Optimization of TILLING system based on capillary electrophoresis for targeted selection of pepper gene mutants

Abstract

Mutation breeding is largely performed by forward and reverse genetics-based approaches. In the latter, Target Induced Local Lesions IN Genomes (TILLING) can be used to screen mutants harboring mutations in a target gene whose function can be predicted with available functional genomic information. In TILLING, it is necessary to use an analytical system for accurate and efficient screening of target gene mutants among several randomly mutagenized lines. Therefore, we optimized a capillary electrophoresis-based TILLING system that can quickly and easily analyze many samples and confirmed its performance by applying it to pepper mutation populations. First, optimal conditions concerning heteroduplex DNA formation, type and concentration of endonucleases, and concentration, pooling depth, and size of PCR amplicons were determined in an analytical system using a capillary electrophoresis apparatus without labeled primers. The results showed that CelII endonuclease (Surveyor (TM)) had higher sensitivity than CelI. While the recommended concentration in the manufacturer's manual was 1x, we obtained higher sensitivity when the amount of PCR product and concentration of the endonucleases were at 180 ng and 0.5x for CelII and 120 ng and 2x for CelI. Meanwhile, the maximum pooling depth for DNA from individuals in mutant populations inversely correlated to PCR amplicon size. The limitation of DNA pooling was 4x for a 1872-bp amplicon, while at least 16x pooling was possible for a 672-bp amplicon. Using this optimized system, TILLING was performed on 2615 individuals from the mutant populations developed in a Korean pepper landrace, "Yuwolcho," to screen lines bearing a mutation in CCS (capsanthin/capsorbin synthase). We were able to select a mutant line with a substituted base pair in the translational start codon of CCS. The capillary electrophoresis-based TILLING system optimized in this study is expected to be useful in practical applications for reverse genetics-based breeding and functional genomics studies.