Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations

In total, 66 populations of cyst nematodes representing six species were obtained from different locations in Germany, France, and Norway (Table 1). The molecular identification at the level of species was conducted by PCR amplification and sequencing of the cytochrome c oxidase subunit I (COI). Thirteen of those 66 populations (i.e., Vechelde, Bodenstedt, Titz-Kalrath, Hottorf, Artenay, Ingeleben, Söllingen, Peine, Sonnenhof, Vanikum, Acholshausen, Großgoltern, Köchingen) were collected from soil samples by centrifugal floatation with a MgSO₄ solution of 1.28 g/cm³ (Müller, 1980). Nine out of those 13 populations were previously propagated for two generations on oilseed rape (Brassica napus ‘NK-fair’) under greenhouse conditions (20/16°C), starting from either single cysts or second stage juveniles (J2) according to the objective of this study as described below. The nematode materials of the further 53 populations were obtained from the German institutions of Julius Kühn Institut in Braunschweig, Münster and Elsdorf. Table 1

Sampling locations of 66 cyst nematodes populations

To identify and amplify gene fragments specific for H. schachtii, the vap1 sequence of the six different cyst nematode species had to be determined. To achieve this aim, 12 populations of H. schachtii originating from Vechelde, Bodenstedt, Titz-Kalrath, Asperden and 8 ones from Münster, 1 population of H. filipjevi, 1 population of H. avenae, 2 populations of H. betae, 12 populations of Globodera pallida, and 28 populations of G. rostochiensis were processed. From each population a sample of 1 to 10 cysts was used for DNA extraction. Cysts of each sample were squashed with a micro pestle in a 1.5 ml tube containing 20 µl of water.

To study the genetic variation within and among H. schachtii populations, three approaches were followed. For the first approach, single J2 were used that hatched from single cysts collected from six sites within the same field. The six soil samples were randomly taken from an infested sugar beet field near Hottorf, Germany. For each sample, one core was taken containing 600 g of soil. Cysts were extracted by centrifugal flotation as described above. A single cyst from each of the six samples was transferred into a 1.5 ml tube containing 300 µl 2 mM ZnCl₂ as hatching solution. Five freshly hatched juveniles were individually fished from each sample into 1.5 ml tubes containing 10 µl water and squashed with a micro pestle for subsequent DNA extraction (see below). The second approach used single cysts derived from a single cyst propagation. This was done independently for six populations. For each population, composite soil samples of about 2 kg each were obtained from sugar beet fields located near Ingeleben, Söllingen, Peine, Sonnenhof and Vanikum, all in Germany, and Artenay in France. Cysts were extracted by centrifugal flotation and a single cyst culture was established for each population on oilseed rape. Since the single cyst culture failed in case of the Peine population, 10 cysts were used instead for propagation. Sixteen weeks after inoculation, two life cycles representing two generations of H. schachtii were completed. Cysts from each population were collected by washing the growth substrate under running tap water through a 250-µm aperture sieve. The content of the sieve was then transferred to a filter paper. Five individual cysts were picked per population and separately transferred into 1.5 ml tubes containing 20 µl water. Finally, cysts were crushed with a micro pestle for subsequent DNA extraction.

The third approach was for testing the DNA patterns of pooled cysts of H. schachtii populations in DGGE fingerprinting of vap1, and to achieve this purpose, three populations were selected. Composite soil samples were collected from infested sugar beet fields at Acholshausen, Köchingen and Großgoltern in Germany. Cysts were extracted from the soil by the above-described centrifugal flotation method. J2 of all three populations were gained by hatching in 2 mM ZnCl₂ using the modified Baermann funnel technique (Baermann, 1917). In total, 240 J2 were obtained for the population from Acholshausen, 470 J2 for Köchingen, and 740 J2 for Großgoltern. To best conserve the natural variability within a population, the total of hatched juveniles from each sample was used for further propagation on oilseed radish in loess substrate. After completing two generations, cysts were separated from the loess substrate by washing the substrate through a 250-µm aperture sieve. Cysts collected on the sieve were transferred to filter paper.

