From rice to orphan crops: robust high throughput genetic markers for all the grasses.

为了充分理解和操纵控制作物生长重要方面的遗传机制，如产量，抗病性和适应气候变化，我们需要确定控制这些性状的基因。这需要分子标记，用于标记含有这些基因的染色体区域。在过去的十年中，选择的标记是简单序列重复（SSR），也称为微卫星。SSR是很好的遗传标记，但由于许多原因需要更换。它们不能在物种之间转移，小麦的SSR图谱不能与大麦等近亲的图谱相比。它们的开发成本很高，不能针对特定区域，并且不能用于绘制足够密度的地图以用于许多目的。这项工作的目的是启动SSR的替代，并为尚未拥有SSR的物种提供一套分子标记。最好的可用标记系统是保守正射序列（COS）系统。与SSR相似，COS标记是基于PCR的，但与SSR不同的是，PCR引物锚定在高度保守的外显子序列中。因此，这些标记在各属中同样有效（扩增）。在这项研究的试点工作中，标记在玉米、水稻、小米、小麦、羊茅和短柄草中工作良好。尽管引物锚定在外显子（最终翻译成蛋白质的基因部分）中，但它们从内含子（非蛋白质编码）扩增DNA。与外显子相比，内含子具有相对较高的差异发生率（单核苷酸多态性，SNP），因此是内含子允许基因的变体（等位基因）作为分子标记被区分，但是是外显子赋予系统这种稳定性。本研究中使用的SNP鉴别方法是单链构象多态性（SSCP），其中SNP导致DNA在毛细管电泳过程中以不同的速率折叠和迁移。该方法已被证明是高度可重复的一个大的一组标志物在相同的条件下，所以很少优化是必需的。许多标记物可以同时在每个毛细管上运行（多路复用）。这意味着COS/SSCP具有比SSR更高的潜在吞吐量。这项工作将提供940个COS/SSCP标记，并将为所有在水稻基因组中具有等同物的小麦EST设计引物。广泛使用的现有小麦遗传图谱将增加100个COS/SSCP标记，这意味着所有广泛使用的SSR标记将相对于新标记进行定位。SSCP也是SNP发现的途径。将对100个映射的SNP变体进行测序。所有数据将立即公开。这个标记集和图谱的存在将意味着任何研究，从英国的小麦工作到发展中国家的小米，都可以在没有开发成本的情况下部署基于基因的标记。此外，他们制作的地图可以受益于测序基因组的巨大预测价值，特别是水稻和短柄草。因此，感兴趣的区域可以用新的标记饱和，并且可以将水稻中精细定位或克隆的基因鉴定为COS定位物种中感兴趣性状的候选者。

To fully understand and manipulate the genetic mechanisms controlling important aspects of crop growth such as yield, disease resistance, and adaptation to climate change we need to identify the genes that control these traits. This requires molecular markers, which are used to tag the chromosomal regions containing these genes. Over the last ten years the markers of choice have been the simple sequence repeat (SSR) also known as microsatellites. SSRs are excellent genetic markers but need to be replaced for a number of reasons. They are not transferable between species, an SSR map for wheat is not comparable to the map of close relatives such as barley. They are expensive to develop, cannot be targeted to specific regions, and they cannot be used to map to a sufficient density for many purposes. The aim of this work is to kickstart the replacement of SSRs and to provide a set of molecular markers for species which, as yet, have none. The best available marker system for this is the Conserved Orthologous Sequence (COS) system. Like SSRs COS markers are PCR based but unlike SSRs the PCR primers are anchored in highly conserved exon sequence. Consequently the markers work (amplify) equally well across genera. In the pilot work for this study markers worked well in maize, rice, millet wheat, festuca, and Brachypodium. Although the primers are anchored in exons (the parts of the gene that are eventually translated into protein) they amplify DNA from introns (non protein coding). Introns have a relatively high occurrence of differences (Single Nucleotide Polymorphisms, SNPs) compared to exons and so it is the introns that allow variants (alleles) of the genes to be discriminated as molecular markers but it is the exons which give the system such stability. The method of SNP discrimination used in this study will be Single Strand Conformation Polymorphism (SSCP) in which SNPs cause DNA to fold differently and migrate at a different rate during capillary electrophoresis. The method has been shown to be highly reproducible for a large set of markers under the same set of conditions, so very little optimisation is required. Many markers can be run on each capillary simultaneously (multiplexed). This means that COS/SSCPs have a higher potential throughput than SSRs. This work will deliver 940 COS/SSCP markers and primers will be designed for all wheat ESTs that have equivalents in the rice genome. Widely used existing wheat genetic maps will have 100 COS/SSCP markers added to them, this means that all the widely used SSR markers will be mapped relative to the new markers. SSCP is also a route to SNP discovery. The 100 mapped SNP variants will be sequenced. All data will be made public immediately. The existence of this marker set and map will mean that any research, from UK wheat work to developing world millet, can deploy gene based markers with no development costs. Moreover, the maps they produce can benefit from the immense predictive value of the sequenced genomes, especially rice and Brachypodium. So regions of interest can be saturated with new markers and finely mapped or cloned genes in rice can be identified as candidates for the traits of interest in the COS mapped species.