Gene discovery using mutagen-induced polymorphisms and deep sequencing: application to plant disease resistance

• Published in: Genetics (Austin) Vol. 192; no. 1; pp. 139 - 146
• Main Authors: Zhu, Ying, Mang, Hyung-gon, Sun, Qi, Qian, Jun, Hipps, Ashley, Hua, Jian
• Format: Journal Article
• Language: English
• Published: United States Genetics Society of America 01-09-2012
• Subjects: Active Transport, Cell Nucleus - drug effects; Arabidopsis - cytology; Arabidopsis - drug effects; Arabidopsis - genetics; Arabidopsis - immunology; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Deoxyribonucleic acid; Disease Resistance - drug effects; Disease Resistance - genetics; DNA; Genes, Plant - genetics; Genetic Loci - drug effects; Genetic Loci - genetics; Genomics; High-Throughput Nucleotide Sequencing; Hybridization, Genetic; Investigations; Mutagenesis; Mutagenesis - drug effects; Mutagens - pharmacology; Plant Diseases - immunology; Plant Proteins - genetics; Plant Proteins - metabolism; Polymorphism; Polymorphism, Genetic - drug effects; Polymorphism, Genetic - immunology; Sequence Analysis, DNA; Temperature
• ISSN: 1943-2631; 0016-6731; 1943-2631
• DOI: 10.1534/genetics.112.141986

Next-generation sequencing technologies are accelerating gene discovery by combining multiple steps of mapping and cloning used in the traditional map-based approach into one step using DNA sequence polymorphisms existing between two different accessions/strains/backgrounds of the same species. The existing next-generation sequencing method, like the traditional one, requires the use of a segregating population from a cross of a mutant organism in one accession with a wild-type (WT) organism in a different accession. It therefore could potentially be limited by modification of mutant phenotypes in different accessions and/or by the lengthy process required to construct a particular mapping parent in a second accession. Here we present mapping and cloning of an enhancer mutation with next-generation sequencing on bulked segregants in the same accession using sequence polymorphisms induced by a chemical mutagen. This method complements the conventional cloning approach and makes forward genetics more feasible and powerful in molecularly dissecting biological processes in any organisms. The pipeline developed in this study can be used to clone causal genes in background of single mutants or higher order of mutants and in species with or without sequence information on multiple accessions.