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

Pi：雨果·K·杜纳(罗格斯大学)Copi：Charles Du(蒙特克莱尔州立大学)单一基因被破坏的突变系的可用性为生物学家提供了一个强大的工具来理解该基因的作用。因此，序列索引的单插入片段集合是阐明具有测序基因组的生物体中基因功能的关键资源，并被生物界认为是充分利用玉米基因组序列的关键。最近，将高通量测序与多维池策略相结合以一次对数百个新插入进行排序和索引已成为可能。这项工作将完成基于转座子D的反向遗传资源的产生，使社区能够产生和建立玉米的单基因敲除资源。具体地说，这项工作将：(1)表征上一次赠款期间农杆菌转化产生的78个单一DS-GFP(或DS*)发射平台的转座频率，并将它们映射到基因组。将确定高度活跃的平台，并将其存放在股票中心，以补充已经绘制的82个平台。这些平台将允许对基因组许多区域的转座进行可视化选择，从而使研究人员能够创建区域性基因敲除集合；(2)从位于全部10条染色体上的24个DS*启动平台产生480个D*转座集合，这些集合与手头现有的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；以及(3)通过高通量MiSeq Illumina三维DNA池测序对这些集合进行序列索引。为此，开发了一个特殊的软件包(InsertionMapper)来从大量的测序数据中提取DS*转座子连接，并将它们映射到玉米基因组。该项目将把罗格斯大学的高通量测序与新泽西州蒙特克莱尔州立大学的生物信息序列分析结合起来。密歇根州立大学的学生将对NextGen测序仪生成的所有DS*相邻序列进行注释。该项目将为该机构的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都将在这个项目中作为暑期实习生在PI的分子生物学实验室和玉米遗传学苗圃工作。国际和平协会和COPI是代表人数不足的团体的成员，并且有富有成效的先前合作的记录。他们在以前的NSF PGRP资助的项目中进行了合作，这些项目导致了生物信息学工具的开发和三篇联合论文的发表。在密歇根州立大学参与该项目的基因组学学生中，约有三分之一是少数族裔代表不足的成员。密歇根州立大学已经将这一项目的研究机会添加到其吸引和留住少数族裔高中毕业生的外联活动中。该项目与美国农业相关，因为它涉及玉米，玉米是当今美国最重要的作物。它解决了一个关键需求，因为它将提供一个按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的关键。所有数据将通过可在网上搜索的插入位点序列数据库(http://www.acdsinsertions.org/)，与玉米遗传合作库存中心(http://maizecoop.cropsci.uiuc.edu).)提供的约10000个种子库交叉参考)获得所有来自该项目的相关信息将通过http://www.acdsinsertions.org/和长期通过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).