Development and validation of a flexible genotyping platform for wheat

通过标记辅助选择（MAS）开发操纵产量和抗病虫害的新策略是英国培育改良小麦品种战略的基础。学术实验室，基因分型服务提供商和育种公司使用MAS来跟踪控制理想性状（如抗病性，耐旱性和产量）的许多基因座的遗传。直到最近，大多数实验室都在他们的MAS项目中使用微卫星标记，而这些标记继续使用，对于许多物种，单核苷酸多态性（SNP）由于其易于使用和评分以及其相对容易自动化的能力而成为首选标记。然而，在异源六倍体小麦中，由于来自A、B和D基因组的同源物的出现，鉴定相似的有用序列多态性的任务是有问题的。在这项提案中，John Innes中心、IDna Genetics和KBioscience以及布里斯托大学将合作开发小麦SNP数据库和灵活的小麦基因分型平台。两者的效用将通过将工具和技术应用于几个示范项目来验证。如果成功，用户将能够通过两种途径获得该技术：第一种是内部设置，只需要基本的分子生物学设备（PCR机和荧光板阅读器）。在这种情况下，用户将使用数据库来帮助他们决定运行哪些测定和订购哪些引物/试剂（所有都可通过数据库公开获得）以及关于在哪里可以获得这些的建议。这构成了一个非常低的采用障碍，因为大多数用户将能够以最适合其目的的规模在内部进行这些SNP测定。这一层平台将用于在JIC进行的工作。KASPar平台将取代JIC现有的SSR基因分型服务，并与现有服务一样，可用于外部工作。全套KASPar引物的等分试样将保存在JIC，并通过该服务提供。第二种形式的获取包括外包给有竞争力的服务提供商，如KBioscience和IDna Genetics。这两家公司都安装了高通量平台，这将有助于降低成本，两家公司都将被设置为运行本项目中开发的所有KASPar检测。用户将仅需要提供基因组DNA和要运行的测定的列表。除了KASPar，社区成员将能够使用该数据库访问一系列替代基因分型技术，如Illumina GoldernGate/Infinium/SureSelect等。例如，该数据库将使研究人员能够设计合适的探针，从适当的技术供应商订购，或者向他们提供详细信息，说明谁在商业基础上提供所需的技术。该数据库将至少更新至2017年。

Developing new strategies to manipulate yield and pest and disease resistance by marker-assisted selection (MAS) underpins the UK's strategy to generate improved wheat varieties. Academic laboratories, genotyping service providers and breeding companies use MAS to track the inheritance of a host of loci controlling desirable traits such as disease resistance, drought tolerance and yield. Until recently most laboratories have used microsatellite markers in their MAS projects, while these markers continue to be used, for many species Single Nucleotide Polymorphisms (SNPs) have become the marker of choice due to their ease of use and scoring and their ability to be automated with relative ease. However, in allohexaploid wheat the task of identifying similar useful sequence polymorphisms is problematical due to the occurrence of homoeologs from the A, B and D genomes. In this proposal, in collaboration, the John Innes Centre, IDna Genetics and KBioscience and Bristol University will develop both a wheat SNP database and a flexible genotyping platform for wheat. The utility of both will be validated by applying the tools and technologies to several exemplar projects. If successful users will be able to access the technology via two routes: The first consists of an in-house set-up requiring only basic molecular biology equipment (PCR machine and fluorescence plate reader). In this case, users would use the database to help them decide which assays to run and which primers/reagents (all publicly available through the database) to order and suggestions as to where these might be obtained. This constitutes a very low barrier for adoption as most users will be able to perform these SNP assays in house at the scale that best fits their purpose. This level of platform will be employed for the work carried out at JIC. The KASPar platform will replace JICs existing SSR genotyping service and, like the existing service, will be available for external work. An aliquot of the full set of KASPar primers will be kept at JIC and made available via this service. The second form of access consists of outsourcing to competitive service providers, such as KBioscience and IDna Genetics. Both companies have high-throughput platforms installed which will help lower costs and both will be set up to run all the KASPar assays developed in this project. The user would only need to provide genomic DNA and a list of which assays to run. In addition to KASPar, members of the community will be able to use the database to access a range of alternative genotyping technologies such as Illumina GoldernGate/Infinium/SureSelect, etc. For instance, the database will enable researcher to either design suitable probes for ordering from the appropriate technology supplier or they will be provided with details as to who offers the required technology on a commercial bases. This database will then be updated until at least 2017.