雌雄异型异熟青钱柳性别分化及其异熟机理研究

Cyclocarya paliurus is the sole species in its genus and one of endangered tree in China. It shows great application potential for the development of a new woody plant used for medicinal and health care resources, wood products and ornamental species. At present, the method of reproduction in Cyclocarya paliurus mainly focuses on sexual propagation by seeds, which is the main restrict factor of industrial development because of its unavailablity. Based on observations over several years, we guess the key factors inhibiting seed yield and quality is flowering character of heterdichogamy in Cyclocarya paliurus. In this program, we attempt to construct the fingerprint map on basis of specific molecular markers (SRAP or SSR etc.)for mating types of Cyclocarya paliurus. Then, the inheritance mode of mating types will be investigated by identifying controlled-pollination hybrid offspring between different mating types. In order to disclose theory of sex determination by hormonal regulation and the function of genes related to sex determination, changing patten of hormones during period of sexual differentiation will be detected by High efficiency liquid chromatography (HPLC), and genes related to sex determination will be screened using GeneFishing technique. Moreover, we try to elucidate the mechnism of dischogamy based on knowledge of physiology and biochemical, and development-specific miRNAs profiles sequened by Solexa technique during sexual differential and development. In short, we not only attempt to demonstrate theoretical system involving the genetic mode of maing type, as well as the mechnism of sex determination and dichogamy, but also to provide technical instruction for establishment and management of seed orchards and plantation for heterodichogamous Cyclocarya paliurus.

青钱柳是我国重点保护的单种属濒危植物和多用途珍贵树种，具有很高的保健和药用开发价值。目前发展青钱柳人工林资源主要通过种子繁殖，但种子来源不足严重制约了其产业化进程，究其原因可能是其特有的雌雄异型异熟的开花机制。本项目旨在运用分子手段筛选交配类型基因特异性标记（SSR、SRAP等）并建立指纹图谱，通过鉴定控制授粉杂交子代的基因型来探讨其遗传方式。在雌雄花性别分化时期，利用高效液相色谱技术（HLPC）监测激素生理变化，同时采用GeneFishing技术筛选性别分化相关基因，以探讨性别分化的激素控制理论及性别分化基因的作用。结合花芽分化和发育各阶段的生理生化研究，运用Solexa深度测序挖掘阶段特异性miRNAs，从不同角度探索雌雄花异熟机理。通过上述研究，可从理论上系统阐述青钱柳交配类型的遗传方式、雌雄花性别分化及其异熟机理，也可为种子园经营和人工林栽培提供技术支撑。

青钱柳为重要的木本药用植物，同时又是典型的雌雄异型异熟植物。本研究首次系统阐述了其开花习性，即林分群体表现为2个开花阶段，但2个阶段的花期有一定程度的重叠。典型的雌雄异型异熟植物表现为2个开花阶段几乎无重叠，青钱柳与其存在较大差异。林分群体的散粉特性与开花特性一致，2个开花阶段花粉密度和花粉活力无显著差异，但均出现花粉活力较低的现象（<30%）。两种交配类型在年度间表达稳定，说明其雌雄异型异熟特性是由遗传控制，而非环境因素造成的，进一步证实了雌雄异型异熟植物的遗传控制理论。. 通过解剖学结合形态变化观察，将花芽发育分为5个阶段：生理分化期(S0)、休眠期(S1)、萌动期(S2)、花序伸长期(S3)和成熟期(S4)。花期激素研究发现调控其花芽分化和发育的主要激素为GA1和GA3、ABA、IAA；其中，较高含量GA1、GA3和低含量ABA有助于打破休眠，促进萌动；高含量IAA和低含量ABA有利于花序的伸长生长；较高含量ABA促进了花芽的生理分化。. 不同交配类型上的雌雄花分化和发育时序上存在差异，其所需养分来源各不相同。研究发现第1开花阶段的花发育所需养分主要来其自身或周围营养器官的贮藏养分，而第2开花阶段的花发育所需养分则主要来自光合产物的输入。青钱柳花发育时序上的差异不仅体现在主要激素在不同发育阶段雌雄花上的差异性积累，而且各发育阶段花芽（序）的转录组测序分析获得差异性基因的表达，则从分子水平上提供了更充足的证据。因此来自生理水平、解剖水平和分子水平上的证据可充分阐述了青钱柳雌雄异型异熟的机理。. 基因组survey分析和细胞流式仪的倍性分析发现，林分群体主要由四倍体组成(95%)，少有二倍体和三倍体的存在。这是首次发现多倍体的雌雄异型异熟植物，且其多倍体特性也可能是导致开花特异性、低花粉活力和低种子饱满度的主要原因；该发现也对雌雄异型异熟的孟德你遗传理论提出了挑战。.