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、抗病性和对气候变化的适应，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。SSRs是优秀的遗传标记，但由于许多原因需要被取代。它们不能在物种间转移，小麦的SSR图谱不能与大麦等近亲的SSR图谱相比较。它们的开发成本很高，不能针对特定的区域，也不能用于绘制许多用途所需的足够密度的地图。这项工作的目的是启动ssr的替代，并为尚未有ssr的物种提供一套分子标记。最好的标记系统是保守同源序列（COS）系统。与SSRs一样，COS标记基于PCR，但与SSRs不同的是，PCR引物锚定在高度保守的外显子序列上。因此，这些标记在不同的属中同样有效（放大）。在本研究的试点工作中，标记在玉米、水稻、小米、小麦、羊茅和短柄茅中表现良好。虽然引物被固定在外显子（基因最终被翻译成蛋白质的部分），但它们从内含子（非蛋白质编码）中扩增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，SNPs），因此内含子允许基因的变体（等位基因）被区分为分子标记，但它是外显子给予系统这样的稳定性。本研究中使用的SNP鉴别方法将是单链构象多态性（Single Strand构象Polymorphism， SSCP），在毛细管电泳过程中，SNP导致DNA折叠不同，并以不同的速率迁移。该方法已被证明在相同条件下对大量标记具有高度可重复性，因此需要很少的优化。许多标记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，并为所有在水稻基因组中具有等价物的小麦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也是发现SNP的途径之一。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，从英国的小麦研究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，他们绘制的图谱可以受益于测序基因组的巨大预测价值，尤其是水稻和短柄茅。因此，感兴趣的区域可以被新的标记所饱和，精细定位或克隆的水稻基因可以被识别为COS定位物种感兴趣的性状的候选基因。

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.