【作者】 丁广大；

【导师】 徐芳森；

【作者基本信息】 华中农业大学 ， 植物营养学， 2011， 博士

【摘要】 油菜是产油效率最高的油料作物之一,我国油菜的种植面积和总产均居世界首位。甘蓝型油菜占我国油菜种植面积的80%以上。甘蓝型油菜对缺磷敏感,其栽培地区又处于我国土壤有效磷缺乏或严重缺乏的地区。因此,研究甘蓝型油菜磷营养高效的遗传机理,然后通过遗传改良的方法培育甘蓝型油菜磷高效品种对促进油菜持续稳定的发展、实现磷资源的可持续利用有着重要的意义。本实验室利用甘蓝型油菜磷高效品种鄂油长荚和磷低效品种B104-2为亲本,构建了一个含124个株系的高度纯合的重组自交系群体(命名为BE-RIL)。本研究以BE-RIL群体为材料,首先利用GBM、SRAP、SSR等标记加密BE-RIL遗传图谱,构建了一张高密度的新版本遗传连锁图。然后通过两批苗期水培试验和两年成熟期大田试验,调查了磷正常和低磷条件下群体苗期的干重及磷含量、成熟期的产量及产量相关性状、种子中七种矿质离子(磷、钙、镁、铁、锰、铜、锌)的含量等性状的表型变异。结合高密度的磷高效分子整合图谱,对所考察的性状在全基因组水平上进行了QTL定位及分析。获得的主要研究结果如下：1.甘蓝型油菜磷高效分子整合图谱的构建及其与拟南芥基因组的比较作图：利用172个白菜的BAC序列自主设计了243对SSR标记,利用模式植物拟南芥中与磷吸收代谢相关的46个功能基因自主设计了256对GBM标记,然后利用BE-RIL群体124个株系的基因型,通过Joinmap 4.0软件构建了一张含840个分子标记的高密度BE-RIL遗传图谱。该图谱含62个AFLP标记,257个SRAP标记,472个SSR标记以及49个GBM标记。该图谱总长为1913.6 cM,标记的平均间距为2.3 cM。以已鉴定的拟南芥24保守区段为基础,利用BE-RIL遗传图谱上304个有DNA序列信息的标记与拟南芥基因组进行比对,在BE-RIL遗传图谱上共定位了38个共线性区段和77个保守岛。从发表的文献和拟南芥网站中收集了789个与目标性状相关的基因,利用电子定位的方法将它们的3162个同源基因定位到BE-RIL遗传图谱上。2.亲本及BE-RIL群体在不同磷水平下的表型分析：对苗期调查的各性状而言,在缺磷的条件下,磷高效亲本鄂油长荚相比磷低效亲本B104-2表现出较大的优势。对成熟期各性状的调查表明,在缺磷的条件下,磷高效亲本鄂油长荚的籽粒产量、每荚粒数、每株荚果数、分枝数、株高等性状与磷低效亲本B104-2相比具有显著的差异,而千粒重则刚好相反。磷高效亲本在缺磷条件下比磷低效亲本能累积更多的矿质养分。在磷正常条件,两亲本间上述所有性状的差异不显著。研究结果表明无论是苗期还是在成熟期,BE-RIL群体植株的所有性状都呈现连续分布,并表现出超亲分离的现象。证实这些性状为数量性状,受到多基因的调控。性状间的遗传相关系数在同一磷水平下表现出较大的差异,而且其遗传力也表现出显著的差异,表明不同性状其遗传调控位点存在很大的差异,且受环境影响的程度不一。3.控制成熟期产量及相关性状的QTL检测及分析：在磷正常和缺磷的水平下,对BE-RIL群体两批实验的成熟期产量、千粒重、每荚粒数、单株荚果数、分枝数、株高、磷效率系数等性状进行QTL分析,利用WinQTL Cartographer 2.5软件共检测到95个显著性QTL,其中磷正常条件下48个,缺磷条件下41个,6个QTL控制磷效率系数。这些QTL分布在16条连锁群上。有19个QTL可以在两批实验中重复检测到。单个QTL解释表型的范围为7.3-25.4%。利用BioMercator 2.1软件,我们将22个在其它研究中报导的控制产量及相关性状的QTL映射到本研究所构建的遗传图谱上,这些QTL与本研究报导的18个QTL相互重叠。4.控制种子矿质养分含量的QTL检测及分析：在磷正常和缺磷的水平下,对BE-RIL群体两批实验的种子磷、钙、镁、铁、锰、铜、锌等七种矿质养分的含量进行QTL分析,共检测到78个显著性QTL。其中磷正常条件下检测到45个QTL,缺磷条件下检测到33个QTL。这些QTL分布在17条连锁群上,单个QTL解释表型的范围为7.8-18.1%。16个QTL可以在两批实验中重复检测到。5.控制苗期各性状的QTL检测及分析：在磷正常和缺磷的条件下,对BE-RIL群体三批苗期实验的地上部干重、根干重、磷含量等性状进行QTL分析,共检测到109个显著性QTL,其中磷正常条件下检测到49个QTL,缺磷条件下检测到60个QTL。这些QTL在除A10和C2之外的其它各个连锁群上均有分布。单个QTL解释表型的范围为6.9-26.7%。在三个实验环境中只检测到一个一致表达的QTL,表明不同的生长介质及培养环境对QTL的表达有较大的影响。通过对大田苗期和成熟期定位的QTL进行比较分析,发现在苗期检测到的部分QTL在成熟期中有表达。6.QTL的综合分析：通过对苗期三批实验和成熟期两批实验检测到的282个QTL进行综合分析发现,部分QTL成簇地分布在某些连锁群的特定位置,并与本室之前分析得到的QTL簇相互重叠。例如A1、A3、A6和C3等连锁群上的QTL簇。这表明在染色体的某些区段存在QTL的热点区域控制不同的性状。通过电子定位,最终将拟南芥198个与磷吸收代谢及产量等相关基因的224个同源基因定位到50个控制产量及产量相关性状QTL的置信区间内。将21个参与离子养分吸收转运的同源基因定位在13个控制籽粒中矿质离子含量QTL的置信区间内。另外,有14个GBM标记位于QTL置信区间内,其中5个标记位于低磷条件下检测到的QTL置信区间内。这一结果将为甘蓝型油菜磷高效相关QTL的深入研究提供大量的遗传信息。更多还原

【Abstract】 Rapeseed is one of the most effective oil crops for oil production. China is the largest rapeseed producer in the world, in which more than 80% of the plant area is occupied by Brassica napus. In the middle and lower valley of the Yangtze River in South China, the largest cultivated region for rapeseed, the soil available P concentration is usually lower than 10 mg/kg. However, B. napus has high P requirement for its optimal seed yield and quality. Hence, the development of cultivars with enhanced phosphorus (P) use efficiency would be an economical and sustainable way for the management of P nutrition in B. napus production. In our previous research, a set of 124 F10 B. napus recombinant inbred line (RIL) population named