TABLE CONTENTS

A Real-time PCR Assay for Detection and Quantification of the Burrowing
Nematode, Radopholus similis
Athman, S. & P. Agudelo
Department of Entomology, Soils, and Plant Sciences, Clemson University, 210 Long Hall, Clemson, South
Carolina 29634, USA.
A real-time PCR method using SYBR Green was developed for the detection and relative
quantification of the burrowing nematode, Radopholus similis, a major pest in bananagrowing
regions worldwide. Primers were designed to amplify a portion of the internal
transcribed spacer (ITS2) region of the rDNA and used for both conventional and real-time
PCR. A standard curve was generated using three replicates of independent DNA extractions
from 2000 mixed stages of R. similis and ten-fold serial dilutions from 1 to 0.0001. Precise
numbers of nematodes were used to determine the sensitivity and accuracy of the assay. This
real time PCR SYBR green method can be used to accurately detect and quantify 1 to 2,000
individuals of R. similis extracted from root or soil samples. The assay described is highly
specific and has several advantages over conventional PCR.
Genetic Diversity in Portuguese Potato Cyst Nematode Isolates assessed by
AFLP and Massive Parallel Sequencing
Feio, G. (1), C. Egas (2,3), B. Santos (2), J.L. Oliveira (2), I.L. Conceição (1), M.J.M. Cunha
(1,4), I. Abrantes (1) & M.S. Santos (1)
(1) IMAR – CIC and Departamento de Zoologia, Faculdade de Ciências e Tecnologia, Universidade de
Coimbra, 3004-517 Coimbra, Portugal; (2) Biocant, Centro de Inovação em Biotecnologia, BiocantPark, Núcleo
04, Lote 03, 3060-197 Cantanhede, Portugal; (3) Centro de Neurociências e Biologia Celular and Departamento
de Zoologia, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal; (4)
Escola Superior Agrária de Coimbra, Bencanta, 3040-316 Coimbra, Portugal
Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are widely distributed
among potato production areas in Portugal, where infestations are often associated with
significant economic losses. As both species and populations within species have different
spectra of virulence towards host resistance genes, it is important to know the genetic
diversity that is present in the field populations. Among molecular methods, AFLP, a DNA
fingerprinting technique based on selective PCR amplification of restriction fragments from a
total digest of genomic DNA, has been widely used to provide molecular markers for
organisms that still lack genome sequence information. The discrimination power of AFLP
technology, in combination with massive parallel sequencing, was used to obtain sequence
profiles for PCN isolates in order to find molecular markers of known sequence that might
later be used with simpler methodology to assess the virulence of the PCN field populations.
Genomic DNA was extracted from cysts of eight isolates of G. rostochiensis and two of
G. pallida, adaptor-ligated, and pre-amplified. Selective amplification was performed with
primer set E63-M33 and subjected to 454 sequencing, following the standard protocol.
Overall sequencing yielded 58,180 reads, which clustered into a minimum of 63 and a
maximum of 408 sequence markers. Comparative analysis of the sequences is under way to
determine differences among isolates.
5 th International Congress of Nematology, 2008 329