«Master thesis: Development of a species-specific PCR to detect the Cereal Cyst Nematode Heterodera latipons By Fateh Farah Toumi Faculty of Science ...»
Development of a species-specific PCR to detect the Cereal Cyst Nematode
Fateh Farah Toumi
Faculty of Science
EUMAINE – European Master in Nematology
Prof. Dr. ir. Maurice Moens
Dr. ir. Nicole Viaene
Ghent, June, 2010
Development of a species-specific PCR to detect the Cereal Cyst Nematode
Wheat and barley are the most important crops within the cereals (Nicol et al., 2002). In general, cereal crops are exposed to biotic and abiotic stresses. Among the biotic stress, plantparasitic nematodes have an important role in decreasing the yield (Brown et al., 1985; Nicol et al., 2002). The cereal cyst nematodes (CCN) are widespread and one of the most important group of nematodes in the world (Rivoal and Cook, 1993). The genus Heterodera includes 62 species (Wouts & Baldwin, 1998). Twelve species affect roots of cereals and grasses (Yan & Smiley, 2009). Three of them (H. avenae, H. filipjevi and H. latipons) are among the economically most important cyst nematodes (Rivoal & Cook. 1993; McDonald & Nicol. 2005;
Yan & Smiley, 2009). The identification of Heterodera species using morphological and morphometrical characteristics is time consuming and requires great skill and training by the observer. However, there are many other characteristics allowing to discriminate between different species of nematodes e.g. biotechnological tools (Romero et al., 1996; Rumpenhorst et al., 1996; Rivoal et al., 2003; Subbotin et al., 2003). As a consequence, the development and use of new tools to identify nematodes using molecular technologies increases exponentially (Rivoal et al., 2003). The analysis of coding and non-coding regions of ribosomal DNA (rDNA) became a favorite way for nematode identification (Vrain et al., 1992; Wendt et al., 1993; Zijlstra et al., 1995). The internal transcribed spacer region (ITS) is variable and therefore useful for nematode identification and phylogenetic studies at species level. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based on ITS-regions of the rDNA repeat units has provided a reliable tool for quick and precise identification of cyst nematode species and subspecies (Bekal et al., 1997; Subbotin et al., 1999; Subbotin et al., 2000; Rivoal et al., 2003;
Madani et al., 2004; Abidou et al., 2005; Smiley et al., 2008). This was a start of many studies leading to an explosion of RFLP-patterns and sequences. Comparisons of sequences of the ITSrDNA of unknown nematodes with those published or deposited in GenBank (Ferris et al., 1994; Orui, 1997; Szalanski et al., 1997; Subbotin et al., 1999, 2000, 2001; Sabo et al., 2001;
Tanha Maaﬁ et al., 2003) facilitated the quick identiﬁcation of most species of cyst nematodes.
In a later stadium, the sequences were used to develop species-specific primers (Amiri et al., 2001). By a simple PCR-reaction, it was then possible to detect the nematode species for which the primers were designed.
Although the ITS-regions are very useful for species identification, several species cannot be separated from each other by ITS-RFLP, e.g. H. avenae (type A) and H. arenaria, H. ciceri and H. trifolii and also H. carotae and H. cruciferae due to the same restriction patterns obtained in each pair of these species (Subbotin et al., 2000). Sequences of these and other Heterodera species, like H. trifolii, H. schachtii and H. betae, were compared and found to be almost identical. Moreover, polymorphism occurs between rDNA repeats within one species resulting in different RFLP-patterns which can overlap with the RFLP-pattern for another species (Wouts et al., 2001). I therefore concluded that in some cases it is impossible to design reliable RFLPpatterns or species-specific primers using the ITS-region. Therefore I decided to investigate other DNA-regions to determine their usefulness for the development of additional identification methods. The objectives of this study were (i) to screen different DNA-regions (PCR) not belonging to rDNA and mtDNA for assessing their usefulness to develop species-specific primers for H. latipons, (ii) to optimize the species-specific PCR.
Materials & Methods Nematode samples. A survey was conducted in Syria and Turkey in July 2008-2009 after the harvesting time of cereals. Cysts were extracted from soil by Seinhorst method (Seinhorst, 1964). Extracted cysts, which were retained on the 250 µm sieve, were hand-picked with a needle under a dissecting stereomicroscope. More Heterodera cysts (different species) were obtained from colleagues in different countries (Table 1). All cysts were stored at 4°C for molecular and morphological identification.
Morphological identification. For each population, vulval cones of several mature cysts were mounted in glycerin jelly. Underbridge structure, shape of semifenestra in the fenestral area and development of bullae were observed under a microscope and depending on all these structures the identifications was done (Shurtleff & Averre. 2000; Handoo. 2002).
