菊花头状花序发育的遗传调控机理

Flower shape is an important quality trait for ornamental plants. The capitulum of chrysanthemum consists of ray florets and disc florets, which together form the various flower shapes via their differentiation and development under the same genetic background, as well as their specific distribution on the cephalophorum. At present, the directional breeding for novel flower shape in chrysanthemum is especially difficult due to the limited information regarding the developmental mechanism of the two types of florets. Our previous studies showed that significant differences existed in the sexual determination, symmetry, tissue structure, and expression of genes related to floral development between the two florets. In this project, typical chrysanthemum varieties will be selected as experimental materials. Transcriptome analysis will be used to screen the different expressed genes. Key genes which regulate the floret development will be ascertained by in situ hybridization, over expression, RNAi and CRISPR/Cas9. Then, the mechanism of these genes regulating the boundary shifting and florets development will be investigated from the aspects of gene expression networks and epigenetic regulation. Finally, gene regulatory networks for the development of both disc and ray florets are expected to be established to reveal the genetic regulation mechanism of capitulum development in chrysanthemum. The present study is of practical implications for the directional breeding on novel flower shape in chrysanthemum, and also possesses theoretical significance for the evolutionary and developmental biological studies on the flowers of higher plants.

花型是观赏植物重要的品质性状。菊花的花是由管状花和舌状花两种单花构成的头状花序。两种小花在相同遗传背景下的分化和发育及其在花托上的分布是菊花千姿百态花型形成的基础。迄今由于对两类小花发育机制不清楚，菊花花型育种始终难于稳定定向。本课题组前期对大量菊花品种及其近缘野生种研究发现，两种小花在性别、对称性和组织结构上等存在明显差异。初步分析发现，两类小花发育过程中一些花器官特征基因表达存在差异。本项目拟以收集到的典型样本为材料，利用转录组数据，从筛选差异表达基因入手，确定调控两类小花发育的关键基因，继而从基因调控表达和表观遗传修饰两个方面，探讨这些关键基因调控两类小花在头状花序上定位发育并产生形态差异的作用机理，进而构建两类小花发育的基因调控网络，从而阐明菊花头状花序发育的遗传调控机制。这一研究不仅对人工调控菊花花型改良的育种工作具有实际指导意义，对高等植物花的进化发育生物学研究也具有理论价值。

花型是观赏植物重要观赏性状之一。菊花（Chrysanthemum × morifolium）的花型受到头状花序上舌状花和管状花的相对数量、排列位置及花冠形态等多方面的影响，千姿百态的花型堪称园艺植物中的奇迹。但目前对菊花头状花序上两类小花发育的遗传调控机制尚不清晰，阻碍了定向调控菊花花型的育种实践。本研究以具有异型头状花序的菊花近缘野生种甘菊（C. lavandulifolium）为主要研究材料，结合具有同型头状花序的芳香堆心菊（Helenium aromaticum）和贺兰山女蒿（Hippolytia alashanensis）为材料，全面探究了头状花序发育过程及其分子调控机制。主要取得如下成果：.（1）基于扫描电镜和石蜡切片观察发现，甘菊头状花序上各原基的发生顺序为典型的非螺旋向心方式，小花原基形成初期（S5）和小花原基形成中期（S6）为两类小花原基分别起始发生的关键时期。仅具管状花的芳香堆心菊和贺兰山女蒿头状花序发育过程中，管状花原基发生是其花序发育的关键时期。.（2）建立了甘菊下胚轴及叶片的高效遗传转化体系和芳香堆心菊的高效再生体系，为关键基因的功能验证奠定了基础。.（3）对甘菊头状花序不同发育阶段和两类小花不同发育阶段的转录组分析，筛选出了可能参与头状花序发育调控的基因，包括MADS-box、CYC2以及NAM三类转录因子。.（4）甘菊转ClNAM基因株系的头状花序上舌状花减少甚至消失，或者出现畸形。表明ClNAM在舌状花形成过程中发挥重要作用。.（5）酵母双杂交分析发现，ClNAM和ClPI、ClNAM和ClCYC2d、DenFUL和ClAG2、ClAP2和ClSEP1、ClAP3和ClPI、ClCYC1和ClCYC2、DenFUL和ClCYC2、ClAP2和ClCYC1均存在双向互作。据此构建了基于ClNAM、MADS-box、B类和C类以及ClCYC2类基因的甘菊头状花序发育的遗传调控网络。.（6）利用MSAP和BSP技术以及miRNA测序分析发现，DNA甲基化通过影响ClCYC2-like基因在两类小花中的可变剪接形式和转录水平发挥作用；miR159、miR164、miR167、miR172和miR396通过调控靶基因表达水平发挥作用。.上述研究成果不仅为解析菊花花型形成的机制提供了重要线索，对解析高等植物花进化发育生物学研究也具有参考价值。