Developing RAD markers as a resource for plant breeding

为了提高植物育种方案的能力，以实现人口增长和气候变化所要求的农业增长，必须开发新技术，以快速发现遗传标记并进行基因分型。由俄勒冈州大学的埃里克约翰逊教授开发的RAD（限制性位点相关DNA）测序（RADSeq）技术，从基因组DNA中产生了数万个遗传“标签”。由于现代DNA测序仪的能力，可以多次对这些标签中的每一个进行测序，从而可靠地发现两个个体之间的遗传差异。第二代测序仪的能力使得来自多个个体的标签可以在单个测序运行中合并，同时仍然保持每个标签的足够高的覆盖率以识别遗传差异。来自每个个体的标签可以通过在测序之前向DNA添加独特的“分子标识符”来识别。通过仔细选择要生成的标签的正确数量，可以在单次运行中筛选足够的个体以覆盖整个遗传作图群体。因此，RAD测序将遗传标记的发现、基因分型和作图结合到一个步骤中。此外，如果样品的表型是已知的，则数据可用于鉴定与表型一起沿着分离的标记，从而有助于基因作图和潜在的基因鉴定。迄今为止，RADSeq主要用于动物或微生物系统。我们建议将RADSeq技术应用于模式谷物物种多年生黑麦草（多年生黑麦草），以确定该技术对改善植物育种工作的适用性。作为一个测试案例，我们将使用现有的映射人口设计，以确定两个遗传位点控制的自交不亲和系统在黑麦草（黑麦草）。我们将使用两个标签密度在高覆盖率下在该群体的亲本中进行RADSeq。然后，我们将从四个分离基因型（每个基因座两个）中的每一个筛选合并的作图群体后代，以鉴定对每个基因型来说独特的RADSeq标记。最后，我们将使用RADSeq标记信息构建该人群的遗传图谱，并使用常规基因分型确认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确定RADSeq技术作为作物快速标记发现和基因分型方法的表现如何，使用最困难的例子之一-高度杂合的远系繁殖物种。如果RADSeq在这些条件下表现良好，它应该很容易适用于其他作物系统。RADSeq作为遗传基因座作图工具的有用性也将通过试图绘制与禾本科植物自交不亲和基因座相关的多态性来评估。识别这些基因对草育种者非常重要，因为它们可以在育种计划中更好地控制交配。

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.