DNA extraction. A selection of Heterodera populations comprising 9 different species and 24 isolates was selected for DNA extraction (Table 1). For each population, one cyst was transferred into 45 µl of double distilled water (ddH2O) in an Eppendorf tube and crashed using a microhomogeniser (Vibro Mixer). After centrifugation of the squashed cyst content, 40 µl of the mix were transferred to a PCR tube (0.2 ml). 50 µl of worm lysis buffer (WLB) and 10 µl of Proteinase K (20mg/ml) were added and the tubes were frozen at -80 ºC for at least 10 min and then incubated at 65 ºC for 1 hour and 95 ºC for 10 minutes consecutively in a thermocycler.
After incubation, the tubes were centrifuged for 1 min at 14 000 rpm and kept at -20 ºC until use (Maafi et al., 2002; Waeyenberge et al., 2009).
Genome Amplification (Gphi). Due to the small amount of the extracted DNA (cysts with only a few juveniles), genome amplification was done. One μl of the DNA extract was used for genome amplification, followed by a purification step using the alcohol precipitation method as described in the manufacturer’s instructions (Illustra™ GenomiPhi V2 DNA Amplification Kit, GE Healthcare, Chalfont St Giles, UK; Skantar & Carta, 2005). The DNA concentration was measured using a UV spectrophotometer (Nanodrop ND1000, Isogen Life Sciences, Sint-PietersLeeuw, Belgium) and 1 ng DNA was used for PCR. The remainder of the crude and amplified DNA extract was stored at − 20◦C for future use.
PCR amplification. For molecular identification, the ITS-rDNA region was amplified. One nanogram of DNA was added to the PCR reaction mixture containing 23 µl ddH2O, 25µl 2X DreamTaq PCR Master Mix (Fermentas Life Sciences, Germany) and 0.5 µl of forward primer (5-CGTAACAAGGTAGCTGTAG-3) and reverse primer (5-TCCTCCGCTAAATGATATG-3) (Ferris et al., 1993). The DNA thermal cycler program consisted of 5 min at 95 °C; 40 cycles of 94 °C for 30 s, 45 °C for 45 s and 72 °C for 45 s; followed by a final elongation step of 8 min at 72 °C. To be able to design species-specific primers, other DNA-regions were amplified as well.
Amplification for those regions was done with suitable primers in the same PCR reaction mixture and program with appropriate and adaptation of the annealing temperature (Table2).
After PCR amplification, 5 µl of each PCR product was mixed with 1 µl of 6× loading buffer (Fermentas Life Sciences, Germany) and loaded on a 1.5% standard TAE buffered agarose gel.
After electrophoresis (100 V for 40 min) the gel was stained with ethidium bromide (0.1 µg/ml) for 15 min, visualized and photographed under UV-light. The remaining PCR product was stored at –20 °C.
Sequencing. The remainder (two times 45 µl) of the PCR product was loaded on a 1% agarose gel for electrophoresis (100 V, 40 min). The purification process was done as described in the manufacturer’s instructions (Wizard® SV Gel and PCR Clean-Up System Kit, Promega).
DNA from each sample was sequenced (Macrogen, Seoul, South Korea) in both directions to obtain overlapping sequences of both DNA strand. The sequences were edited and analysed using software packages Chromas 2.00 (Technelysium, Helensvale, QLD, Australia) and BioEdit 188.8.131.52 (Hall, 1999). Finally all sequences were blasted in GenBank (Sequin v. 9.00, http://www.ncbi.nlm.nih.gov/) to reveal its origin (Heterodera species and DNA-region).
Species-specific primer design. An alignment of all the obtained sequences of the actin gene together with sequences present in Genbank was made using Clustal X 1.64 (Thompson et al., 1997). This alignment was then used to determine putative species-specific DNA fragments that could be used as species-specific primers for the identification of H. latipons. Two speciesspecific primers, one forward and one reverse (sp-spec ActF & sp-specActR) were designed.
Next to these, another reverse primer for the actin gene was developed as well (Act R ILVO).
During the selection of the fragments, certain parameters (internal complementarity, intercomplementarity, melting temperature and length) were taken into account by using the software program DNA calculator (www.sigma-genosys.com/calc/DNAcalc.asp, Sigma-Aldrich, St Louis, MO, USA). The selected potential species-specific primers were further screened by looking for their presence in sequences stored in GenBank (BLAST option, http://www.ncbi.nlm.nih.gov/).
