High-throughput RFLP genotyping method for large genomes based on a chemiluminescent detection system

摘要

Genetic linkage mapping based on RFLPs is a valuable genomics tool for studying organisms with no genome sequence information. However, the generally used Southern hybridization method based on the radioisotope32P is not ideal for genotyping large mapping populations. We have overcome limitations of the alternative chemiluminescent detection system and developed a high-throughput RFLP genotyping method suitable for large-scale mapping studies of large genomes. Important elements in our process are PCR labeling of probes, complete removal of post-PCR unincorporated nucleotides via column-based purification methods, use of a 1:4 DIG-[11]-dUTP:dTTP ratio, and using a rocker instead of an orbital shaker during hybridization and post-hybridization processing of membranes. Using this method, we mapped the large genome of the homosporous fern speciesCeratopteris richardii by genotyping a mapping population of 513 doubled haploid line (DHL) progeny of a cross between two completely homozygous parental lines. Our genotyping method can robustly detect sub-picogram quantities of DNA fragments from a large number of samples and can be applied to linkage mapping studies of other organisms with large genomes.