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的重要方面的遗传机制，如产量、抗病和适应气候变化，我们需要确定控制这些性状的基因。这需要分子标记，用来标记包含这些基因的染色体区域。在过去的十年里，选择的标记是简单序列重复(SSR)，也被称为微卫星。SSR是很好的遗传标记，但由于多种原因需要被取代。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较。它们的开发成本很高，不能针对特定地区，也不能用来为许多目的绘制足够的密度地图。这项工作的目的是启动SSR的更新换代，并为目前还没有SSR的物种提供一套分子标记。最好的标记系统是保守的同源序列(COS)系统。与SSR一样，COS标记是基于聚合酶链式反应的，但与SSR不同的是，聚合酶链式反应的引物锚定在高度保守的外显子序列中。因此，这些标记在属间的作用(扩增)同样好。在这项研究的试点工作中，标记在玉米、水稻、谷子小麦、高羊茅和短梗上效果很好。虽然这些引物位于外显子(基因中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(单核苷酸多态，SNPs)，因此是内含子允许将基因的变异(等位基因)区分为分子标记，但正是外显子使系统如此稳定。本研究中使用的SNP识别方法将是单链构象多态(SSCP)，在毛细管电泳法中，SNP导致DNA以不同的方式折叠并以不同的速度迁移。该方法已被证明在相同的条件下对大的标记集具有高度的重复性，因此几乎不需要优化。多个标记可以同时在每个毛细管上运行(多路传输)。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标记都将相对于新的标记进行定位。SSCP也是SNP发现的途径。将对100个映射的SNP变体进行测序。所有数据将立即公之于众。这一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谷子，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，他们制作的图谱可以受益于测序基因组的巨大预测价值，特别是水稻和短柄蕨属。因此，感兴趣的区域可以被新的标记饱和，在水稻中精细定位或克隆的基因可以被确定为COS定位物种中感兴趣的特征的候选。

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.