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

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

Journal of Nematology
Volume 50 (2018): Issue 4 (January 2018)
By: Rasha Haj Nuaima,  Johannes Roeb,  Johannes Hallmann,  Matthias Daub,  Sandra Otte and  Holger Heuer  
Open Access
|Dec 2018

Figures & Tables

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 nematodeNumberPopulation/locationOrigin
Heterodera schachtii 8Köchingen, Grossgoltern, Ingeleben, Peine, Söllingen, Sonnenhof, Vechelde, BodenstedtLower Saxony/Germany
Heterodera schachtii 12Asperden, Hottorf, Münster, Vanikum, Titz-KalrathNorth Rhine-Westphalia/Germany
Heterodera schachtii 1ArtenayCentre-Val de Loire/France
Heterodera schachtii 1AcholshausenBavaria/Germany
Heterodera betae 1AsperdenNorth Rhine-Westphalia/Germany
Heterodera betae 1BraunschweigLower Saxony/Germany
Heterodera avenae 1MünsterNorth Rhine-Westphalia/Germany
Heterodera filipjevi 1MünsterNorth Rhine-Westphalia/Germany
Globodera spp.29diverseLower Saxony/Germany
Globodera spp.11diverseNorway

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 geneSequence 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 accessionPrimer 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…….
DOI: https://doi.org/10.21307/jofnem-2018-055 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
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Language: English
Page range: 517 - 528
Published on: Dec 3, 2018
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
Beet cyst nematode,
Genetic fingerprinting technique,
Method,
Electrophoresis,
gene,
Venom allergen like protein
Related subjects:
Life sciences,
Life sciences, other

© 2018 Rasha Haj Nuaima, Johannes Roeb, Johannes Hallmann, Matthias Daub, Sandra Otte, Holger Heuer, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

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