Developing RAD markers as a resource for plant breeding

为了提高植物育种计划的能力，以实现人口增长和气候变化所要求的农业增长，必须开发新的技术来快速发现和分型遗传标记。俄勒冈大学的Eric Johnson教授开发的RAD（限制性位相关DNA）测序（RADSeq）技术可以从基因组DNA中产生数万个遗传“标签”。由于现代DNA测序仪的能力，可以对每个标签进行多次测序，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，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。每个个体的标签可以通过在测序之前向DNA中添加唯一的“分子标识符”来识别。通过仔细选择要生成的正确数量的标签，就有可能在一次运行中筛选足够的个体，以覆盖整个遗传图谱种群。因此，RAD测序将遗传标记的发现、基因分型和定位结合为一个步骤。此外，如果样品的表型是已知的，数据可用于识别与表型分离的标记，协助基因定位和潜在的基因鉴定。迄今为止，RADSeq主要用于动物或微生物系统。我们建议将RADSeq技术应用于一种模式谷物——多年生黑麦草（Lolium perenne），以确定该技术在提高植物育种工作中的适用性。作为测试案例，我们将使用一个现有的定位群体来确定控制黑麦草自交不亲和系统的两个遗传位点。我们将使用两个标签密度对该种群的高覆盖率父母进行RADSeq。然后，我们将从四个分离基因型（每个位点两个）中筛选汇总的定位群体后代，以确定每个基因型独有的RADSeq标记。最后，我们将利用RADSeq标记信息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。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效果如何，使用最困难的例子之一-高度杂合的远交物种。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作为基因位点定位工具的实用性也将通过尝试绘制与草的自交不亲和位点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.