Faster marker-assisted selection of pollination-constant non-astringent persimmon offspring by crude-sample PCR

Marker-assisted selection in persimmon is carried out at National Agriculture and Food Research Organization (NARO), Japan, using an astringency-linked marker, but DNA purification by the cetyltrimethylammonium bromide (CTAB) method is time-consuming. Rapid DNA extraction and either direct or crude-sample PCR have been established in other plant species, but not yet in persimmon. Here, we developed a protocol for crude-sample PCR by testing DNA polymerases in persimmon. Crude DNA was prepared by heating crushed leaves in a solution. Amplification efficiency varied widely depending on the polymerase; among the three DNA polymerases tested, only KOD FX Neo produced strong clear fragments. With KOD FX Neo, 95% of samples showed consistent genotypes between crude and purified DNA when we considered only the presence/absence of astringency-linked fragments. We confirmed the utility of the method to select non-astringent offspring in six crossbred populations. Crude DNA was prepared from 96 samples within 5 h, while the conventional CTAB method takes 25 h. Our method will help breeders to efficiently select non-astringent offspring from many persimmon seedlings.