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

Rasha Haj Nuaima

Rasha Haj Nuaima

  • Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen DiagnosticsBraunschweig, Germany
  • Department of Plant Protection, Faculty of Agriculture, Euphrates UniversityTurkey, Syria
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Nuaima, Rasha Haj
, 
Johannes Roeb

Johannes Roeb

Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen DiagnosticsMünster, Germany
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Roeb, Johannes
, 
Johannes Hallmann

Johannes Hallmann

Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen DiagnosticsMünster, Germany
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Hallmann, Johannes
, 
Matthias Daub

Matthias Daub

Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Field Crops and GrasslandElsdorf, Germany
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Daub, Matthias
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Sandra Otte

Sandra Otte

Strube Research GmbH & Co. KGSöllingen, Germany
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Otte, Sandra
 und 
Holger Heuer

Holger Heuer

Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen DiagnosticsBraunschweig, Germany
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Heuer, Holger
  
03. Dez. 2018
Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations's Cover Image
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Online veröffentlicht: 03. Dez. 2018
Seitenbereich: 517 - 528
DOI: https://doi.org/10.21307/jofnem-2018-055
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© 2018 Rasha Haj Nuaima et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Dendrogram generated from the alignment of amplified and sequenced vap1 gene fragments (ca. 600 bp) of various cyst nematode species and populations (MUSCLE alignment and Neighbor-Joining tree with 1,000 bootstraps done with MEGA6). The triangles represent the vap1 sequences gained from the populations of each species. Note that there exist two triangles for H. schachtii within which the sequences represent higher variability of vap1 genes than for Globodera spp.
Dendrogram generated from the alignment of amplified and sequenced vap1 gene fragments (ca. 600 bp) of various cyst nematode species and populations (MUSCLE alignment and Neighbor-Joining tree with 1,000 bootstraps done with MEGA6). The triangles represent the vap1 sequences gained from the populations of each species. Note that there exist two triangles for H. schachtii within which the sequences represent higher variability of vap1 genes than for Globodera spp.

Figure 2

GC-clamped PCR products amplified from cloned fragments of vap1 genes obtained from populations of Heterodera schachtii originating from Vechelde, Bodenstedt, Titz-Kalrath, Asperden, and Münster. (A) Size separation in 1.5% agarose gel electrophoresis; L: 1 kb ladder. (B) Separation in DGGE; M: marker composed of all 14 fragments.
GC-clamped PCR products amplified from cloned fragments of vap1 genes obtained from populations of Heterodera schachtii originating from Vechelde, Bodenstedt, Titz-Kalrath, Asperden, and Münster. (A) Size separation in 1.5% agarose gel electrophoresis; L: 1 kb ladder. (B) Separation in DGGE; M: marker composed of all 14 fragments.

Figure 3

GC-clamped PCR products amplified from the genomic DNA of three single cysts of Heterodera avenae. (A) 1.5% agarose gel electrophoresis; L: 1 kb ladder. (B) DGGE profiles of vap1 gene fragments of Heterodera avenae from three single cysts compared to the distinct gene fragments of Heterodera schachtii in the marker (L).
GC-clamped PCR products amplified from the genomic DNA of three single cysts of Heterodera avenae. (A) 1.5% agarose gel electrophoresis; L: 1 kb ladder. (B) DGGE profiles of vap1 gene fragments of Heterodera avenae from three single cysts compared to the distinct gene fragments of Heterodera schachtii in the marker (L).

Figure 4

Sequence logos of nucleotide sequences (A) and translated protein (B) of 14 vap1 gene variants of Heterodera schachtii in the region used for PCR-DGGE. The exon region included in the gene fragment is underlined. Sequence logos were generated by CLC Main Workbench version (7.8.1).
Sequence logos of nucleotide sequences (A) and translated protein (B) of 14 vap1 gene variants of Heterodera schachtii in the region used for PCR-DGGE. The exon region included in the gene fragment is underlined. Sequence logos were generated by CLC Main Workbench version (7.8.1).

Figure 5

Fingerprints of single second stage juveniles from six cysts of Heterodera schachtii from the field population at Hottorf, North Rhine-Westphalia, Germany. The fingerprints were derived by PCR amplification of vap1 variants and electrophoretic separation by DGGE. The profiles were significantly different among cysts except for cyst2-cyst4, cyst2-cyst5, cyst3-cyst4, cyst4-cyst6 (permutation test on Pearson correlations, p < 0.04, d = 18). M: marker of cloned vap1 variants.
Fingerprints of single second stage juveniles from six cysts of Heterodera schachtii from the field population at Hottorf, North Rhine-Westphalia, Germany. The fingerprints were derived by PCR amplification of vap1 variants and electrophoretic separation by DGGE. The profiles were significantly different among cysts except for cyst2-cyst4, cyst2-cyst5, cyst3-cyst4, cyst4-cyst6 (permutation test on Pearson correlations, p < 0.04, d = 18). M: marker of cloned vap1 variants.

