【作者】 刘任重；

【导师】 张天真； 章元明；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 湘杂棉2号是我国上世纪90年代选育的棉花杂交种,在棉花产量方面具有较高的杂种优势。亲本CRI12是高产、优质、抗病的棉花品种,J8891是高产棉花品系。本研究利用以湘杂棉2号构建的重组自交系群体以及重组自交系间两两杂交构建的永久性F2群体,分别进行了主要农艺性状和纤维品质性状的QTL定位；通过杂种优势QTL的定位分析,探讨了该杂交种杂种优势形成的遗传机理；用cDNA-AFLP技术构建了叶片转录图谱,进一步进行了性状的QTL定位和候选基因分析。主要结果如下：1、在前人工作的基础上,用重组自交系群体构建了一张以SSR标记为主的分子标记遗传图谱。该图谱总长度941.2cM,含22条染色体和1个连锁群,标记位点总数为181个,大约覆盖棉花基因组的20.20%。标记位点间平均遗传距离6.67cM,最大间距26.18cM。标记的分布不很均匀,有些标记在染色体上成簇存在。2、以重组自交系3年共8个环境下的资料,通过复合区间作图法对产量和产量因子等7个农艺性状进行了QTL定位,共定位了42个加性效应显著的QTL,其中18个在A亚组、24个在D亚组染色体上。对11个主要的纤维品质性状进行了QTL定位,共检测到59个加性效应显著的QTL,其中22个分布在A亚组、36个分布在D亚组染色体上。对株高、果枝数等7个形态性状共检测到16个QTL,1个在A亚组、其余均在D亚组染色体上。这些QTL在染色体上大多数成簇分布,其中染色体D2、D6、D9、A5和A11上QTL非常集中,存在多个QTL热点区域。3、利用永久性F2群体3年共6个环境的数据以复合区间作图法(CIM)、多区间作图法(MIM)和多标记联合分析法对9个主要农艺性状进行了QTL定位。通过CIM检测到的104个QTL中,有37个QTL可在两个以上的环境下检测到,70个可由MIM同时检测到,43个由多标记联合分析法同时检测到。许多在不同环境中检测到的QTL具有共同的邻近标记。用三种方法同时检测到的相对稳定的QTL达28个,其中单株籽棉1个；单株皮棉1个；单株铃数3个；铃重5个；衣分6个；籽指3个；衣指3个；株高2个；果枝数3个。有26个QTL与以前的报道位置相同或相近。利用MIM对农艺性状进行了位点间上位性QTL分析,共检测到47个上位性QTL位点对,其中参与加性与加性(AA)、加性与显性(AD)、显性与加性(DA)和显性与显性(DD)互作的位点对分别为26、9、12和19个。因此互作类型以AA和DD居多,有些双位点间具有两种以上的显著性互作。一些位点与其它多个位点间有互作,说明在基因组中存在参与互作的热点区域。利用多标记联合分析法对农艺性状进行了位点与环境的上位性互作分析。共检测到28个环境上位性QTL,其中10个在A亚组、18个在D亚组染色体上。有15个环境上位性QTL在复合区间作图中检测到,表明在以前研究中用复合区间作图法定位的QTL至少有一部分实际上是环境上位性QTL。位点间上位性和位点与环境间上位性QTL的大量存在,表明上位性在湘杂棉2号杂种优势表现中发挥了重要作用。4、利用多标记联合分析法以永久性F2群体四环境中亲优势数据进行了农艺性状杂种优势的QTL分析。共检测到60个杂种优势位点,其中19个表现加性效应,31个表现显性和部分显性效应,10个表现超显性效应。43个杂种优势位点与相应性状的QTL位置相同,表明这些位点可能通过控制性状的表达来影响杂种优势表现。同数量性状的QTL一样,性状杂种优势的QTL也具有多效性,研究发现在杂种优势的QTL中,有16个QTL同时控制多个性状的杂种优势表现。检测到农艺性状杂种优势的环境上位性QTL75个以及杂种优势上位性互作位点对75对。在加性、显性、超显性和上位性QTL中,上位性QTL解释的杂种优势变异最大,说明上位性效应包括基因间互作和基因与环境的互作效应是湘杂棉2号杂种优势的主要遗传基础。基因位点内的加性、显性和超显性对杂种优势的产生也发挥了一定的作用。5、以现蕾期叶片为材料,用cDNA-AFLP技术构建了一张湘杂棉2号永久性F2群体的转录图谱。该图谱含26个连锁群,总长度2747.O1cM,包含302个cDNA-AFLP标记,连锁群平均长度95.27cM。平均标记间距8.23cM。所有标记均匀分布在不同的连锁群上。以该图谱以及永久性F2群体的四环境数据,用复合区间作图法定位了单株籽棉产量等9个主要农艺性状的76个QTL和9个纤维品质性状的61个QTL。QTL也具有成簇分布现象。大部分QTL表现为显性和超显性效应。对与QTL共分离的大部分长度在200bp以上的cDNA-AFLP片段(TDFs)进行了回收、克隆和测序。通过同源性序列搜索,对61个TDFs进行了候选基因的蛋白质功能预测和分析。这些基因大多在转录和翻译调控、信号转导、运输、纤维素合成、光合作用、碳水化合物及脂肪代谢、合成代谢等方面起作用,是具有重要生物学功能的基因。通过共分离TDFs的表达与产量等农艺性状的相关分析,鉴定出多个与产量及产量杂种优势相关的候选基因。可通过RACE技术或图位克隆技术,将这些功能基因进行克隆,或者将与QTL共分离的cDNA-AFLP标记转化为CAPS标记应用于分子标记辅助育种。更多还原

【Abstract】 XZM2 is a hybrid cotton variety with high yield heterosis. The parent CRI12 is an elite variety of high yield, good fiber quality and disease resistance and J8891 is a germplasm line with high yield potential. A recombinant inbred lines (RILs) population constructed from XZM2 and an immortalized F2 (IF2) population derived from crosses between the RILs were used to map QTL for agronomic and fiber quality traits; the genetic bases of heterosis were dissected by mapping of heterotic loci; and a transcriptome map of the IF2 population was constructed by cDNA-AFLP techniques, followed by QTL and candidate gene analyses for agronomic and fiber quality traits. The results are as follows:1. A genetic map mainly consisting of SSR markers was constructed using the RILs population, which was 941.2cM in recombined length, including 22 chromosomes and 1 linkage group, and covered 20.20% of cotton genome. The average interval between two markers was 6.67cM. Some of the markers were clustered on certain chromosomes.2. Based on data of RILs population in 8 environments in 3 years, QTL analysis was conducted using Composite Interval Mapping (CIM) procedure of Windows QTL cartographer 2.5. Totally 42 additively significant QTL for 7 yield and yield components were identified,18 and 24 of which were mapped on A and D subgenome respectively; Fifty nine QTL for 11 fiber quality traits were detected, with 22 distributed on A and 36 on D subgenome. Sixteen QTL for 7 morphological traits were mapped, with 1 on A and 15 on D subgenome. Some of these QTL were clustered on chromosome D2, D6, D9, A5 and A11, implying the existence of hot QTL region on the cotton genome.3. Utilizing the IF2 data in 6 environments in 3 years, QTL for 9 agronomic traits were analyzed by CIM and multiple interval mapping (MIM) of Cartographer 2.5 and multi-marker joint analysis methods respectively. Of the total 104 QTL detected by CIM, 37 can be detected in more than 2 environments,70 were also identified by MIM, and 43 were detected simultaneously by multi-marker joint analysis. Many of the QTL detected in different environments shared common