Developing RAD markers as a resource for plant breeding

为了提高植物育种方案实现人口增长和气候变化所要求的农业增长的能力，必须开发快速发现遗传标记和对遗传标记进行基因分型的新技术。俄勒冈大学的埃里克·约翰逊教授开发的RAD(限制性内切酶相关DNA)测序技术(RADSeq)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，有可能对这些标签中的每一个进行多次排序，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是这样的，来自多个个体的标签可以在单个测序运行中汇集在一起，同时仍然保持对每个标签的足够高的覆盖率以识别遗传差异。每个个体的标签可以在测序前通过在DNA上添加唯一的分子识别符来识别。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来覆盖整个基因图谱群体。因此，RAD测序将遗传标记的发现、基因分型和图谱绘制结合到一个步骤中。此外，如果样本的表型已知，这些数据可以用来识别与表型分离的标记，帮助基因图谱和潜在的基因识别。到目前为止，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)，以确定该技术在改进植物育种工作中的适用性。作为测试案例，我们将使用现有的定位群体来识别控制黑麦草自交不亲和系统的两个遗传位点。我们将使用两种标签密度在这一群体的父母中进行高覆盖率的RADSeq。然后，我们将从四个分离的基因类型(每个基因座两个)中的每一个中筛选出汇集的作图群体后代，以确定每个基因独有的RADSeq标记。最后，我们将使用RADSeq标记信息来构建该群体的遗传图谱，并使用常规基因分型确认一小部分遗传标记的生物信息学鉴定。这项拟议的工作将使我们能够确定RADSeq技术作为一种快速标记发现和作物基因分型方法的表现如何，使用最困难的例子之一--高度杂合的近亲繁殖物种。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作为绘制遗传基因座图谱的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图谱来评估。识别这些基因对牧草育种者非常重要，因为它们可以在育种过程中更好地控制交配。

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.