Abstract
There is a dire need of alternative reliable and non-laborious methods with high resolution for identification of the root-knot nematodes at the species level where molecular techniques have shown the potentials. This study assessed four DNA extraction protocols, (<em>i.e</em>. TNES buffer method, Modified CTAB method, Phenol / Chloroform Method and 1% SDS method) to extract DNA from egg masses of root knot nematodes. Egg masses were collected from pure cultures of nematodes maintained in tomato cultivation under net house condition. After extraction of genomic DNA, Polymerase Chain Reaction was performed to determine the success of the method of DNA extraction. The 1% SDS method with incubation at -20 0C for 1hr and incubated at 65 0C for 1 hr and again incubated for 95 0C for 10 min, was a better method to result in intense PCR bands of the expected sizes (720 bp and 999 bp) when amplified by <em>Meloidogyne </em>genus and <em>Meloidogyne incognita </em>specific primers. This DNA extraction procedure could contribute as an effective method to molecular identification of species and other downstream applications of population studies of root knot nematodes.
Highlights
IntroductionAccording to Eisenback (1985) and Cliff et al, (1985), precise and reliable identification at species level is a difficult task for the genus Meloidogyne, even for well qualified taxonomists
Nematodes are ubiquitously present multicellular organisms which belong to a diversified taxonomic group of the phylum Nematoda
The OD values lower than 1.8 for 260/280 and 260/230 are due to contamination of the extracted DNA with proteins and polysaccharides (Lucena-Aguilar et al.,2016). Both quantitative and qualitative parameters were at an acceptable level in the DNA extracted by 1% SDS method (Table 2)
Summary
According to Eisenback (1985) and Cliff et al, (1985), precise and reliable identification at species level is a difficult task for the genus Meloidogyne, even for well qualified taxonomists. In this connection, as an alternative and a polyphasic approach, a wide range of molecular methods (i.e., DNA-based and protein-based) have been introduced (Bogale et al, 2020). As reviewed by Bogale et al, (2020), DNA analyses based on genomic fingerprints and nucleotide sequences are in heavy use for identification of species of these nematodes To this end, DNA of high quality, quantity and integrity is an essential prerequisite
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