Bruce Myron, Hess Ann, Bai Jianfa, Mauleon Ramil, Diaz M Genaleen, Sugiyama Nobuko, Bordeos Alicia, Wang Guo-Liang, Leung Hei, Leach Jan E
Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA.
BMC Genomics. 2009 Mar 25;10:129. doi: 10.1186/1471-2164-10-129.
The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions.
We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to approximately 500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/.
We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue to grow with further hybridizations, allowing researchers to quickly identify mutants that harbor deletions in candidate genomic regions, for example, regions containing QTL of interest.
利用物理或化学试剂诱导基因组缺失是一种简单且经济的方法，可用于构建覆盖整个基因组的突变体库。可以选择不同的诱变剂，以确保获得具有一系列缺失大小的突变体库。这将有助于鉴定单个基因中的突变，或者鉴定可能共同控制某一性状的一组缺失基因（例如，数量性状位点，QTL）。然而，缺失突变体在功能基因组学中尚未得到广泛应用，因为突变基因没有被标记，因此难以鉴定。在此，我们提出一种基于微阵列的方法，用于鉴定从由伽马射线或快中子处理产生的大量水稻突变体中筛选出的突变体中缺失的基因组区域。我们的研究不仅关注该方法在正向遗传学中的应用，还关注其作为反向遗传学工具的潜力，即通过积累含有多个遗传损伤的缺失突变体库的杂交数据来实现。
我们证明，将标记的基因组DNA直接与Affymetrix水稻基因芯片杂交，可以快速定位水稻突变体中的缺失区域。缺失大小从一个基因模型到约500 kb不等，在水稻的所有12条染色体上均有预测。该技术作为正向遗传学工具的实用性，通过与一系列等位基因突变体结合得以证明，可快速缩小基因组区域，最终鉴定出导致类病斑表型的候选基因。最后，在一个用户友好的基因组浏览器中将14个突变体中的突变位置定位到水稻假分子上，以便快速鉴定未标记的突变（http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/）。
我们证明了寡核苷酸阵列在发现水稻中缺失基因方面的实用性。缺失的密度和分布表明，建立一个覆盖水稻全基因组缺失的数据库是可行的。随着进一步的杂交，这个公共资源可以不断扩充，使研究人员能够快速鉴定在候选基因组区域（例如，含有感兴趣QTL的区域）中存在缺失的突变体。
