A Sequence-Indexed Reverse Genetics Resource for Maize: A Set of Lines with Single Ds-GFP Insertions Spread throughout the Genome

作者：Hugo K. Dooner （Rutgers大学）作者：Charles Du （Montclair州立大学）单个基因被破坏的突变系为生物学家了解该基因的作用提供了有力的工具。因此，单插入序列索引的集合是阐明基因组测序生物体中基因功能的关键资源，并且被认为是充分利用玉米基因组序列的必要条件。最近，将高通量测序与多维池策略相结合，一次对数百个新插入进行测序和索引已经成为可能。这项工作将完成基于转座子d的反向遗传资源的生产，这将使社区能够产生和建立玉米的单基因敲除资源。具体而言，该工作将：(1)表征在先前资助期间由农杆菌转化产生的78个单Ds- gfp（或Ds*）启动平台的转座频率，并将其映射到基因组中。将确定高度活跃的平台并将其存放在库存中心，以补充已经绘制的82个平台。这些平台将允许从基因组的许多区域对转位进行视觉选择，从而使研究人员能够创建区域基因敲除集合；(2)从位于所有10条染色体上的24个Ds*启动平台生成480个Ds*转位集合，这些集合与现有的9个集合一起，将作为基于Ds*的玉米单基因敲除资源的基础；(3)利用高通量MiSeq Illumina对3- d DNA池进行测序，对这些集合进行序列索引。为此，开发了一个特定的软件包（InsertionMapper），从大量测序数据中提取Ds*转座子连接并将其映射到玉米基因组中。该项目将整合罗格斯大学的高通量测序和新泽西州蒙特克莱尔州立大学的生物信息学序列分析。密歇根州立大学的学生将注释由NextGen测序器生成的所有Ds*相邻序列。该项目将为该校信息学和分子生物学专业的学生提供直接参与玉米研究的机会，并在毕业前完成他们的独立研究要求。罗格斯大学和密歇根州立大学的学生将作为暑期实习生在PI的分子生物学实验室和玉米遗传苗圃工作。PI和coPI是代表性不足的群体的成员，以前的合作记录卓有成效。他们曾在NSF pgrp资助的项目中合作，开发了一种生物信息学工具，并发表了三篇联合论文。参与密歇根州立大学基因组学项目的学生中，约有三分之一是未被充分代表的少数族裔。密歇根州立大学将这个项目的研究机会加入到吸引和留住少数民族高中毕业生的推广活动中。这个项目与美国农业有关，因为它涉及玉米，这是当今美国最重要的作物。它解决了一个关键的需求，因为它将提供一个序列索引的反向遗传资源，这被认为是研究人员充分利用玉米基因组序列的必要条件。所有数据将通过插入位点序列的网络检索数据库（http://www.acdsinsertions.org/）提供，该数据库与玉米遗传合作库存中心（http://maizecoop.cropsci.uiuc.edu）提供的约10000种种子库存进行交叉引用。该项目的所有相关信息均可通过http://www.acdsinsertions.org/访问，长期可通过MaizeGDB （http://www.maizegdb.org）访问。

PI: Hugo K. Dooner (Rutgers University)CoPI: Charles Du (Montclair State University)The availability of a mutant line in which a single gene has been disrupted gives biologists a powerful tool in understanding the action of that gene. Thus, sequence-indexed collections of single insertions are critical resources for elucidating gene function in organisms with sequenced genomes and are deemed essential by the community to fully exploit the maize genome sequence. It has recently become feasible to combine high-throughput sequencing with multi-dimensional pooling strategies to sequence and index hundreds of new insertions at a time. This work will complete the production of a reverse genetics resource based on the transposon Ds that will enable the community to generate and build up a single-gene knockout resource for maize. Specifically, the work will: (1) characterize the transposition frequency of 78 single Ds-GFP (or Ds*) launching platforms generated by Agrobacterium transformation during the previous grant period and map them to the genome. Highly active platforms will be identified and deposited in the Stock Center to complement the 82 already mapped. These platforms will allow visual selection of transpositions from many regions of the genome and, thus, enable researchers to create regional gene knock-out collections; (2) generate collections of 480 Ds* transpositions each from 24 Ds* launching platforms located on all 10 chromosomes which, together with the 9 collections currently at hand, will serve as the foundation for a Ds*-based maize single-gene knockout resource; and (3) sequence-index these collections by high throughput MiSeq Illumina sequencing of 3-D DNA pools. To this end, a specific software package (InsertionMapper) was developed to extract Ds* transposon junctions from the large amount of sequencing data and map them to the maize genome.This project will integrate high throughput sequencing at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. MSU students will annotate all Ds*-adjacent sequences generated by the NextGen sequencer. The project will provide informatics and molecular biology students at that institution with the opportunity to participate directly in maize research and fulfill their independent research requirement for graduation. Students at both Rutgers and MSU will work in the project as summer interns in the molecular biology lab and maize genetics nursery of the PI. The PI and coPI are members of underrepresented groups and have a record of fruitful prior collaborations. They have collaborated in previous NSF PGRP-funded projects which led to the development of a bioinformatics tool and the publication of three joint papers. About a third of the Genomics students participating in the project at MSU are members of underrepresented minorities. MSU has added the research opportunity from this project to its outreach campaign to attract and retain minority high school graduates. This project is relevant to U.S. agriculture in that it deals with maize, the most important American crop today. It 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. All data will be made available through a web-searchable database of insertion site sequences (http://www.acdsinsertions.org/) that are cross-referenced to ~10000 seed stocks available from the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project will be accessible through http://www.acdsinsertions.org/ and long-term through MaizeGDB (http://www.maizegdb.org).