Developing RAD markers as a resource for plant breeding

开发 RAD 标记作为植物育种资源

• 批准号: BB/H023844/1
• 负责人: Mark Blaxter
• 金额: $ 9.11万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH023844%2F1
• 关键词: Developing; RAD; markers; resource; plant

In order to improve the ability of plant breeding programmes to deliver the agricultural increases mandated by a growing population and changing climate, new techniques must be developed for rapid discovery and genotyping of genetic markers. The RAD (Restriction-site Associated DNA) sequencing (RADSeq) technique developed by Professor Eric Johnson of the University of Oregon, generates tens of thousands of genetic 'tags' from genomic DNA. Due to the capabilities of modern DNA sequencers, it is possible to sequence each of these tags many times and thus reliably spot genetic differences between two individuals. The capacity of second-generation sequencers is such that tags from multiple individuals can be pooled within a single sequencing run whilst still maintaining a high enough coverage of each tag to identify genetic differences. Tags from each individual can be identified by adding a unique 'molecular identifier' to the DNA prior to sequencing. By carefully selecting the right number of tags to be generated, it becomes possible to screen enough individuals within a single run to cover an entire genetic mapping population. RAD sequencing therefore combines the discovery, genotyping and mapping of genetic markers into a single step. Furthermore, if the phenotype of the samples is known, the data can be used to identify markers which segregate along with the phenotype, assisting in gene mapping and potentially gene identification. To date, RADSeq has primarily been used in animal or microbial systems. We propose to apply the RADSeq technique to a model cereal species, Lolium perenne (perennial ryegrass), in order to determine the applicability of this technique to improving plant breeding efforts. As a test case, we will use an existing mapping population designed to identify the two genetic loci controlling a self-incompatibility system in Lolium (ryegrass). We will perform RADSeq in the parents of this population at high coverage using two tag densities. We will then screen pooled mapping population progeny from each of four segregating genotypes (two per locus) in order to identify RADSeq markers which appear unique to each genotype. Finally, we will use the RADSeq marker information to construct a genetic map for this population and confirm the bioinformatic identification of a small subset of genetic markers using conventional genotyping. The proposed work will enable us to determine how well the RADSeq technique performs as a method for rapid marker discovery and genotyping in crops, using one of the most difficult examples - a highly heterozygous, outbreeding species. If RADSeq performs well under these conditions, it should easily be applicable to other crop systems. The usefulness of RADSeq as a tool for mapping of genetic loci will also be assessed by attempting to map polymorphisms associated with the self-incompatiblity loci of grasses. Identifying these genes is of high importance to grass breeders as they would allow greater control of mating during breeding programmes.

为了提高植物育种计划的能力提供由人口增长和气候变化所规定的农业增长的能力，必须开发新技术，以快速发现和基因分型遗传标记。俄勒冈大学埃里克·约翰逊（Eric Johnson）教授开发的RAD（限制位置相关的DNA）测序（RADSEQ）技术从基因组DNA产生了数万个遗传“标签”。由于现代DNA测序仪的能力，可以多次对每个标签进行测序，从而可靠地发现两个个体之间的遗传差异。第二代序列仪的容量使得可以将来自多个个体的标签在单个测序中汇总，同时仍然保持足够高的每个标签覆盖范围以识别遗传差异。可以通过在测序前向DNA添加唯一的“分子标识符”来识别每个人的标签。通过仔细选择要生成的正​​确数量的标签，可以在一次运行中筛选足够的个体以覆盖整个遗传映射种群。因此，RAD测序将遗传标记的发现，基因分型和映射结合到一个步骤中。此外，如果已知样品的表型，则可以使用数据来识别与表型一起分离的标记，从而有助于基因映射和潜在的基因鉴定。迄今为止，RadSeq主要用于动物或微生物系统。我们建议将RadSeq技术应用于谷物模型，Lolium Perenne（多年生黑麦草），以确定该技术在改善植物育种工作中的适用性。作为测试案例，我们将使用旨在确定控制Lolium（Ryegrass）自我兼容系统的两个遗传基因座的现有映射种群。我们将使用两个标签密度在高覆盖范围内在该人群的父母中执行RADSEQ。然后，我们将从四个分离的基因型（每个基因座）中的每一个中的每一个筛选汇总的映射种群后代，以识别每种基因型看起来独特的RadSeq标记。最后，我们将使用RADSEQ标记信息为该人群构建遗传图，并使用常规基因分型确认对遗传标记的一小部分的生物信息学鉴定。拟议的工作将使我们能够使用最困难的例子之一（一种高度杂合的，异生的物种）来确定RadSeq技术在农作物中的快速标记和基因分型的表现。如果RadSeq在这些条件下表现良好，则应很容易适用于其他作物系统。 RadSeq作为遗传基因座映射的工具的有用性也将通过尝试映射与草的自我兼容基因座相关的多态性来评估。识别这些基因对草育种者至关重要，因为它们可以在育种计划中对交配的更多控制。

项目成果

Special features of RAD Sequencing data: implications for genotyping.

• DOI: 10.1111/mec.12084
• 发表时间: 2013-06
• 期刊: Molecular ecology
• 影响因子: 4.9
• 作者: Davey JW;Cezard T;Fuentes-Utrilla P;Eland C;Gharbi K;Blaxter ML
• 通讯作者: Blaxter ML