Facile Production and Efficient Indexing of Transposon-tagged Lines Using Next-generation Sequencing Technology for Maize

Pi：雨果·K·杜纳(罗格斯大学)Copis：春光杜(蒙特克莱尔州立大学)和Randall A.Kerstetter(罗格斯大学)转座元件(TE)插入导致突变，帮助研究人员阐明基因功能。已经被序列索引的插入片段的集合(即，其中与插入片段相邻的宿主DNA是已知的)是具有已测序基因组的生物中的宝贵资源，并且已经被玉米界确定为充分利用刚刚产生的玉米基因组序列所必需的。最近，将高通量DNA测序与高效的多维池策略相结合以快速、同时对数百个新插入片段进行测序和索引的可行性已被证明。该项目将利用下一代测序技术，开发和使用一种快速、准确和经济的通用方法，用于产生、测序和索引玉米中的Ac/DS转座子插入。具体地说，该项目将：(1)对包含唯一转座Ac元件的现有系的集合进行序列索引；(2)完成一组120个大致等距的转基因ds元件作为启动平台，并携带易于评分的标记，该标记将允许从基因组的任何区域对元件转座进行简单的视觉选择，从而使研究人员能够产生区域基因敲除集合；(3)使用一种新的方法对模型平台中的10,000个DS元件插入位点进行序列索引，该新方法应该适用于在共同遗传背景中产生的任何插入集合；以及(4)开发与可从玉米遗传资源中心(http://maizecoop.cropsci.uiuc.edu).)免费获得的品系交叉引用的插入位点序列的网络可搜索数据库该项目的所有相关信息将可从MaizeGDB获取(http://www.maizegdb.org).This项目解决了一个关键需求，因为它将提供一个序列索引的反向遗传学资源，这被认为是研究人员充分利用玉米基因组序列所必需的。它将把罗格斯大学的下一代测序技术与蒙特克莱尔州立大学的生物信息序列分析结合起来，蒙特克莱尔州立大学是新泽西州的一所以本科生为主的机构。密歇根州立大学的学生将协助组装、注释和定位作为假定的相邻序列的短阅读片段，并协助设计用于验证的引物。该项目还将为密歇根州立大学信息学和分子生物学学生提供研究培训机会，他们中的许多人来自代表性不足的群体。罗格斯大学和密歇根州立大学的学生也将作为暑期实习生参加这个项目，在分子生物学实验室和玉米遗传学苗圃实习。

PI: Hugo K. Dooner (Rutgers University)CoPIs: Chunguang Du (Montclair State University) and Randall A. Kerstetter (Rutgers University)Transposable element (TE) insertions cause mutations that help researchers to elucidate gene function. Collections of insertions that have been sequence-indexed (i.e., in which the host DNA adjacent to the insertion is known) are valuable resources in organisms with a sequenced genome and have been identified by the maize community as essential to fully exploit the maize genome sequence just generated. Recently, the feasibility of combining high-throughput DNA sequencing with efficient multi-dimensional pooling strategies to rapidly and simultaneously sequence and index hundreds of new insertions has been demonstrated. This project will develop and use a general method for generating, sequencing, and indexing Ac/Ds transposable element insertions in maize that is rapid, accurate, and cost-effective by taking advantage of next-generation sequencing technology. Specifically, the project will: (1) sequence-index a collection of existing lines that contain a unique transposed Ac element; (2) complete a set of 120 roughly equidistant transgenic Ds elements that serve as launching platforms and carry easily scored markers that will allow simple visual selection of element transposition from any region of the genome and, thus, enable researchers to generate regional gene knock-out collections, (3) sequence-index 10,000 Ds element insertion sites from model platforms using a novel method that should be applicable to any collection of insertions produced in a common genetic background; and (4) develop a web-searchable database of insertion site sequences cross-referenced to lines that will be freely available from the Maize Genetics Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project will be accessible from MaizeGDB (http://www.maizegdb.org).This project addresses a critical need in that it will deliver a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. It will integrate next-generation sequencing technology at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. Students at MSU will assist with the assembly, annotation, and mapping of short reads as putative Ac-adjacent sequences and with the design of primers for validation. The project will also provide research training opportunities for MSU informatics and molecular biology students, many of whom are from underrepresented groups. Students at both Rutgers and MSU will also participate in the project as summer interns in the molecular biology lab and maize genetics nursery.