【作者】 龙艳；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 发育生物学， 2008， 博士

【摘要】 开花是高等植物由营养生长向生殖生长转变的标志之一,决定着植物的有性生殖。适时开花以避开不利外界环境,是植物得以繁衍的前提,也是决定作物能否获得丰收的优良农艺性状。植物的开花是由多基因所控制的各种生理生化代谢途径所决定的,是复杂的数量性状,同时也受各种外界环境因素的调节,其中温度和光照是关键的环境因子。油菜是重要的油料作物,根据开花是否需要低温春化,分为冬油菜和春油菜两种类型。人们对油菜开花的机制知之甚少,对决定油菜冬、春性分化的机制更不得而知。甘蓝型冬油菜品种Tapidor和半冬性油菜品种Ningyou7对低温春化需求不同。本实验室以这两个品种为亲本构建了TN DH群体并在9个冬油菜环境下种植了该群体。实验室还在TN DH群体基础上衍生了RC-F₂群体。本论文报告了用TN DH群体做的两年春油菜开花试验,分析整理了DH群体和RC-F₂群体多年多点共13个冬油菜实验的开花期数据;用100个MS-AFLP分子标记参与构建了含352个标记的初级的TN遗传连锁图,在此基础上整合了230个SSR标记,构建了含621个标记的高密度TN遗传连锁图;利用高密度图谱上229个有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）,在TN遗传图谱上定位了40个共线性区段和84个保守岛;然后采用QTL分析软件WinQTLcart2.5、QTLmapper2.0进行油菜开花期QTL及互作位点的检测和遗传效应分析,目的是在基因组水平上构建甘蓝型油菜开花性状的基因网络图。研究结果表明,在所有冬油菜环境中群体植株的开花期均表现超亲分离,证实TN DN群体的开花期为数量性状。为了充分揭示数量性状的多基因特征,本文不仅综合分析了两个群体13个冬、春油菜环境中在显著水平上（P=0.05）检测出的开花期QTL（称为显著水平QTL,significant level QTL,SL-QTL）,还将效应较小、显著性程度较低（0.5＜P＜0.05）但能重复检测到的QTL鉴别为微效真实QTL（micro-realQTL,MR-QTL）。结果共检测到42个开花期QTL,其中包括36个SL-QTL和6个MR-QTL。这42个QTL中,过半的QTL仅能在冬油菜种植环境检测到,其中第16连锁群上的一个QTL簇对冬油菜环境开花早晚有决定性作用。仅有1个QTL能在春油菜环境条件下检测到,为春油菜环境特异QTL。另外还检测到了63个互作对。通过电子比对,将拟南芥120个开花基因的548个同源基因定位到TN遗传图谱上,发现其中27%和9%的同源基因被分别定位到油菜开花期QTL置信区间和互作位点的标记区间,它们在很大程度上代表着油菜相应区间的开花功能基因。由此,一个由上百个QTL、互作位点和区间功能基因构成的甘蓝型油菜开花调控网络图初步形成。它不仅为进一步研究油菜开花性状提供了坚实的基础,同时为其它复杂农艺性状的解析拓宽了研究思路。为了进一步检验QTL定位的准确性和电子比对定位功能基因的可靠性,也为了为油菜的遗传改良提供重要的基因资源,本文对TN DH群体中表现春油菜环境特异性的主效应QTL作了较深入的解析。该QTL位于N10连锁群上,命名为qFT10-4。它在缺乏春化的环境条件下解释群体开花期变异（方差）的52%,而在越冬的冬油菜条件下对群体植株开花期表型变化无贡献。前人在拟南芥中已经揭示了开花的春化途径,而本试验开展的比较基因组学分析表明,春化途径中的关键抑制基因FLC在拟南芥基因组中所处的位置,正对应于qFT10-4的置信区间。以TN DH群体两亲本之一Tapidor中FLC的同源序列为探针与TN DH群体的总DNA进行Southern杂交,结果将FLC的一个RFLP多态性标记定位到TN遗传图谱上,正位于qFF10-4置信区间的峰值处,于是将该区间的同源FLC基因命名为BnFLC10。从双亲中分离出BnFLC10的长4.5kb的基因组序列,发现等位基因的第一个内含子中存在着三处碱基的差异。根据其中两个差异位点设计等位基因特异性的PCR引物M1和M2,对用半冬性油菜品种Ningyou7为轮回亲本、BnFLC10为目标基因构建的BC₃F₂群体,在春油菜环境下进行开花期表型和基因型的分析。卡方测验表明开花期表型分离符合3:1的分离比（晚花或不开花:早花）,同时基因型与表型共分离,表明BnFLC10为春油菜环境中控制TN DH群体开花早晚的主效基因。基因表达实验发现春化确实能使BnFLC10的表达下降,春化前BnFLC10的基因表达量在亲本Ningyou7中居于春油菜品种和冬油菜品种之间,冬油菜亲本Tapidor及其它冬油菜品种的表达量最高。对73个不同来源的油菜品种资源进行BnFLC10与油菜冬春性关联分析,结果M2扩增位点的多态性与开花早晚所表现的冬春性表型相关达到极显著水平（P=0.007）。这些研究均表明qFT10-4的候选基因BnFLC10为控制油菜品种冬、春性分化的关键基因。本论文提出了三个进一步研究课题:1.微效真实QTL的证实及QTL检测体系的改进;2.油菜冬、春性分化关键基因BnFLC10决定油菜种性的分子机理;3.冬油菜环境特异开花期QTL簇的解析。更多还原

【Abstract】 Flowering is the symbol of the transition from vegetative to reproductive stage for plants,and it’s a determining factor for sexual reproduction of plant.Flowering in time can make plants avoid bad environments,such as harsh winters or hot summers,to get good harvest.Flowering of plants is not only regulated by multiple endogenous genetic factors,also controlled by complex environmental factors.Temperature and light are the most two important environmental factors.Rapeseed is an important oil plants and flowering time is both an important agronomical and a complex quantitative trait. Rapeseeds can be divided into two types,the winter annuals and the spring annuals, according to the requirement of vernalization or not.Until now it’s known a little about the mechanism of controlling flowering time and differentiation of the winter- and spring-typed cultivars in Brassica napus.The winter-typed B.napus variety Tapidor and the semi-winter typed variety Ningyou7 have different requirement for vernalization.A DH population named with TN DH population was constructed with these two varieties,and a RC-F2 population was constructed based on TN DH population in our lab.The field trial of one location in two years for spring environments was designed and the flowering time data was investigated. Besides the data,the flowering time data of 9 winter environments of TN DH population and 2 winter environments of RC-F₂ population were collected.About one hundred methylation sensitive AFLP markers were added to the TN basic linkage map which including 352 markers.Besides that,about 230 SSR markers were collected and a high density linkage map including 621 markers was constructed by using the software joinmap3.0.After that,229 markers with known DNA sequence information were used as anchored markers to do comparative mapping between TN linkage map and the genome of Arabidopsis.There were 40 synteny blocks and 84 islands were mapped to TN linkage map by using in silico mapping analysis.Then,QTL mapping analysis and epistatic interaction detection were done by using the softwares WinQTLcart2.5 and QTLmapper2.0.The aim of the study was to construct a genetic network controlling flowering time in B.napus.Transgressive segregation of flowering time