RESEARCH-PGR: Deciphering the link between RNA directed DNA methylation and reproduction in Brassicaceae

种子是人类和牲畜的主要卡路里来源，因此，种子开发是农业生产力最关键的方面之一。即使是种子发育的微小变化也会对生产力产生深远的影响，并对整个食物链产生连锁影响。这项建议阐述了DNA甲基化在种子发育中的作用，特别是那些影响种子结实、大小和生存能力的标记。我们最近在重要的油料作物油菜中发现了DNA甲基化途径的三个突变。每一种突变在种子生产中都有严重和特定的缺陷。使用先进的DNA测序技术，我们将确定这些突变的遗传、分子和基因组后果。这些发现将有助于理解调节种子发育的内部机制。该项目还将为多名研究生和本科生提供培训机会。这次培训将培养他们在诸如基因组编辑和高通量测序等尖端技术方面的专业知识。这项工作建立在几个关键观察的基础上，这些观察将小RNA指导的DNA甲基化(RdDM)与适当的种子发育联系起来，可能是通过建立和维护：1)基因组印迹(等位基因表达中的亲本偏见)，和/或2)基因组平衡(全基因组复制后显性亚基因组的优先表达)。这个项目将首先探索RdDM在种子发育中的转录和发育后果，方法是分析甘蓝型油菜中RNA导向的DNA甲基化突变体的小RNA、转录本和表观基因组，每一个突变体都极大地减少了结实率。此外，我们还将研究白菜型油菜基因组印记和基因组优势之间的联系。最后，我们将检验RdDM通过改变基因组印记和/或通过在十字花科其他成员中产生同源突变来影响种子结实的假设。这项研究的结果将为更好地理解印记和基因组优势提供更好的理解，这些优势可以转化为十字花科中密切相关的物种，包括种子作物甘蓝型油菜。

Seeds are the primary source of calories for humans and livestock, and thus one of the most critical aspects of agricultural productivity is seed development. Even small changes in seed development can have profound impacts on productivity with cascading repercussions through the entire food chain. This proposal addresses the role of DNA methylation, a chemical mark placed on DNA, in the development of seeds, particularly those marks that impact seed set, size, and viability. We have recently uncovered three mutations of a DNA methylation pathway in the important oil crop Brassica rapa. Each mutation has severe and specific defects in seed production. Using advanced DNA sequencing technologies, we will identify the genetic, molecular, and genomic consequences of these mutations. These findings will help understand the internal mechanisms that regulate seed development. This project will also provide training opportunities for multiple graduate and undergraduate students. This training will develop their expertise in cutting-edge techniques such as genome editing and high-throughput sequencing.This work builds on several key observations that link small-RNA directed DNA methylation (RdDM) with proper seed development, likely through establishment and maintenance of: 1) genomic imprinting (parent-of-origin bias in allele expression), and/or 2) genome balance (preferential expression from a dominant subgenome following whole genome duplication). This project will first explore the transcriptional and developmental consequences of RdDM in seed development by profiling small RNAs, transcriptomes, and epigenomes of RNA-directed DNA methylation mutants in Brassica rapa, each of which dramatically reduces seed set. In addition, we will investigate links between genomic imprinting and genome dominance in B. rapa. Finally, we will test the hypothesis that RdDM influences seed set by altering genomic imprinting and/or genome dominance by generating cognate mutations in other members of the Brassicaceae family. Results from this study will provide a better understanding of both imprinting and genome dominance that can be translated to closely related species in the family Brassicaceae, including the seed crop B. napus.