【作者】 杨美；

【导师】 徐芳森；

【作者基本信息】 华中农业大学 ， 植物营养学， 2010， 博士

【摘要】 油菜是我国的主要油料作物之一,种植面积和总产均居世界首位。位于长江中下游的甘蓝型油菜主产区磷素缺乏,而甘蓝型油菜对缺磷敏感,限制了此产区油菜产量的提高。通过遗传育种的方法,开发和培育高效吸收利用磷营养的油菜品种具有很好的发展前景。根系形态构型的适应性变化是植物响应低磷胁迫的重要机制。为阐明油菜磷高效遗传特性,本研究以磷高效亲本鄂油长角和磷低效亲本B104-2所构建的重组自交系(命名为BE-RILs,F10)为材料,采用纸培试验,调查了高磷和低磷条件下BE-RILs的苗期干物重,磷含量和根系形态性状。在利用SSR、AFLP、SRAP等分子标记所构建的遗传连锁图谱的基础上,对BE-RILs表型性状进行数量遗传位点(QTL)的定位,并利用QTL元分析和比较作图对油菜适应低磷胁迫的遗传机制进行了解析。主要结果如下：1甘蓝型油菜磷效率遗传连锁图的构建及与拟南芥基因组的比较作图分析以BE-RILs各株系的基因组DNA为模板,利用SSR、AFLP、SRAP等分子标记分析群体株系基因型,通过作图软件Jionmap4.0,构建了一张含有553个分子标记的遗传连锁图,包括202个SSR,62个AFLP,234个SRAP和55个功能标记。此遗传连锁图长度为1592.7 cM,两相邻标记的平均距离为2.9 cM。利用BE-RILs遗传图谱上已知序列信息的标记作为锚定标记,将该遗传图谱与拟南芥基因组进行比较作图分析,在BE-RILs遗传图谱的10个连锁群的部分区段定位了27个保守区段。2布置三次纸培试验,调查两亲本和BE-RILs在高磷和低磷条件下的表型性状设计以蓝色无磷吸水纸为支撑的纸培系统,在根系收获后,利用WinRhizo根系分析软件测定根系总根长,根系表面积,根体积和平均根系直径,同时测定植株干重和磷含量。结果发现,在低磷条件下,相对于磷低效亲本B104-2,磷高效亲本鄂油长角不但形成了较大根系系统,而且吸收了较多磷和生成了较多干物质,但有较低的磷利用效率。说明鄂油长角的高效机制在于低磷条件下形成发达根系,从而获得较多磷和形成较高干物质。BE-RILs群体各表型性状在低磷和高磷条件下均呈连续的分布,并存在着广泛的变异,变异系数处于20.5-40.6%之间,这表明BE-RILs群体的双亲对这些性状所贡献的等位基因在其后代群体中有广泛的分离。3磷效率QTL的检测和分析’3.1两个磷水平下苗期各性状显著性QTL检测利用Wincart2.5的复合区间作图法对三次纸培试验两个磷水平下的地上部干重,根系干重,总干重,根冠比,总根长,根系表面积,平均根系直径,根体积,根尖数,磷含量,磷吸收累积量和磷利用效率12个性状进行了QTL定位。共检测到136个显著性QTL,其中高磷条件下63个和低磷条件下73个。这些QTL主要集中分布在A1, A3, A6, C1, C2, C3, C6和C8这8个连锁群上,单个QTL解释的表型遗传变异率在7.95%-22.00%之间。3.2磷效率QTL的整合对置信区间重叠的显著性QTL利用QTL元分析软件进行整合。首先对同一性状在不同试验中重叠显著性QTL进行整合,得到94个(高磷44个和低磷50)一致性QTL。第二步对不同性状相互重叠的一致性QTL进行整合,得到37个特异性QTL,包括高磷特异的QTL 10个,低磷特异的QTL 15个,稳定表达的QTL 12个。3.3功能标记在QTL区间的分布有10个功能标记定位在特异性QTL的置信区间内,低磷特异QTL置信区间内的有5个,高磷特异QTL置信区间内的只有1个,其他4个位于稳定表达的QTL置信区间内。这5个位于低磷特异QTL区间内的标记为BnSQD1-C1, BnGPT2-C1, BnPHO1-C1, BnIPS2-C3和BnGPT1-C3,其相应的低磷特异QTL为uq.C1a, uq.C1b, uq.C1b, uq.C3a和uq.C3c。利用这些低磷特异QTL区间内的标记对BE-RILs各株系进行筛选,得到聚合不同优良等位基因的株系。3.4同源基因在BE-RILs图谱上的in silico定位在完成与拟南芥基因组比较作图的基础上,把拟南芥中423个磷代谢途径相关基因,响应低磷胁迫的转录因子,根系发育和激素传导相关基因的位置信息,用电子作图方法定位在BE-RILs遗传图谱的810个基因座位上,其中67个基因位于特异性QTL区间所对应的保守区段内,预测为QTL候选基因。这些QTL信息和候选基因为解析油菜适应低磷胁迫机制奠定了基础,为下一步定位克隆这些基因提供了丰富的信息。更多还原

