Jordan Journal of Agricultural Sciences, Volume 6, No.3, 2010

DNA Extraction and PCR-Based Diagnosis of the Root-Knot Nematodes

(Meloidogyne Species and Races) of Jordan

Muwaffaq R. Karajeh1 ,Walid I. Abu-Gharbieh2 and Sameer A. Masoud3

1) Department of Plant Protection and IPM, Faculty of Agriculture, Mu'tah University, Karak 7, Jordan.

2) Plant Protection Department, Faculty of Agriculture, University of Jordan, Amman 11942, Jordan.

3) Department of Biotechnology and Genetic Engineering, Faculty of Science, Philadelphia University, Amman 19392

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ABSTRACT

Several methods of genomic DNA extraction from different developmental stages of Jordanian populations of root-knot nematodes species viz., Meloidogyne javanica, M. incognita (races 1 and 2) and M. arenaria (race 2), were evaluated. Two assays of DNA fingerprinting viz., Sequence Characterized Amplified Regions (SCAR) and Random Amplified Polymorphic DNA (RAPD) based Polymerase Chain Reaction (PCR) were used. Among the tested DNA extraction methods, a minipreparation method was the most efficient, cost and time effective for SCAR-PCR. Methods used for DNA extraction from single juveniles or females were more suitable for RAPDPCR. Typical DNA products of 670, 420, or 1200 bp in size were specifically amplified by SCAR-PCR when DNA extracts of M. javanica, M. arenaria (race 2), or M. incognita (races 1 or 2), respectively, were used. Accordingly, Meloidogyne species in Jordan could be most reliably identified by using SCAR-PCR assay. Using RAPD-PCR primer PA-01 had produced RAPD DNA patterns with clear bands that clearly distinguished one species from the others and so allowed the identification of the three Meloidogyne species. Molecular biology techniques for the identification of Meloidogyne spp. could be particularly useful in cases of mixed populations of the three species and as a reliable quarantine tests.