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.

VII International Symposium on Persimmon

N. Onoue, A. Kono, A. Sato, R. Matsuzaki, A. Azuma, T. Shimizu, T. Saito

CTAB, DNA extraction, DNA marker, DNA polymerase, fruit tree, kaki, MAS

https://doi.org/10.17660/ActaHortic.2022.1338.9