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

PI：Hugo K.杜纳（罗格斯大学）CoPI：杜春光（蒙特克莱尔州立大学）和兰德尔A。转座因子（TE）的插入导致突变，帮助研究人员阐明基因功能。 已被序列索引的插入的集合（即，其中邻近插入的宿主DNA是已知的）是具有测序基因组的生物体中的宝贵资源，并且已经被玉米群体鉴定为充分利用刚刚产生的玉米基因组序列所必需的。 最近，已经证明了将高通量DNA测序与有效的多维汇集策略相结合以快速且同时对数百个新插入进行测序和索引的可行性。 该项目将开发和使用一种通用方法，用于产生，测序和索引玉米中的Ac/Ds转座因子插入，该方法利用下一代测序技术快速，准确和具有成本效益。具体地说，该项目将：（1）序列索引包含一个独特的转座Ac元件的现有线的集合;（2）完成一组120个大致等距的转基因Ds元件，这些元件用作发射平台并携带容易标记的标记，这些标记将允许从基因组的任何区域简单地视觉选择元件转座，因此，使研究人员能够产生区域性基因敲除集合，（3）使用新方法对来自模型平台的10，000个Ds元件插入位点进行序列索引，所述新方法应当适用于在共同遗传背景中产生的任何插入集合;和（4）开发一个插入位点序列的网络可检索数据库，该数据库与玉米遗传学储备中心（http：//www.example.com）免费提供的品系交叉引用maizecoop.cropsci.uiuc.edu。 该项目的所有相关信息都可以从MaizeGDB（http：//www.example.com）获得。该项目解决了一个关键需求，即它将提供一个序列索引的反向遗传学资源，这对研究人员充分利用玉米基因组序列至关重要。www.maizegdb.org 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学序列分析结合起来，后者是新泽西州一所主要的本科院校。 在MSU的学生将协助装配，注释，并与验证引物的设计作为推定的Ac相邻序列的短读段的映射。 该项目还将为MSU信息学和分子生物学学生提供研究培训机会，其中许多学生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该项目，在分子生物学实验室和玉米遗传学苗圃。

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.