High Resolution Melt (HRM) Genotyping for Detection of Induced Mutations in Coffee (Coffea arabica L. var. Catuaí)

Arabica coffee (C. arabica L.) is a highly valued agricultural commodity on the world market. Tons of products are traded internationally, and it has become an extremely valuable resource. However, the species is threatened by the alarmingly low genetic diversity present among its wild populations and agronomic varieties. It is highly relevant to exploit different mechanisms to increase genetic variability in coffee. One of such methods is the induction of variability through chemical or physical mutagenesis. In this work, a population of 320 coffee plants (Coffea arabica L. var. Catuaí) originated from chemically mutagenized embryogenic callus was analysed. Here we describe a protocol for detection of induced mutations using High Resolution Melting (HRM) on a Real Time PCR machine with HRM capabilities. The protocol allows to detect mutations in pooled DNA samples of up to four M2 mutant plants. The procedures and example data are presented for mutation detection in the CaWRKY1 gene. This procedure can be applied for mutation detection in other genes of interest to coffee breeders and scientists.