Figure 6

Fingerprints of single cysts from six field populations of Heterodera schachtii analyzed by PCR amplification of vap1 variants and electrophoretic separation in DGGE. M: marker of cloned vap1 variants.
Fingerprints of single cysts from six field populations of Heterodera schachtii analyzed by PCR amplification of vap1 variants and electrophoretic separation in DGGE. M: marker of cloned vap1 variants.

Figure 7

Fingerprints from three populations of Heterodera schachtii derived by PCR amplification of vap1 variants from DNA of 20 pooled cysts per replicate sample, and electrophoretic separation by DGGE. The profiles were significantly different among all populations (permutation test on Pearson correlations, P < 0.001, d = 33). M: marker of cloned vap1 variants.
Fingerprints from three populations of Heterodera schachtii derived by PCR amplification of vap1 variants from DNA of 20 pooled cysts per replicate sample, and electrophoretic separation by DGGE. The profiles were significantly different among all populations (permutation test on Pearson correlations, P < 0.001, d = 33). M: marker of cloned vap1 variants.

Figure 8

Box-Whisker Plots of the Shannon indices calculated for gene diversity of vap1 based on DGGE profiles of three field populations of Heterodera schachtii. Different letters indicate significant differences in diversity among populations (Tukey’s test, P < 0.05, n = 8).
Box-Whisker Plots of the Shannon indices calculated for gene diversity of vap1 based on DGGE profiles of three field populations of Heterodera schachtii. Different letters indicate significant differences in diversity among populations (Tukey’s test, P < 0.05, n = 8).

Sampling locations of 66 cyst nematodes populations

Cyst nematode Number Population/location Origin
Heterodera schachtii 8 Köchingen, Grossgoltern, Ingeleben, Peine, Söllingen, Sonnenhof, Vechelde, Bodenstedt Lower Saxony/Germany
Heterodera schachtii 12 Asperden, Hottorf, Münster, Vanikum, Titz-Kalrath North Rhine-Westphalia/Germany
Heterodera schachtii 1 Artenay Centre-Val de Loire/France
Heterodera schachtii 1 Acholshausen Bavaria/Germany
Heterodera betae 1 Asperden North Rhine-Westphalia/Germany
Heterodera betae 1 Braunschweig Lower Saxony/Germany
Heterodera avenae 1 Münster North Rhine-Westphalia/Germany
Heterodera filipjevi 1 Münster North Rhine-Westphalia/Germany
Globodera spp. 29 diverse Lower Saxony/Germany
Globodera spp. 11 diverse Norway

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_

PCR primer or gene Sequence 5′-3′
Forward primer HSvap244f AGT TCG TCG ACA ATT TCG GAA GG
Heterodera schachtii vap1 … ..R … … … … … ..
Heterodera betae vap1 … … … … … … … ..
Heterodera filipjevi vap1 … ..A ..T ..C … … … ..
Globodera spp. vap1 ..C ..A … ..G ..C W.C … ..
Reverse primer HSvap548rGC a clamp- GCC TGG CTC CAA TGT CCG ATG
Heterodera schachtii vap1 … … … … … … …
Heterodera betae vap1 … … … … ..A … …
Heterodera filipjevi vap1 … … … … … … …
Globodera spp. vap1 A….A … … ..C ..A …

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_

Primer or GenBank accession Primer or vap1 gene DNA sequence 5′-3′a
Forward primer HGvap657f CCA TGC TCT GTT TTG GCW CTT TCT G
H. glycines AF374388 … … … … … ..T … … .
H. schachtii CF101080 ..G … … … … ..A … … .
G. rostochiensis AJ536826 … .T…C … … ..A … … .
G. pallida BM416493 … ..G ..C … … ..A … … .
Reverse primer HGvap1238r AGT GGA GGC CCA TGC TTG CTG
Reverse primer HSvap1238r … .TT … … B……..
H. glycines AF374388 … … … … … … …
H. schachtii CF101080 … .TT … … C……..
G. rostochiensis AJ536826 C…TT … … G……..
G. pallida BM416493 C…TT … … AC…….
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