Molecular Identification Of Cyst Forming Nematodes (Heterodera And Globodera)

The study describes the development of species-specific pairs of PCR primers for the cyst forming nematodes Heterodera glycines and Globodera rostochiensis that amplify species-specific RAPD fragments. Random primers OPA02, OPA03, OPA06, OPA09, OPA13, OPA18, OPB15, OPC06, OPD13, OPG06, OPG08, OPK16, acquiring the species-specific fragment that OPA06 amplified. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-charaterized amplified regions (SCARs). Using the developed pairs of SCAR primers. SCAR fragments of H.glycines and G. rostochiensis were easily amplified from DNA extractes from single cyst or juveniles of the particular nematode species investigated. The SCAR-PCR-based assays desribed have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.The amplification of the rDNA-ITS region of each population of H. avenae (CCN)from Zhengzhou, Henan province,China respectively, yielded one fragment of approximately 1060bp. No PCR products were obtained in the control lacking the DNA template. A total 18 scored fragments were obtained with 8 enzymes used for analysis . AluI did digest CCN ITS products of the nine Henan ,China populations and yieled two fragments, Rsal digested the PCR products of all populations and yielded two fragments. CofI\ MvaI, HaeIII, HinfI, AluI and RsaI produced restriction profiles identical for the all CCN populations. HindIII and AvaI were the two enzymes that did not restrict any the ITS products of cereal cyst nematode.