was found in all the environments and it showed that flowering time of TN DH population was a quantitative trait.In order to fully discover the characters of the flowering time trait,two types of QTL were collected.One type is the QTL attached to the significant level（P=0.05） in each of the 13 different environments for the two populations,the other type is the MR-QTL（Micro-real QTL） with small effect under low significant level（0.5＜P＜0.05） while could be repeatedly deteced in two or more environments.Totally there were 42 unique flowering time QTL detected,including 6 MR-QTL.Among the QTL,there were 55%of the QTL could only be detected in the winter-cropped environments,and one QTL could only be deteced in the spring-cropped environments,which showed that it was a spring-cropped environment specific QTL.Sixty-three interacting loci pairs were detected.Five hundred and forty-eight homolougous genes representing 120 flowering time genes of Arabidopsis were mapped to TN linkage map by in silico mapping analysis.Among these genes,there were 27%and 9%mapped to the QTL regions and the marker interval of interacting loci pairs.To a large extent,these homologous genes represented the flowering functional genes of the QTL regions in B.napus.Therefore,the genetic network of flowering time composed of hundreds of QTL,interacting loci pairs and the functional genes of the regions in B.napus was constructed.It not only provided the further research basis of the flowering time in B.napus,also provided the research clues for dissecting other complex agronomical traits.In order to certify the accuracy of QTL mapping results and the reliability of the mapped functional genes by in silico mapping analysis,meanwhile provide the important genetic resources for genetic improvement for rapeseed,the spring-cropped environment specific QTL was further analyzed.This QTL was located on N10 linkage group and named as qFT10-4.It could explain the 52%of phenotypic variation in the spring-cropped environments,while it could not be detected in any of the winter-cropped environment.Former researches have discoved the vernalization pathway controlling the flowering time in Arabidopsis.The comparative mapping analysis in this study showed that the position of the key gene FLC in the vernalization pathway was homologous to the confidence interval of qFT10-4.The probe designed according to the homologous sequence of FLC in one of the parent Tapidor was hybrized with the total genomic DNA of TN DH population.As a result,the RFLP maker of the gene was mapped to the peak of the QTL region.So the gene located in N10 linkage group was named as BnFLC10.The genomic sequence of the BnFLC10 alleles of the two parents in TN DH population was isolated and they were about 4.5kb long.There were only three differences existing in the region of intron 1 and the gene specific primers M1 and M2 were designed according to the two differences.The primers were used to analyze the genotype of BC₃F₂ population with the recurrent parent Ningyou7 and harboring the target gene BnFLC10.The flowering time data was also surveyed in spring-cropped environment.The Chi-square analysis showed that the ratio of flowering time was fit to the ratio of 3:1（late flowering or not flowering:early flowering）.The genotype of all the early flowering plants was consistent with the early flowering parent Ningyou7.These results showed that BnFLC10 was the major gene controlling the flowering time in TN DH population.Gene expression experiment result showed that vernalization could repress the gene expression of BnFLC10.The gene expression amount was stronger in winter-typed cultivar Tapidor than that in spring-type under the same condition,and the gene expression amount of semi-winter cultivar Ningyou7 was in the middle. Seventy-three different cultivars were used to do association mapping alaysis of the BnFLC10 by using the primers M1 and M2.The polymorphic marker of M2 showed significant relative to the flowering time（P=0.007）.All the above results showed that the candidate gene BnFLC10 of the QTL qFT10-4 was the key gene and controlling the differentiation of the winter- and spring-typed rapeseed.Three aspects of research work are listed in the last section of the thesis.They are as follows:1.Certification of MR-QTL and the improvement of QTL detecting system;2. Dissecting the mechanism of BnFLC10 regulates the differentiation of winter- and spring-typed rapeseed;3.Dissecting the winter-cropped environment specific QTL cluster.更多还原