Optimization of DNA extraction methods for genomic analysis of rice root-knot nematode (Meloidogyne graminicola) using PCR (polymerase chain reaction) and sanger sequencing

Abstract

The root-knot nematode Meloidogyne graminicola is an economically important pest in rice production. The identification of a nematode species is an important basis in nematode management to reduce yield losses by extracting nematode DNA as an early step in molecular identification. This study aimed to investigate the optimal extraction method and number of M. graminicola for nematode genomic analysis based on PCR (polymerase chain reaction) and Sanger sequencing. The DNA extraction methods used in this study were the CTAB, SDS, and commercial kit (GeneAidTM Tissue/Blood DNA Mini Kit). The results revealed that the three DNA extraction methods could be used to analyze the nematode genomics based on PCR and Sanger sequencing using one nematode, both in a second-stage juvenile and a female, equipped with the process of nematode destruction by freezing. This finding was shown by the amplification of all DNA templates with Mg-F3 and Mg-R2 primers through PCR with a size of 370 bp, while Sanger sequencing obtained 372 bp.