Efficient Identification of Induced Mutations in Crop Species by Ultra-High Throughput DNA Sequencing

主要研究者：Luca Comai（加州大学-戴维斯分校）CoPI：Jorge Dubcovsky（加州大学-戴维斯分校）和Steven Henikoff（弗雷德哈钦森癌症研究中心，西雅图）合作者：Robert Tran和Dawei Lin（加州大学-戴维斯分校）。基因序列和功能的信息不断增加，激发了人们对重要农艺性状和品质性状产生重大影响的途径的兴趣。然而，尽管越来越多的基因是有价值的失活靶标，但可以灵活地靶向每个作物物种50至3000个基因的作物基因修饰方法是有限的。该项目将超高通量测序应用于TILLING（靶向诱导的基因组局部病变），这是一种发现感兴趣基因中诱导突变的通用方法。与水稻和小麦TILLING系合作，它将采用PCR介导的扩增技术，从合并的基因组模板中靶向数十至数百个基因。该研究将测试和实施几种技术和计算方法，以解决高度复杂模板中错误的真实的突变。为了靶向数百到数千个基因，该项目将使用序列条形码从数百或数千个现有的拟南芥TILLING系以及后来的水稻和小麦中创建短接头连接随机片段的DNA池。使用定制可编程阵列或连接至微阵列或微珠的长寡核苷酸对来自扩增池的等分试样进行捕获程序，并对洗脱的片段进行Solexa测序。该项目将建立一个经济有效的管道，作为用户支持的服务，取代更费力和昂贵的核苷酸错配检测技术。水稻和小麦跨越了关键特征的范围，因此可以作为所有作物的模型。它们在倍性、二倍体与异源多倍体、序列信息（高与低）、基因组大小（小与大）和突变密度（中等与非常高）方面存在差异。在开发TILLING的测序方法的同时，该项目将使用现有的和经过测试的群体来靶向每种作物中的一组基因，这些基因将对科学家和育种家社区有用。因此，这项研究将通过提供简便的分离和变异分析来促进改良作物品种的选育。这项研究将为作物的有效分蘖提供方法。通过这样做，它将刺激基础发现以及有针对性的性状改进。例如，在小麦中，在育种之后对同源异型基因组进行独立失活可以解决许多重要的农艺和品质性状。因为通过直接测序发现突变将允许研究人员从头开始选择哪种突变体来表征，所以诱导突变的计算机收集将为育种社区提供强大的资源。所开发的方法将不仅是有用的TILLING，但也发现自然变异，或确定数量性状基因座的等位基因的变化。连接外展计划结合了本科和专业培训。它基于三种方法：实习计划，将来自代表性不足的人群的学生和教师与实验室到田间的实验基因组管道配对，数据库和方法网站，以及培训作物科学家的年度研讨会。公众可通过项目网站（http：//www.example.com）和已建立的基因组学数据库（http：//www.gramene.org/; http：//www.arabidopsis.org/）获得数据和方法。tilling.ucdavis.edu

PI: Luca Comai (University of California - Davis)CoPIs: Jorge Dubcovsky (University of California - Davis) and Steven Henikoff (Fred Hutchinson Cancer Research Center, Seattle)Collaborator: Robert Tran and Dawei Lin (University of California - Davis). The growing information on gene sequence and function stimulates interest in pathways expected to have large effects on important agronomic and quality traits. Nevertheless, while more and more genes are valuable inactivation targets, approaches for crop gene modification that can flexibly target 50 to 3000 genes per crop species are limited. This project will apply ultra-high throughput sequencing to TILLING (Targeting Induced Local Lesions IN Genomes), a general method for the discovery of induced mutations in genes of interest. Working with rice and wheat TILLING lines, it will employ PCR-mediated amplification from pooled genomic templates to target dozens to hundreds of genes. The research will test and implement several technical and computational methods to resolve real mutations from errors in highly complex templates. To target hundreds to thousands of genes, the project will use sequence barcoding to create DNA pools of short adapter-ligated random fragments from hundreds or thousands of existing TILLING lines of Arabidopsis, and later rice and wheat. Aliquots from an amplified pool will be subjected to a capture procedure using custom programmable arrays or long oligos attached to either microarrays or beads, and the eluted fragments subjected to Solexa sequencing. The project will build an economical and effective pipeline that will be sustainable as a user-supported service, superseding more laborious and expensive nucleotide mismatch-detection technology. Rice and wheat span the range of critical characteristics and can therefore serve as models for all crops. They differ in ploidy, diploid versus allopolyploid, sequence information, high versus low, genome size, small versus large, and mutation density, moderate versus very high. While developing a sequencing approach to TILLING, the project will use already existing and tested populations to target a set of genes in each crop that will be useful to the community of scientist and breeders. Thus, this research will facilitate breeding of improved crop varieties by providing facile isolation and analysis of variants. This research will provide methods for efficient TILLING of crop plants. By doing so, it will stimulate both basic discovery as well as targeted trait improvement. For example, in wheat independent inactivation of homeologous gene sets followed by breeding can address a host of important agronomic and quality traits. Because discovery of mutations by direct sequencing will allow researchers to choose ab initio which mutant to characterize, in silico collections of induced mutations will provide powerful resources for breeding communities. The developed methods will not only be useful for TILLING but also to discover natural variation, or to identify allelic changes underlying quantitative trait loci. The connected outreach program combines the undergraduate and professional training. It is based on three approaches: an internship program that will pair students and teachers from underrepresented populations with laboratory-to-field experimental genomic pipeline, a database and methods web site, and a yearly workshop for training crop scientists. Data and methods will be accessible to the public through the project web site (http://tilling.ucdavis.edu) and through established genomics databases (http://www.gramene.org/; http://www.arabidopsis.org/).