To study the gene patterns of pooled cysts for each of the three populations, eight replicates of 20 cysts each were collected. Cysts were then soaked in 4% sodium hypochlorite (NaOCl) for about 6 min to sterilize and clean the outer layer of the cysts. Following three washes with distilled water, cysts were transferred into 1.5 ml tubes containing 50 µl water and squeezed with the help of sharp forceps for subsequent DNA extraction. Additionally, the vap1 patterns of the single cysts for those three populations were analyzed in DGGE. The similar approach was followed for preparing the single cysts for DNA extraction, where five single cysts were picked separately after washing the loess substrate for each population and prepared as described above.

For DNA extraction, one volume of lysis buffer containing 0.2 M NaCl, 0.2 M Tris-HCl (pH 8.0), 1% (vol/vol) β-mercaptoethanol, and 0.8 mg/ml proteinase K (≥30 mAnson Units/mg) was added to one volume of the mechanically treated nematode suspension. The tubes were incubated in a thermal mixer (Eppendorf, Hamburg, Germany) at 750 rpm and 66°C for two hours. Proteinase K was inactivated in a water bath at 100°C for 5 min. After each incubation step, the tubes were centrifuged for 2 min at 13,000 rpm. Lysates were stored at −20°C until molecular analyses.

The GenBank accessions of H. glycines AF374388, H. schachtii CF101080, G. rostochiensis AJ536826, and G. pallida BM416493 were aligned in MEGA6 software to design degenerate primers of HGvap657f, HGvap1238r, and HSvap1238r, flanking the vap1 gene fragments to obtain a PCR-amplicon with an expected size of approx. 600 bp (Table 2). PCR amplification was performed in 25 µl volume containing 1 µl template DNA extracted from 1 to 10 cysts, 5x GoTaq buffer, 2.5 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphates, 0.1 mg/ml BSA, 0.6 mM forward primer, 0.3 mM of each reverse primer, and 1 U GoTaq Flexi polymerase (Promega, Mannheim, Germany). A touchdown PCR was applied to increase the specificity of the primers: 95°C for 5 min, then 10 cycles of 45 sec 95°C, 45 sections 68°C - 1°C / cycle, 90 sec 72°C, then 30 cycles of 45 sec 95°C, 45 sec 57°C, 60 sec 72°C, final extension at 72°C for 5 min, and cooling to 4°C. To confirm the success of the PCR amplification, 5 µl of the PCR product was loaded on a 1.5% (w/v) agarose gel and electrophoresis was performed at 80 V for 90 min. The agarose gel was then stained with 0.5 µg/ml ethidium bromide and bands were visualized under ultraviolet light. The PCR-amplified vap1 fragments were inserted into the vector pGEM-T and cloned in Escherichia coli JM109 high efficiency cells following the protocol provided by the manufacturer of the TA-cloning kit (Promega, Mannheim, Germany). The plasmids each carrying a single gene fragment were extracted from the bacterial clones using the Gene JET Plasmid Miniprep kit (Thermo Fisher Scientific, Waltham, MA), and sent for sequencing using vector primers T7 or SP6. A multiple sequence alignment of the partial regions of the vap1 gene was created by MUSCLE and Neighbor-Joining tree with 1,000 bootstraps using the software of MEGA6 and CLC Main Workbench version 7.8.1. Table 2

Primers to amplify an approx. 600 bp fragment of the vap1 genes from genomic DNA of Heterodera spp. and Globodera spp. for sequencing, and comparison to the corresponding priming sites in vap1 genes of cyst nematode species.

