New Tools for Genetic Analysis in Arabidopsis thaliana

拟南芥遗传分析的新工具

• 批准号: 1243754
• 负责人: R. Poethig
• 金额: $ 40万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1243754&HistoricalAwards=false
• 关键词: New; Tools; Genetic; Analysis; Arabidopsis; New; Tools; Genetic; Analysis; Arabidopsis

Intellectual MeritResearch on Arabidopsis thaliana has produced major advances in plant biology because of the many advantages of this system for genetic analysis. But, while it is straightforward to study mutations of Arabidopsis that have obvious, viable, fully penetrant phenotypes, it is still difficult and time-consuming to study mutations that cannot be maintained in homozygous condition, and mutations that have invisible, weak or highly variable phenotypes. This project will produce three resources that facilitate analyses of this large class of mutations: 1) marked chromosome segments, 2) introgression lines and 3) induced polymorphic lines. A marked chromosome segment (MCS) is a chromosome region marked at one end by pNAPIN::dsRED and at the other end by pNAPIN::eGFP. These seed-specific, dosage- sensitive, fluorescent transgenes make it possible to visually identify seeds carrying the marked chromosome segment, as well as seeds homozygous or heterozygous for genes in trans to the MCS. Using an MCS of an appropriate size and location, seeds homozygous or heterozygous for genes/mutations of interest can be accurately identified prior to planting, greatly simplifying genetic and phenotypic studies. MCS lines can also be used to identify recombination events in the vicinity of a gene of interest, facilitating the introgression of mutations and natural polymorphisms into a common genetic background. MCS lines will be generated in both the Columbia (Col) and Landsberg erecta (Ler) ecotypes. Approximately 20 Ler MCS lines covering the entire genome will be introgressed into Col by multiple backcrosses. These MCS introgression lines will be genetically identical to Col, except in the region encompassed by the MCS, and will facilitate the map-based cloning of mutations whose phenotype is only obvious in a Col genetic background. Induced polymorphic (IP) lines offer a similar advantage, but differ from MCS introgression lines in that they involve induced, rather than natural, polymorphisms. IP lines of the Col ecotype will be generated by 3 rounds of sequential mutagenesis, accompanied by selection for lines with a wild-type Col phenotype. IP lines will be made homozygous and the identity of the induced polymorphisms will be determined by next generation sequencing. These new genetic tools will enable a wide variety of experiments that are currently difficult or impossible to perform, adding to the value of Arabidopsis thaliana as a model system for research in plant biology. Broader ImpactsIn principle, Arabidopsis thaliana is an ideal organism for teaching plant genetics: it is small, easy to grow, has short life cycle, and its seeds mature in only two weeks. However, its utility as a teaching tool is limited by the difficulty of making controlled intercrosses. Male parents must be emasculated to prevent self-fertilization, and this is difficult to do without practice. This limitation can be overcome by using male sterile mutants, but these mutants must be maintained as heterozygotes, and this introduces significant complications. An MCS stock that makes it possible to unambiguously identify seeds homozygous for the dyt1 male-sterile mutation will be generated, and a laboratory exercise that takes advantage of this stock and transgene insertions created in this project will be developed. Specifically, students will cross plants heterozygous for linked pNAPIN::dsRED and pNAPIN::eGFP transgenes to male sterile plants, harvest the resulting seeds, calculate the recombination distance between these transgene insertions, and generate new MCS lines in the process. A stock containing three linked dominant mutations on the same chromosome as dyt1 will also be produced, enabling teachers to readily generate the plants necessary for a laboratory exercise on three point recombination mapping. These resources will make it possible for students to perform plant genetics in 'real time' in high school or undergraduate courses, and will enable the development of new laboratory exercises that take advantage of the numerous genetic resources available in Arabidopsis.

关于拟南芥的知识分子研究研究了植物生物学的重大进步，因为该系统在遗传分析中具有许多优势。 但是，尽管研究具有明显，可行，完全渗透的表型的拟南芥的突变是很简单的，但是研究无法保持在纯合条件下的突变以及具有无形，弱或高度可变的表型的突变仍然很困难且耗时。 该项目将产生三种资源，以促进该大型突变的分析：1）标记的染色体段，2）浸润线和3）诱导的多态线。 一个明显的染色体段（MCS）是PNAPIN :: DSRED在一端标记的染色体区域，另一端由PNAPIN :: EGFP在另一端。这些种子特异性的，敏感的荧光转基因使视觉上识别带有标记染色体段的种子，以及纯合子或杂合的种子，用于trans到MCS中的基因。在播种之前，可以使用适当的大小和位置的MC准确鉴定出纯合子或杂合的种子，以确定感兴趣的基因/突变，从而大大简化了遗传和表型研究。 MCS线还可以用于鉴定感兴趣基因附近的重组事件，从而促进突变和自然多态性的渗透到共同的遗传背景中。 MCS线将在哥伦比亚（Col）和Landsberg Erecta（LER）生态型中产生。覆盖整个基因组的大约20个LER MCS线将被多个反向交叉渗入COL。除了由MCS所包含的区域外，这些MCS渗入线将在遗传上与COL相同，并将促进基于地图的突变克隆，其表型仅在COL遗传背景中很明显。 诱导的多态性（IP）线具有类似的优势，但与MCS渗入线不同，因为它们涉及诱导的，而不是自然的多态性。 Col Ecotype的IP系将通过3轮顺序诱变产生，并伴随着选择具有野生型Col表型的线。 IP线将成为纯合，并通过下一代测序确定诱导的多态性的身份。这些新的遗传工具将使目前难以或无法执行的各种实验增加拟南芥作为植物生物学研究模型系统的价值。更广泛的影响力原则，拟南芥是教授植物遗传学的理想生物：它很小，易于生长，生命周期较短，并且在短短两周内就成熟了种子。但是，其作为教学工具的实用性受到控制互变困难的限制。男性父母必须被剥夺以防止自我施肥，而没有实践，这很难做到。可以通过使用雄性无菌突变体来克服这种局限性，但是这些突变体必须保持为杂合子，这引入了显着的并发症。 将产生一个明确地识别纯合子男性雄性突变种子的MCS股票，并将开发利用该股票和Transgene插入的实验室运动。具体而言，学生将跨植物跨植物进行链接的PNAPIN :: DSRED和PNAPIN :: EGFP转基因到雄性无菌植物，收集所得的种子，计算这些转基因插入之间的重组距离，并在此过程中产生新的MCS线。 还将产生与DYT1同一染色体上的三个连接的主要突变的股票，使教师能够轻松地在三点重组映射上产生实验室运动所需的植物。这些资源将使学生在高中或本科课程中“实时”进行植物遗传学，并能够开发新的实验室练习，以利用拟南芥可用的众多遗传资源。