makers. There were 28 relatively stable QTL concurrently detected by three methods, including 1 for seed-cotton yield; 1 for lint yield; 3 for bolls per plant; 5 for boll weight; 6 for lint percentage; 3 for seed index; 3 for lint index; 2 for plant height and 3 for fruit branch number respectively. About 26 QTL were at the same or close to the position of previously reported QTL of corresponding traits.Forty seven digenic epistatic QTL pairs were detected by MIM procedure. The number of QTL pairs involving in additive by additive (AA), additive by dominance (AD), dominance by additive (DA) and dominance by dominance (DD) interaction was 26,9,12 and 19 respectively, therefore the interaction modes between two loci were predominantly AA and DD, and some of the QTL pairs involved in more than two kinds of significant interaction. Some loci frequently interacted with other multiple loci, indicating there are hot regions of interaction in the cotton genome. Interaction between loci and environment for agronomic traits were analyzed using multi-marker joint analysis method. Totally 28 QE eptistatic QTL were identified including 10 on A and 18 on D subgenome. The results clearly demonstrated that the more susceptible to environment variations the traits, the more QE epistatic QTL were detected. Among these epistatic QTL,15 were also detected by CIM, suggesting that at least some of the QTL generally detected by CIM in previous researches were indeed QE epistatic QTL. Many digenic epistatic QTL pairs and QE epistatic QTL detected in this research implies the significance of epistasis in the constitution of heterosis in the hybrid cotton XZM2.4. QTL for mid-parent heterosis of the 9 agronomic traits were analyzed by multi-marker analysis method based on the data of IF2 in 4 environments. Totally 60 heterotic loci were detected, of which 19 were additive,31 were partial to complete dominant, and 10 were over-dominant. Forty three of these heterotic loci were found to be the QTL position of the corresponding traits, indicating these loci might indirectly affect the performance of heterosis by controlling the expression of traits. Sixteen pleiotropic QTL were found for heterosis of different traits, each simultaneously controlling heterosis of multiple traits. Seventy five QE epistatic heterotic QTL and 75 digenic epistatic QTL pairs were also detected by multi-marker analysis. That the heterosis variation explained by epistatic QTL was the largest among different gene action modes for all trait heterosis demonstrated that epistasis including digenic as well as QE interaction was the main genetic foundation of heterosis in XZM2. Additive, dominance and overdominance also played a role in heterosis.5. A transcriptome map of the IF2 was constructed via cDNA-AFLP techniques, using the top fully opened leaves during budding stage to extract RNA. The map was 2747.01cM in length, with 302 cDNA-AFLP markers distributed among 26 linkage groups. The average length of linkage groups was 95.27 cM, and the average interval between two markers was 8.23 cM. All markers distributed uniformly among linkage groups. Seventy six and 61 QTL were identified by CIM for 9 agronomic traits and 9 fiber quality traits respectively, using the transcriptome map and the 4-environment data. Some of the QTL were clustered on certain linkage groups. The majority of the QTL detected by this map were dominant and over-dormant.Most of the closely collocated or co-segregated candidate TDFs over 200bp in length were excised from the gels, successfully re-amplified and sequenced. Through homology searches, altogether 61 TDFs collocated with QTL of agronomic and fiber quality traits were detected with potential gene products or biological function. The putative gene products of these TDFs involved in controlling of transcription and translation, signal transduction, transport, cellulose synthesis, photosynthesis, metabolism of carbohydrate as well as lipid, and constitution components of cell structure. Several candidate genes for yield and yield heterosis were identified through correlation analysis between differential expression of TDFs and yield related traits. In the future, these functional candidate genes can be cloned by RACE or map based cloning, and the QTL associated TDFs can be converted into CAPS markers for the purpose of marker assisted selection (MAS).更多还原