Development of an HRM-based, safe and high-throughput genotyping system for two low phytic acid mutations in soybean

Two low phytic acid ( lpa ) mutants, Gm - lpa -ZC-2 (ZC- lpa ) and Gm - lpa -TW-1 (TW- lpa ), resulting from a G → A mutation in GmIPK1 and a 2-bp deletion in GmMIPS1 , respectively, were previously developed to increase the nutritional value and environmental friendliness of soybean meal. Two functional CAPS markers were subsequently developed for genotyping plants carrying the two mutant genes; however, both are costly and time consuming and hence unsuitable for large-scale breeding use. In the present work, by integrating a quick DNA extraction protocol with an optimized high-resolution melting curve (HRM) analysis, we developed a fast and high-throughput genotyping system for the two mutations. In this system, (1) DNAs are extracted within half an hour using a protocol that only requires freezing and heating of leaf disks in two non-toxic solutions and can be directly used for PCR; (2) for genotyping, asymmetric PCRs with competitive primers are performed, and the samples are then discriminated and grouped through HRM analysis; and (3) all steps are performed in a 96-well plate, and hence adaptable to high-throughput genotyping. Although the system was developed for two lpa mutations, the general principle should be applicable to any other genes in soybean.