Institute of Experimental Botany (IEB), Czech Acad Sci, Centre of the Region Haná for Biotechnological and Agricultural Research (CRH), Olomouc, Czech Republic.
Methods Mol Biol. 2025;2873:53-70. doi: 10.1007/978-1-0716-4228-3_4.
Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.
正向遗传学筛选在鉴定特定生物过程中涉及的新基因方面非常强大，包括各种染色质调控途径。然而，传统的遗传作图方法既费时又费钱。最近，随着测序作图（MBS）方案的发展，整个过程发生了革命性的变化。在 MBS 中，通过对基于突变体表型从分离群体中选择的大量突变体植物进行全基因组测序和生物信息学分析，可以直接鉴定因果突变及其在基因内的位置。MBS 提高了作图的精度和经济性。在这里，我们以拟南芥正向遗传学筛选为例，描述了一个通用的方案，并提供了关于如何进行测序作图的实用技巧，该筛选旨在鉴定对特定类型 DNA 损伤敏感的突变体。所描述的方案通常适用于具有参考基因组序列的各种自交物种中的广泛的正向遗传学筛选。
