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

Pi：Luca Comai(加州大学戴维斯分校)Copis：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)关于基因序列和功能的不断增长的信息刺激了人们对通路的兴趣，预计这些通路将对重要的农艺和品质性状产生重大影响。然而，尽管越来越多的基因是有价值的失活靶点，但可以灵活地针对每个作物物种50到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(靶向诱导基因组局部损伤)，这是一种发现感兴趣基因诱导突变的通用方法。与水稻和小麦的耕作品系合作，它将使用聚合酶链式反应(PCR)从汇集的基因组模板中进行扩增，以针对数十到数百个基因。这项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的错误造成的真正突变。为了针对数十万个基因，该项目将使用序列条形码来创建短接头连接的随机片段的DNA池，这些片段来自数百或数千个现有的拟南芥以及后来的水稻和小麦的耕作品系。来自扩增池的等份样品将使用定制的可编程阵列或连接到微阵列或珠子上的长寡核苷酸进行捕获程序，并对洗脱片段进行Solexa测序。该项目将建设一条经济有效的管道，作为用户支持的服务将是可持续的，取代更费力和昂贵的核苷酸错配检测技术。水稻和小麦涵盖了一系列关键特性，因此可以作为所有作物的模式。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、小与大以及突变密度、中等与极高等方面存在差异。在开发耕作的测序方法的同时，该项目将使用现有的和测试的种群来定位每种作物中一组对科学家和育种者社区有用的基因。因此，这项研究将通过提供简便的变异分离和分析来促进改良作物品种的育种。这项研究将为农作物的高效耕作提供方法。通过这样做，它将既刺激基本发现，也将促进有针对性的特征改进。例如，在小麦中，对同源基因集进行独立的失活，然后进行育种，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从头开始选择哪个突变来表征，在诱导突变的电子集合中将为育种群体提供强大的资源。所开发的方法不仅对耕作有用，而且对发现自然变异或识别潜在的数量性状座位的等位基因变化也是有用的。互联外展计划将本科生培训和专业培训相结合。它基于三种方法：实习计划，将来自代表性不足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，一个数据库和方法网站，以及每年一次的作物科学家培训研讨会。数据和方法将通过项目网站(http://tilling.ucdavis.edu))和已建立的基因组数据库(http://www.gramene.org/；http://www.arabidopsis.org/).)向公众开放

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/).