【Abstract】 Rapeseed is one of the major oilseed crops in China, and planting area and total yield are the first in the world. The middle and lower valley of the Yangtze River in South China is the largest cultivated region for rapeseed (Brassica napus), where the soil available P concentration is usually lower. B. napus has high P requirement for its optimal seed yield and quality, so low available P concentration in soils seriously limits the production of B. napus in this area. Exploiting or breeding P-efficient cultivars has become an attractive prospect for rapeseed production. Modification of root morphology and architecture is the key adaptive strategy of plant in response to P deficiency. In this study, a B. napus F10 recombinant inbred lines (was named as BE-RILs) population, which developed from a cross between P-efficient rapeseed cv. Eyou Changjia and P-inefficient cv. B104-2, was employed to investigate plant dry weight, P concentration, and root morphology traits at the seedling stage under high P (HP) and low P (LP) conditions in paper culture experiments. Based on a genetic linkage map constructed with SSR, AFLP, SRAP markers, quantitative trait loci (QTL) for these traits were identified, and the genetic basis of P-efficiency was dissected through QTL meta-analysis and comparative mapping method.The main results were as follows:1 Constructing a genetic linkage map for P efficiency in B. napus and comparative mapping with Arabidopsis Used genomes DNA of BE-RILs as template, the genotypes were analyzed with SSR, AFLP, SRAP markers. A genetic linkage map with 553 molecular markers was constructed by Jionmap4.0, including 202 SSR,62 AFLP,234 SRAP and 55 functional markers. The total genetic distance was 1592.7 centimorgans (cM) and an average distance was 2.9 cM between adjacent markers. Futhermore, the comparative genomics analysis between B. napus and Arabidopsis was mapped employing SSR markers with known sequence information as anchored markers. And 27 synteny blocks were aligned with some regions of ten linkage groups of BE-RILs.2 Investigating the phenotypic traits of two parents and BE-RILs under HP and LP conditions by three independent paper culture experiments The paper culture system which employed P-free blue germination paper as support was designed to investigate the phenotypic traits. After harvesting, total root length of plant, root surface area, root volume, and root average diameter were analyzed with WinRhizo root image analysis software. Plant dry weight and plant P concentration were also detected. As results, under low P condition, compared to the P-inefficient parent B104-2, the P-efficient parent Eyou Changjia not only developed bigger root system, but also produced higher biomass and acquired more P. However, Eyou Changjia had lower P use efficiency. This suggested that high P efficiency of Eyou Changjia was mainly attributed to developed root system. The phenotypic traits showed continuous distribution in BE-RILs. The coefficients of variation (CV) for these traits ranged from 20.5% to 40.6%. This indicated that each parent possesses alleles that have both positive and negative effects on the traits.3 Detecting and analyzing the QTL of P efficiency3.1 Detecting the significant-QTL for various traits under two P conditions QTL for twelve traits-SDW, RDW, TDW, RSR, RL, RSA, RD, TIP, PC, PU, and PUE under HP and LP conditions were detected in the three experiments using the composite interval method by Wincart2.5 software. As results, a total of 136 significant-QTL were identified, where 63 and 73 QTL were detected for HP and LP condition, respectively. These significant-QTL were distributed on eight linkage groups-A1, A3, A6, C1, C2, C3, C6 and C8. The contribution to phenotypic variations for the single QTL ranged from 7.95%-22.00%.3.2 Meta-analysis QTL of P efficiency The overlapping QTL for the traits in different experiments were integrated using QTL meta-analysis. First, the overlapping significant-QTL for the same traits in different experiments were integrated, and were got 94 consensus-QTL, which included 44 for HP and 50 for LP, respectively. Second, the overlapping consensus-QTL for different traits were integrated into 37 unique-QTL, which included 10 QTL specific for HP,15 QTL specific for LP and 12 constitutive QTL, respectively.3.3 Distributing of functional markers in the confidence intervals of unique-QTL 10 functional markers were distributed in the confidence intervals of unique-QTL. Five markers were located in the intervals of LP-specific QTL. One marker was located in the interval of HP-specific QTL. Four markers were located in the intervals of constitutive QTL. The five markers located in the intervals of LP-specific QTL were BnSQDl-Cl, BnGPT2-C1, BnPHO1-C1, BnIPS2-C3 and BnGPT1-C3, which correspond to LP-specific QTL uq.C1a, uq.C1b, uq.C1b, uq.C3a and uq.C3c. Some RI lines pyramiding favorable alleles were found using these functional makers in the intervals of LP-specific QTL3.4 Locating the orthologous genes of Arabidopsis on BE-RILs linkage map by in silico mapping On the basis of comparative genomics between B. napus and Arabidopsis,423 orthologous genes for P metabolism, P-responsive transcription factors, root development and auxin transporter in Arabidopsis were located on 850 loci of BE-RILs linkage map by in silico mapping, and 67 orthologous genes were corresponded to the intervals of QTL, which were speculated as the candidate genes of QTL in B. napus. These information of QTL and candidate genes provide a solid foundation for improving the adaptability of P deficiency, and paves the way for map-based cloning of these genes.更多还原