YIA-PGR: Assessment of the use of Oligonucleotide Microarrays for Single Nucleotide Polymorphism Mutation Detection in Maize

大规模的植物基因组测序项目已经产生了丰富的序列信息，可以用来形成关于基因功能的假说。然而，由于缺乏反向遗传学资源，在许多植物物种中很难检验这些假说。该项目将开发乙烷甲基磺酸(EMS)诱变的玉米植株群体，并使用两种SNP突变检测方法对其进行筛选：靶向诱导局部损伤IN基因组(TILLING)，其利用错配特定的内切酶消化来检测SNPs；以及原型寡核苷酸微阵列系统，其使用基于杂交的方法。EMS是一种化学诱变剂，主要导致C-T到A-G的转变突变。生物信息学分析可以用来预测这些转变是否会导致无意义、错误或沉默的突变。可以设计寡核苷酸微阵列来通过掺入与野生型亲本序列相同的寡核苷酸和与过渡突变相同的寡核苷酸来筛选信息性突变。来自突变个体的DNA将被标记并与寡核苷酸微阵列杂交，以识别包含特定突变的个体。这种筛查方法的优点是提高了效率(以更低的成本获得了更多的基因)，在检测到突变时可以准确地识别突变，并使用了具有特征性的遗传背景。该项目中采用的方法是通过使用无掩膜阵列合成器(MAS)设备实现的，该设备允许快速构建可轻松定制的廉价寡核苷酸微阵列。如果使用8个寡核苷酸来筛选每个SNP，则单个微阵列可以用于筛选超过100,000个潜在的突变。该项目的成果将构成公开可用的玉米反向遗传资源的基础，该资源将包括突变体、筛选方案和生物信息学工具。这些资源对于有兴趣为其他植物物种开发反向遗传学资源的研究人员以及对植物和动物基因组DNA的其他SNP检测应用感兴趣的研究人员将是有价值的。

Large-scale genome sequencing projects in plants have yielded a wealth of sequence information that can be used to form hypotheses regarding gene function. However, in many plant species it is difficult to test these hypotheses due to a lack of reverse-genetics resources. This project will develop an Ethane methylsulfonate (EMS)-mutagenized population of maize plants and screen it using two approaches to SNP mutation detection: Targeted Induced Local Lesions IN Genomes (TILLING), which utilizes mismatch-specific endonuclease digestion to detect SNPs, and a prototype oligonucleotide microarray system, which uses a hybridization-based approach. EMS is a chemical mutagen that causes primarily C-T to A-G transition mutations. Bioinformatic analysis can be used to predict whether these transitions result in nonsense, missense, or silent mutations. An oligonucleotide microarray can be designed to screen for the informative mutations by incorporating oligonucleotides identical to the wild-type parental sequence and oligonucleotides identical to the transition mutation. DNA from mutagenized individuals will be labeled and hybridized to the oligonucleotide microarrays to identify individuals containing a specific mutation. The advantages of this screening method are increased efficiency (more genes at lower cost), precise identification of the mutation when it is detected, and the characterized genetic background being used. The approach employed in this project is made possible by the use of a maskless array synthesizer (MAS) device, which allows rapid construction of inexpensive oligonucleotide microarrays that can easily be customized. A single microarray can be used to screen for over 100,000 potential mutations if eight oligonucleotides are used to screen for each SNP. The outcomes of this project will form the foundation of a publicly available reverse-genetic resource for maize that will include mutants, screening protocols, and bioinformatic tools. These resources will be valuable to researchers interested in developing reverse-genetics resources for other plant species as well as researchers interested in other SNP detection applications for plant and animal genomic DNA.