Based on the sequence alignments of the cloned vap1 genes and the GenBank accessions, conserved exon regions flanking variable intron regions were identified to design non-degenerate primers. Resolving genetic variation in DGGE is enhanced for amplicons being less than 500 bp in size and stabilized on one side by a GC-clamp (Myers et al., 1985; Muyzer and Smalla, 1998). To do so, the primer pair HSvap244f/HSvap548r was designed to amplify vap1 fragments of approx. 365 bp from plasmid and genomic DNA obtained from the above mentioned 12 populations of H. schachtii (Table 3). The designed primer pair was also tested to amplify vap1 gene fragments from genomic DNA of single cysts of H. avenae. For a 25 µl PCR reaction, the master mix was prepared by adding 1 µl of target DNA (5-50 ng) or 1 µl of plasmid DNA with cloned vap1 (20 ng), 5 µl 5x GoTaq buffer, 2.5 mM of MgCl₂, 0.2 mM deoxynucleoside triphosphates, 4% acetamide (vol/vol), 0.2 mM each of forward and reverse primer and 1 U GoTaq Flexi polymerase (Promega). Cycling conditions for the PCR reaction were as follows: 94°C for 5 min followed by 35 cycles at 95°C for 30 sec, 54°C for 30 sec and 72°C for 5 min, final extension at 72°C for 5 min, and cooling to 4°C. DGGE analysis was conducted by loading 3-15 µl aliquots of PCR-amplified fragments on a polyacrylamide gel containing denaturing gradients of 23 to 58%, where 100% denaturants corresponded to 7M urea plus 40% formamide. The INGENY PhorU₂ system (Ingeny, Goes, The Netherlands) was used to run the DGGE gel in 1x Tris-acetate-EDTA buffer at 58°C for 16 hr. DGGE profiles were visualized by acid silver staining (Heuer et al., 2001). The multiple sequences obtained from 40 populations of Globodera spp. allowed to design non-degenerate primers specific to amplify vap1 fragments for the purpose of studying the genetic structure of Globodera spp. populations (data not shown). Table 3.

Primers to amplify a vap1 gene fragment of Heterodera spp. for electrophoretic separation of the PCR products by DGGE, and comparison with the corresponding priming sites in vap1 genes of cyst nematode species.

The GelCompar II 6.6 software (Applied Math, Gent, Belgium) was utilized to create a similarity matrix based on the Pearson correlation coefficient, and to determine the Shannon diversity index from DGGE banding patterns of H. schachtii populations. Comparisons of the similarity measures and diversity indices were carried out with PERMTEST (Kropf et al., 2004) and R statistical software packages, respectively.

A sequence alignment of published vap1 cDNA clones and expressed sequence tags (ESTs) (GenBank accessions no. AF374388, CF101080, AJ536826, and BM416493) revealed conserved regions suitable to design degenerate primers (Table 2), which were efficient to amplify vap1 fragments of about 600 bp from the genomic DNA of all cyst nematode species included in this study except H. avenae (data not shown). Gene fragments amplified from DNA of the above mentioned 12 populations of H. schachtii and from populations of the further cyst nematodes studied here were cloned and sequenced. A collection of 76 vap1 sequences was obtained from the cyst nematodes, including one sequence of H. filipjevi, five sequences of H. betae, five sequences of G. pallida, 20 sequences of H. schachtii, and 45 sequences of G. rostochiensis. The phylogenetic analysis of sequence data revealed that the sequence homology of vap1 within the genus is higher than among the genera of cyst nematodes studied here. Furthermore, substantial sequence variation of vap1 was shown among species of the same genus (Fig. 1). Sequence variation among the Globodera species and populations that originated from Norway and Germany was much lower than among Heterodera species and among H. schachtii populations originating from Germany only (Fig. 1). Sequence logos of translated protein for the cloned variants of Globodera spp. is less variable than for H. schachtii. (Fig. 4). The high variability of vap1 genes among the 20 sequences of H. schachtii as shown in the underlined region of the multiple sequence alignment (SD.1) provided an indication of the efficiency of this region as a marker to study the genetic variation within and among populations of H. schachtii.

