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.

智力优点拟南芥研究在植物生物学方面取得了重大进展，因为该系统在遗传分析方面具有许多优点。 但是，虽然研究具有明显、可行、完全渗透表型的拟南芥突变很简单，但研究无法维持纯合条件的突变以及具有不可见、弱​​或高度可变的突变仍然是困难且耗时的。表型。 该项目将产生三种资源，以促进对这一大类突变的分析：1）标记的染色体片段，2）渗入系和3）诱导的多态性系。 标记染色体片段 (MCS) 是一端由 pNAPIN::dsRED 标记、另一端由 pNAPIN::eGFP 标记的染色体区域。这些种子特异性、剂量敏感的荧光转基因使得能够直观地识别携带标记染色体片段的种子，以及反式至MCS的基因纯合或杂合的种子。使用适当大小和位置的 MCS，可以在种植前准确鉴定感兴趣基因/突变的纯合或杂合种子，从而大大简化遗传和表型研究。 MCS 系还可用于识别感兴趣基因附近的重组事件，促进突变和自然多态性渗入共同遗传背景。 MCS 系将在哥伦比亚 (Col) 和兰茨伯格直立 (Ler) 生态型中产生。大约 20 个覆盖整个基因组的 Ler MCS 系将通过多次回交渐渗到 Col 中。这些 MCS 渗入系在遗传上与 Col 相同，除了 MCS 所包含的区域外，并且将促进基于图谱的突变克隆，这些突变的表型仅在 Col 遗传背景中明显。 诱导多态性 (IP) 系具有类似的优点，但与 MCS 渗入系不同，因为它们涉及诱导多态性，而不是自然多态性。 Col生态型的IP品系将通过3轮连续诱变产生，同时选择具有野生型Col表型的品系。 IP 系将成为纯合的，并且诱导的多态性的身份将通过下一代测序来确定。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，从而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，拟南芥是植物遗传学教学的理想生物：它体积小，易于生长，生命周期短，种子仅在两周内成熟。然而，它作为教学工具的实用性由于难以进行受控交叉而受到限制。雄性父母必须被去势以防止自体受精，而如果没有实践，这是很难做到的。这种限制可以通过使用雄性不育突变体来克服，但这些突变体必须保持为杂合子，这会带来严重的并发症。 将生成能够明确识别 dyt1 雄性不育突变纯合种子的 MCS 库存，并将开发利用该库存和本项目中创建的转基因插入的实验室练习。具体来说，学生将连接 pNAPIN::dsRED 和 pNAPIN::eGFP 转基因的杂合植物与雄性不育植物杂交，收获所得种子，计算这些转基因插入之间的重组距离，并在此过程中生成新的 MCS 系。 还将生产出与 dyt1 在同一染色体上包含三个连锁显性突变的库存，使教师能够轻松生成三点重组图谱实验室练习所需的植物。这些资源将使学生能够在高中或本科课程中“实时”进行植物遗传学，并将能够开发利用拟南芥中大量可用遗传资源的新实验室练习。