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

PI：Hugo K.杜纳（罗格斯大学）CoPI：查尔斯杜（蒙特克莱尔州立大学）突变系的可用性，其中一个单一的基因已被破坏给生物学家一个强大的工具，在了解该基因的行动。 因此，序列索引的单一插入集合是阐明基因功能的生物体与测序的基因组的关键资源，并被认为是必不可少的社区，以充分利用玉米基因组序列。 最近，将联合收割机高通量测序与多维池化策略相结合以一次对数百个新插入进行测序和索引已经变得可行。 这项工作将完成基于转座子Ds的反向遗传资源的生产，这将使社区能够产生和建立玉米单基因敲除资源。具体而言，这项工作将：（1）表征上一个授权期内农杆菌转化产生的78个单个Ds-GFP（或Ds*）发射平台的转座频率，并将其映射到基因组。 高度活跃的平台将被确定并存放在库存中心，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视觉选择转座，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* 发射平台产生480个Ds* 易位的集合，连同目前手头的9个集合，将作为基于Ds* 的玉米单基因敲除资源的基础;和（3）通过3-D DNA库的高通量MiSeq Illumina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），从大量的测序数据中提取Ds* 转座子连接点，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蒙特克莱尔州立大学的生物信息学序列分析。 MSU学生将注释NextGen测序仪生成的所有Ds* 相邻序列。 该项目将为该机构的信息学和分子生物学学生提供直接参与玉米研究的机会，并满足他们毕业的独立研究要求。 罗格斯大学和密歇根州立大学的学生将在PI的分子生物学实验室和玉米遗传学苗圃作为暑期实习生参与该项目。 PI和coPI是代表性不足的团体的成员，并且有着富有成效的合作记录。 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出版。 在密歇根州立大学参与该项目的基因组学学生中，约有三分之一是代表性不足的少数民族成员。 密歇根州立大学已经增加了从这个项目的研究机会，其推广活动，以吸引和留住少数民族高中毕业生。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学资源，这对研究人员充分利用玉米基因组序列至关重要。 所有数据将通过插入位点序列的网络可搜索数据库（http：//www.acdsinsertions.org/）提供，该数据库与玉米遗传合作库存中心（http：//www.example.com）提供的约10000种种子库存交叉引用。maizecoop.cropsci.uiuc.edu 该项目的所有相关信息将通过http://www.acdsinsertions.org/和MaizeGDB（http：//www.example.com）长期提供。www.maizegdb.org

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).