Figure 1

The design of the PCR-DGGE primer pair HSvap244f/HSvap548r (Table 3) and the corresponding PCR conditions were optimal to amplify the vap1 segments from the plasmid and genomic DNA of H. schachtii. To establish the vap1 marker of H. schachtii, the gene fragments were amplified from the plasmid DNA of the 20 sequenced variants gained from the 12 populations. Six clones having similar electrophoretic mobility as other variants (variants 5 and 6, variants 11 and 13) were excluded for better separation of the variants of the marker in DGGE. PCR products were slightly varying in size around 360 to 380 bp for 12 cloned variants, and around 270 to 290 bp for two variants (Fig. 2A). From genomic DNA of H. avenae a product of ca. 400 bp was amplified (Fig. 3A). In the DGGE gradient of 23-58%, 8 of 14 vap1 variants obtained from H. schachtii populations could be clearly resolved (Fig. 2B). Variants with high similarity in DNA sequence showed similar electrical mobility. Amplicons with identical electrophoretic mobility differed only by single-nucleotidepolymorphisms (variants 3 and 4), or by two substitutions and a deletion (12 and 13). The PCR products of the 14 vap1 variants were pooled to generate a vap1 marker for H. schachtii, which was used as an external reference in DGGE profiles of H. schachtii populations to represent the most frequent gene variants of H. schachtii. DGGE fingerprinting of vap1 allowed distinguishing between H. schachtii and H. avenae (Fig. 3B). In contrast to the variants of the vap1 marker of H. schachtii, the gene fragments of H. avenae melted earlier in the denaturing gradient. Sequence alignments of the 14 variants revealed that the amplified vap1 segment included intron and exon regions (Fig. 4A). In the sequence logo, the frequency of insertions/deletions and single-nucleotide polymorphisms was higher in the introns than in the exons. The highly variable intronic sequences of the variants were distant from the GC-clamped primer to support separation in DGGE. Interestingly, the translated amino acids of the exons revealed sequence differences among the 14 gene variants of vap1 (Fig. 4B).

Figure 2

Figure 3

Figure 4

Up to six variants of the gene were amplified by PCR from the genomic DNA of each single J2 of H. schachtii, which appeared as separate bands in the DGGE profiles (Fig. 5). DGGE fingerprints of individual J2 from the same cyst varied markedly. Despite this internal variation, the vap1 patterns differed significantly among these cysts derived from the same field population, giving evidence for genetic variation and gene flow within the Hottorf population of H. schachtii.

Figure 5

We first analyzed the variation of vap1 genes among six populations of H. schachtii using DNA of single cysts (Fig. 6). The cysts emerged from a single cyst propagation posing a strong genetic bottleneck. Nevertheless, the gene pattern varied among the five single cysts of each population, providing an indicator for the genetic variation within each population at the level of single cysts. The number and density of the electrophoresis bands representing the gene variants varied among most populations. While for the Sonnenhof population two gene patterns were observed, the other studied H. schachtii populations had 3 to 5 patterns with Artenay being especially heterogeneous (Fig. 6). The profiles were significantly different among populations of Artenay-Ingeleben, Artenay-Söllingen Ingeleben-Söllingen, Ingeleben-Söllingen, Ingeleben-Vanikum, Söllingen-Sonnenhof, Söllingen-Vanikum, Peine-Vanikum, Sonnenhof-Vanikum (permutation test on Pearson correlations, P = 0.04, d = 18).

Figure 6

Considering the observed genetic diversity of field populations, we used DNA of 20 pooled cysts for each replicate sample from three populations. The simple DNA extraction using lysis buffer could be scaled up without problems due to prior cleaning of cyst surfaces by 4% NaOCl. The pattern was highly similar among all eight replicates for each population (Fig. 7). The permutation test on pairwise similarities among populations showed significant differences among vap1 patterns of the three investigated field populations of H. schachtii (permutation test on Pearson correlations, P < 0.001, d = 33). The high value of d indicated a high difference of between-population variance and within-population variance of the vap1 patterns. Furthermore, the gene patterns of single cysts for the three populations were analyzed in DGGE and the profiles were significantly different among the populations (permutation test on Pearson correlations, P < 0.001, d = 26). Even though both profiles of single cysts and pooled cysts were significantly different among the populations, the gene pattern of pooled cysts appeared to be more similar than of the single cysts for the three populations (Fig. 7).

Figure 7

Another way to compare the populations by vap1 DGGE fingerprinting is the possibility to quantify the diversity of gene variants within each of the populations. Within this respect, the calculated vap1 diversity based on Shannon index varied significantly among the studied populations, with the population from Großgoltern being more diverse than the populations from fields in Köchingen and Acholshausen (Fig. 8).

Figure 8