Table 1. Heterodera populations and species used in study other DNA regions
Optimaization and specificity of species-specific PCR. To determine the optimum annealing temperature (Ta), a gradient PCR was performed in one DNA sample of H. latipons (7A-1) with three combinations of primers (A, B and C in Table 3). The temperature ranged from 60 to 70 °C (60-60.3-60.8-61.2-62.9-64.3-66.0-67.5-68.5-69.3-69.8-70 °C). A certain annealing temperature was selected and two additional primer combinations (D and E in Table 3) were used in a duplex PCR to provide for an internal PCR-control. I selected the best primercombination (combination E) and further optimized the duplex PCR with different primers and dNTP concentrations (Table 4). Finally, the specificity of the duplex-PCR was tested for all samples used in this study.
Table 3. Overview of the primer combinations tested for the development of a H.
latipons species-specific duplex PCR.
(*) De Ley et al., (1999).
Results Identification. The survey revealed three species of CCN in infested fields (H. latipons, H.
avenae and H. filipjevi). Heterodera latipons was the most dominant species in Syria and H.
filipjevi in Turkey.
Amplification of the rDNA-ITS regions was successful for all selected samples (fig1). PCR yielded an expected fragment of 1100 bp (Fig1). Sequencing these fragments confirmed the identity of the previously morphologically identified samples (Table 1). Unfortunately, double sequencing-signals in H. latipons sometimes made the analysis difficult but not impossible. No PCR products were obtained in the negative control without nematode DNA template.
Fig. 1. PCR results for all Heterodera samples of the ITS regions (Ferris et al., 1993). 1:Fa1, 2:
Fa3, 3: 4, 5: 9, 6: 15C, 7: 8K, 8: 7A-1, 9: 7A-2, 10: 7A-3, 11: 23.2, 12: 23.4, 13: 7A-4, 14: 7A-5, 15: 7B-1, 16: 7B-2, 17: 42.2, 18: 42.3, 19: negative control. (For code interpretation, see Table 1) L: Fast Ruler DNA Ladder, Low range (Fermentas Life Sciences).
Amplification of other DNA-regions. PCR products were also obtained from Actin, Hsp90 and Tublin genes. No gene-specific bands or no bands (PCR products) were obtained from the remaining genes (Pectate Lyase, Annexin, Chorismate mutase and Aldolase). PCR with the actin gene primers revealed the expected 376 bp fragment (Tytgat et al., 2004; Kovaleva et al., 2005) (Fig. 2). After blasting, the sequences confirmed the actin gene was amplified for all used samples. Based on the alignment (Fig. 3), we were able to construct at least two species-specific primers and additionally a universal actin primer.
Fig. 2. PCR results of actin gene from all samples. 1:Fa1, 2: Fa3, 3: FaC3, 4: 42.2, 5: A36, 6:
E69, 7: DCP1248, 8: H. sch Poland, 9: H.sch NDL, 10: H. gly Riggs, 11: 23.2, N: negative control. (For code interpretation, see Table 1) L: 100 bp DNA ladder (Fermentas Life Sciences).
In some cases PCRs with Tubulin (Sabo and Ferris, 2004) and Hsp90 (Skantar & Carta.
2004) gene primers revealed additional bands next to the expected fragment of 251-382 and 900bp, respectively. After blasting, the sequences of the expected fragments confirmed Tubulin and Hsp90 genes were amplified. After blasting sequences of few of the additional bands, it became clear that Tubulin and Hsp90 genes from various origins were amplified as well (data not shown).
Fig. 3. Alignment of the actin sequences obtained with addition of Heterodera sequences from Genbank. The nucleotides matched to the Fa3 Heterodera latipons sample are represented by dots (.) Species-specific PCR. A gradient PCR was done to set the optimal annealing temperature (Fig. 4). The expected PCR bands were bright over a wide range of temperatures. I decided to keep the annealing temperature at 66 °C.
Fig. 4. Results of the Gradient PCR using Heterodera latipons Fa3. Temperature ranging from 60 to 70°C. A: ActF and sp-spec ActR; B: sp-spec ActF and ActR (ILVO); C: ActF and ActR (ILVO); 100 bp DNA ladder (Fermentas Life Sciences).
Different primer combinations in the duplex-PCR (Table 4) gave various results (Fig. 5).
PCR with combinations A and B showed a clear species-specific band for H. latipons. PCR with primer combination C produced an actin-specific, very bright band. Combination D produced an actin-specific band (400 bp) quite close to the species-specific band (300 bp). For this reason, primer combination E, consisting of ActF and sp-spec ActR with control primers D2A and D3B was retained for further investigations. In this case both PCR-products were easier to separate from each other after electrophoresis due to the larger difference in the length of the PCRproducts.