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

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.

对拟南芥的研究已经在植物生物学方面取得了重大进展，因为这种遗传分析系统具有许多优点。 但是，虽然研究具有明显的、可行的、完全外显的表型的拟南芥突变是简单的，但是研究不能在纯合条件下保持的突变以及具有不可见的、弱的或高度可变的表型的突变仍然是困难和耗时的。 该项目将产生三种资源，以促进这一大类突变的分析：1）标记的染色体片段，2）渐渗系和3）诱导多态系。 标记的染色体片段（MCS）是在一端由pNAPIN：：dsRED标记并且在另一端由pNAPIN：：eGFP标记的染色体区域。这些种子特异性、剂量敏感性、荧光转基因使得可以视觉鉴定携带标记的染色体区段的种子，以及对于MCS反式基因纯合或杂合的种子。使用适当大小和位置的MCS，可以在种植前准确地鉴定目标基因/突变的纯合或杂合种子，从而大大简化遗传和表型研究。 MCS系也可用于鉴定感兴趣的基因附近的重组事件，促进突变和天然多态性渗入共同的遗传背景。 MCS品系将在哥伦比亚（Col）和兰茨贝格直立（Ler）生态型中产生。将通过多次回交将覆盖整个基因组的约20个Ler MCS系渗入Col中。这些MCS渐渗系将与Col在遗传上相同，除了MCS所涵盖的区域之外，并且将促进表型仅在Col遗传背景中明显的突变的基于图位的克隆。 诱导的多态性（IP）线提供了类似的优势，但不同于MCS渗入线，因为它们涉及诱导，而不是自然，多态性。Col生态型的IP系将通过3轮连续诱变产生，同时选择具有野生型Col表型的系。将使IP系纯合，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不可能进行的各种实验成为可能，增加了拟南芥作为植物生物学研究模型系统的价值。更广泛的影响原则上，拟南芥是教授植物遗传学的理想生物：它体积小，易于生长，生命周期短，其种子仅在两周内成熟。然而，它作为教学工具的实用性是有限的，使控制杂交的困难。雄性父母必须被阉割以防止自花受精，而这在没有实践的情况下是很难做到的。这种限制可以通过使用雄性不育突变体来克服，但是这些突变体必须保持为杂合子，这引入了显著的并发症。 将产生一种MCS原种，使其能够明确地识别dyt 1雄性不育突变的纯合种子，并将开发利用该原种和该项目中创建的转基因插入的实验室练习。具体来说，学生将杂交植物杂合的连锁pNAPIN：：dsRED和pNAPIN：：eGFP转基因雄性不育植物，收获产生的种子，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系。 还将产生一个在与dyt 1相同的染色体上含有三个连锁显性突变的原种，使教师能够容易地产生三点重组作图实验室练习所需的植物。这些资源将使学生有可能在高中或本科课程中“真实的时间”进行植物遗传学，并将使新的实验室练习的发展，利用拟南芥中的众多遗传资源。