了35个杂交组合进行人工杂交。采集种子后对杂交种子进行实生繁殖,共得到了7个较大的分离群体。结合表型变异情况及亲本间的AFLP多态性分析,确定在表型及DNA水平均差异较大的小叶×秦仲1号F1群体作为构建杜仲遗传连锁图谱的分离群体。
(3)利用F1作图群体和拟侧交作图策略,构建了杜仲AFLP遗传连锁图谱。亲本Q1遗传连锁图包含12个连锁群,共含有108个标记,连锁位点覆盖基因组总长929.57 cM,连锁图上相邻标记的平均间距为8.61 cM。亲本LF遗传连锁图含有14个连锁群,共含有127个标记位点,连锁位点覆盖LF基因组总长约1116.08cM,连锁图上相邻标记间的平均距离为8.78 cM。
(4)利用构建的杜仲AFLP遗传连锁图和LF×Q1的F1群体的表型数据,对杜仲的生长性状和叶片性状进行了QTL分析。共检测到控制8个性状的29个QTL位点,其中位于Q1连锁群上的有14个,位于LF连锁群上的有15个。
(5)通过对64对AFLP引物组合的筛选及验证,得到了两个杜仲雄性相关分子标记,分别命名为Eum E3M8-350和Eum E8M4-279。经测序和SCAR引物设计,将Eum E3M8-350的350 bp的雄性特有AFLP片段转化成为247 bp的SCAR雄性特有标记。经反复验证,确定该SCAR标记可用于杜仲性别的苗期鉴定。
Eucommia ulmoides Oliv., the single extant species of the genus Eucommia (Eucommiaceae), is endemic to China. The prospects of exploitation and utilization are very extensive, but the breeding programs of Eucommia ulmoides Oliv. is constraining by its narrow genetic background. A powerful tool for early selection of Eucommia ulmoides is provided by development of modern molecular markers. The construction of molecular genetic linkage map and analysis of quantitative traits loci not only provide information for the early selection of important traits but also produce the important significance on map-based cloning and molecular breeding.
Based on the F1 permanent mapping population, useing AFLP marker combined the Pseudo-test-cross strategy, the parent-specific genetic linkage maps in Eucommia ulmoides Oliv. were constructed, some important traits were mapped based on the analysis of phenotype date. And two male-specific AFLP markers were identified. The major results and conclusions are described as follows.
(1) An AFLP reaction system had been established, and it provided technical supporting for the construction of genetic linkage map in Eucommia ulmoides Oliv. With the analysis of main factors including extraction of genomic DNA method, reaction system of digestion and ligation, pre-amplification and selective amplification processes. The result shows extraction of genomic DNA with improved CTAB method, 500ng template DNA should be used in the digestion system with the reaction for 6 h, the time of ligation should be 6 h, 30 times of dilution for the products of pre-amplification for selective amplification. Repeated results show that it is feasible to use the reaction system constructed by the study to conduct the AFLP analysis of Eucommia ulmoides Oliv.
(2) Using 12 selected varieties (clones) as the hybrid parents, a total of 35 crossing combinations were hybridized according to the analysis of mating design. By the way of sowing and culture of seedlings, seven F1 familes have been obtained. Based on the variation analysis of field phenotypic characteristics and detecting the levels of polymorphisms between parents using AFLP, LF×Q1 has been selected as the permanent mapping population for construction the first genetic linkage map in Eucommia ulmoides Oliv..
(3) The first AFLP genetic linkage map in Eucommia ulmoides Oliv. was constructed. For the paternal tree of Q1, 108 markers were ordered in 12 linkage groups, covering the total distance of 929.57 cM of Q1 genome, with an average distance of 8.61cM between adjacent markers. For the maternal tree of LF, 14 linkage groups comprising 127 markers were identified, covering the total distance of 1116.08 cM of LF genome, with an average distance of 8.78cM between adjacent markers.
(4) Based on the parent-specific genetic linkage maps and analysis the data of phenotypic characteristics in the permanent F1mapping population, the Quantitative Trait Loci (QTL) linked to 8 phenotypic traits was analyzed. 29 QTLs was located in the genetic linkage map, there are 14 QTLs located on the Q1 linkage group, and the other 15 QTLs located on the LF linkage group.
(5) Two male specific AFLP markers for Eucommia ulmoides Oliv. which named Eum E3M8-350 and Eum E8M4-279 were identified according to screen 64 primer combinations. The SCAR primers were designed from the sequences of these two determined AFLP fragments. The 350 bp AFLP marker Eum E3M8-350 was converted into a 247 bp male-specific SCAR marker, suggested this SCAR marker can be utilized for early sexual identification in Eucommia ulmoides Oliv. accurately.
引文
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