the BE-RIL population was developed using the single-seed descent method from a cross between P-inefficient cultivar’B104-2 (B)’and P-efficient cultivar’Eyou Changjia (E)’.In this study, an improved BE-RIL genetic linkage map was constructed using gene-based marker (GBM), sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR). Two-year field trials as well as two hydroponic trials were conducted with normal P and low P treatments. Phenotypic variation of seed yield and yield-related traits and seed mineral concentrations (P, Ca, Mg, Zn, Cu, Fe, Mn) at maturing stage, dry weight and P concentration at seedling stage were investigated. Quantitative trait loci (QTL) analysis was performed with the purpose to unravel the genetic network of low-P response of B. napus at seedling and maturing stages.A total of 243 SSR markers were developed from 171 bacterial artificial chromosome (BAC) end sequences and/or B. rapa seed BAC sequences.256 GBMs was developed from 46 functional genes involved in Arabidopsis thaliana P homeostasis. In total,49 GBMs corresponding to 26 genes, assigned to eight functional categories, were integrated into the BE-RIL map. Genotyping data generated in this study were attempted for integrating the marker loci into the framework linkage map using JoinMap software Version 4.0. The present map has a total of 840 loci, including 62 amplified fragment length polymorphism (AFLP),257 SRAP,472 SSR and 49 GBMs. The total map length was 1913.6 cM with an average distance of 2.3 cM between two loci. Based on the identified 24 conserved chromosomal blocks on A. thaliana genome, a total of 115 conserved blocks, including 38 syntenic blocks and 77 insertion segments, were identified using 304 of the 840 linked markers with known sequence information. According to physical positions on the Arabidopsis genome,3162 orthologs of 789 Arabidopsis functional genes related with target traits were mapped onto syntenic blocks and insertion segments. This high-density integrated molecular genetic map will be useful for identifying QTL that control P homeostasis and putative candidate genes for the efficient use of P in B. napus.Compared with P-inefficient cultivar’B104-2’, P-efficient cultivar’Eyou Changjia’ showed a higher value for dry weight and P concentration under low P condition at seedling stage as well as a higher value for seed yield and yield-related traits at maturing stage, except seed weight. In addition, a higher accumulation of minerals was observed in seeds of P-efficient cultivar’Eyou Changjia’than P-inefficient cultivar’B104-2’. However, for all the tested traits, there was no significant difference under normal P condition. The frequency distributions of all the traits showed continuous phenotypic variation for the BE-RIL population, and significant transgressive segregation was observed in both directions, suggesting multiple gene action. Correlation coefficients among traits under low P and normal P condition were different, and a low-to-high broad-sense heritability for each trait was observed in the BE-RIL population. This indicated the genetic loci controlling these traits might