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Title: MULTIPLE DISPLACEMENT AMPLIFICATION (MDA) OF TOTAL GENOMIC DNA FROM MELOIDOGYNE SPP. AND COMPARISON TO CRUDE DNA EXTRACTS IN PCR OF ITS1, 28S D2-D3 RDNA AND HSP90

Author
item Skantar, Andrea
item Carta, Lynn

Submitted to: Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2005
Publication Date: 4/10/2005
Citation: Skantar, A.M., Carta, L.K. 2005. Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90. Nematology 7(2): 285-293.

Interpretive Summary: Nematodes are microscopic worms that cause ten billion dollars of crop losses in the United States each year. Root-knot nematodes are very important root parasites that seriously damage many economically important plants worldwide. The absence of accurate diagnostic tests sensitive enough to distinguish similar nematodes from each other is a major problem that hinders efforts to control the spread of highly damaging or invasive nematode species. In the present study, ARS scientists from Beltsville, Maryland used a method called multiple displacement amplification (MDA) to generate large quantities of genomic DNA from one to nine nematodes, and then verified that the DNA could be used for nematode diagnostic polymerase chain reactions (PCR)tests. These steps made it possible for them to develop a new diagnostic test for root-knot nematodes, based upon the single copy a gene known as the heat shock protein 90 (Hsp90) gene. The results are significant because they provide a quick assay for scientists to accurately identify new and potentially invasive root-knot nematodes. These experiments also demonstrate the feasibility of the MDA method for preserving the genetic material from rare nematode specimens and provides a way for labs to share identical genetic material to be used as testing standards. This research will be used by scientists, action agencies, and extension agencies engaged in nematode research and control.

Technical Abstract: Molecular identification and phylogenetic analysis of nematodes are often performed with limited or rare specimens, which constrains the number of analyses possible with the available DNA. Multiple displacement amplification (MDA) was assessed for whole genome amplification of crude genomic DNA from several species of Meloidogyne. MDA produced microgram quantities of template that resulted in successful amplification of the ribosomal internal transcribed spacer (ITS1) and LSU D2-D3 expansion regions. MDA gave comparable PCR results as compared to template generated by the single nematode smash extraction method. MDA also provided ample template for the amplification of two regions within Hsp90, obviating the need for multiple rounds of PCR for this single copy gene. Hsp90 amplification products, ranging from 313 to 426 bp, were amplified from seven root-knot nematode species. Digestion of Hsp90 PCR products with either HindIII or EcoRI revealed an unexpected restriction pattern for Meloidogyne arenaria and two different patterns for M. incognita and M mayaguensis. These results suggest the presence of intraspecific variation in these species. MDA should be useful for archiving DNA from valuable nematode specimens and provide a way for labs to share identical genetic material to be used as testing standards.