be different under two P levels, and environmental influence for each trait might be different.Phenotype data for seed yield, seed weight, seed number, pod number, plant height, branch number and phosphorus efficiency coefficient (PEC) from two field trials was used for QTL detection by WinQTL cartographer 2.5 software as well as the mean values of the two crop seasons. A total of 95 putative QTL distributed across 16 chromosomes were identified, including 6 QTL for PEC,48 and 41 QTL under normal and low P conditions for the other six traits, respectively. These QTL accounted for a range of 7.3-25.4% of the total phenotypic variation.19 (20%) were detected in two seasons and in the mean value of two season. Based on the common molecular markers on different genetic maps,22 reported QTL, which were related to the same traits and identified from other genetic populations of B. napus, were projected onto the BE-RIL genetic map using the map projection function of BioMercator 2.1 software. These QTL co-located with 18 QTL for corresponding traits detected in the present study, indicating a potential QTL hotspot for each trait in different genetic backgrounds and environmentsA total of 78 putative QTL, including 45 under normal P conditions and 33 under low P conditions, were detected in the two-year field trials for seven mineral traits. These QTL were mainly located on 17 linkage groups, explaining 7.84%to 18.1% of the phenotypic variation. About 65% of these QTL were co-located with at least one other QTL.16 QTL were identified in at least two environments, but the rest were detected in only one environment.109 putative QTL were identified for shoot dry weight, root dry weight, P concentration at seedling stage, including 49 under normal P conditions and 60 under low P conditions. These QTL scattered across 17 chromosomes except A10 and C2, and accounted for a range of 6.9-26.7% of the total phenotypic variation. Only one QTL on A6 was observed in three trials under high P level, suggesting culture environment and growth media might show heavy influence on QTL expression for the same trait. Some QTL also expressed at both seedling and maturing stages, suggesting the same gene action during plant development.Analysis by synthesis of 282 putative QTL, we found QTL were clustered on the location of each linkage group, and co-located with QTL detected previously in our lab, such as on chrosome A0, A3, A6, C3 etc. This indicated QTL hotspots existed on different linkage groups controlling different traits. By comparative mapping between Arabidopsis and B. napus,224 orthologs of 198 genes involved in Arabidopsis P homeostasis and/or controlling yield-related traits were associated with 50 QTL, and 21 genes involved in ion homeostasis in Arabidopsis were mapped to 13 QTL intervals. In addition,14 GBMs developed from genes involved in Arabidopsis P homeostasis were mapped to target QTL intervals, of which five were located in the confidence intervals of QTL detected under low P condition. These results may provide useful information for improving P efficiency of B. napus in soils with low P availability.更多还原