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Additional file 1: Figure S1 - BlobTools-based interrogation of genome assembly of Gr-Line 19 to verify for single-taxon origin of the original sequences. Panel A: Each Gr-Line19 scaffold is represented by a single filled circle. Each scaffold is positioned in the main panel based on its GC proportion (x-axis) and coverage by reads from PacBio sequences (y-axis). On the top right the colours of the individual blobs are linked to their taxonomic origin. At the bottom of the main Blobtool figure, the size of the circles is linked to scaffold size. Panel B: on the left the % of unmapped versus mapped Gr-Line19 PacBio reads are presented, and the right the taxonomic origin of the reads. Figure S2 - BlobTools-based interrogation of genome assembly of Gr-Line 22 to verify for single-taxon origin of the original sequences. Panel A: Each Gr-Line22 scaffold is represented by a single filled circle. Each scaffold is positioned in the main panel based on its GC proportion (x-axis) and coverage by reads from PacBio sequences (y-axis). On the top right the colours of the individual blobs are linked to their taxonomic origin. At the bottom of the main Blobtool figure, the size of the circles is linked to scaffold size. Panel B: on the left the % of unmapped versus mapped Gr-Line22 PacBio reads are presented, and the right the taxonomic origin of the reads. Figure S3 - Multiple sequence alignment of Gr-CLE-1 protein sequences to verify the conservation of the CLE domain in putative CLE-1 genes. Each gene is represented by a gene identifier. Two genes are included for Gr-Line19 (Gros19_g2219.t1 and Gros19_g2223.t1) and two for Gr-Line22 (Gros22_g4869.t1 and Gros22_g4874.t1). The CLE1 sequence identified in Heterodera glycines (Q9BN21) is included as an outgroup.

Additional file 4 245 regulated genes in wrky19–1 during M. incognita infection with significance > 7 or with a relative expression of 0.03.

Additional file 6. Overview of primers used in qRT-PCR for a set of selected genes in Table 1

Additional file 2 Number of root tips for bat5–2 on 14-day-old seedlings. Statistically tested with ANOVA and post hoc Tukey test (p = 0.05). Data represents two biological replicates.

Additional file 1: Figure S1. Confirmation of T-DNA insert in line bat5–2, dsc1–1 and wrky19–1 with PCR. A, allele specific PCRs on genomic DNA isolated from each Arabidopsis mutant line. PCR amplification products using primer combinations for only the wildtype gene allele (P1) and for the wildtype allele including the T-DNA insert (P2). B-D, Relative gene expression of the genes harbouring the T-DNA insert in the mutant lines as compared to the wildtype Arabidopsis Col-0 using quantitative RT-PCR on roots of 14-day old seedlings. B, represents the relative gene expression of BAT5 in bat5–2. C, represents the relative gene expression of DSC1 in dsc1–1 and wrky19–1. D, represents the relative gene expression of WKRY19 in dsc1–1 and wrky19–1. Data (B-D) was generated with three independent biological replicates with three technical replicates each.

The DOG box is not a quantitative predictor of temporal expression. A. In G. pallida, genes with four or more motifs and a signal peptide are primarily expressed during the infective stage. Each line represents the expression pattern of a different gene. B. The number of motifs does not correlate with gene expression at J2 (black, all numbers of motifs; red, four or more motifs). (PDF 1707 kb)

Phylogenetic analysis of G. rostochiensis pathotypes. A. A maximum likelihood phylogeny based on 730,705 genome wide SNPs. Two distinct groups of Ro1 are together separated from Ro2, Ro3, Ro4 and Ro5. Node labels indicate bootstrap support values for 100 iterations. B. Principle component analysis (PCA) based on the same dataset suggest that for pathotype Ro1, intra-pathotype variation is similar to inter-pathotype variation. REF Ro1 = Reference strain Ro1 assembly, MAR149 Ro1 = British Columbia, QC Ro1 = Quebec, Ro19 Ro1 = European, NFLD Ro1 = Newfoundland, SCRI_Ro2, SCRI_Ro3, SCRI_Ro4, SCRI_Ro5 are pathotype populations from the James Hutton Institute collection. (PDF 1636 kb)

Transposable elements in G. rostochiensis, G. pallida and Meloidogyne spp. Quantities of transposable elements from (A) DNA, (B) LTR, (C) LINE and (D) SINE super-familes. Globodera spp. contain notably more Jockey (LINE) and Alu (SINE) than the Meloidogyne spp. (PDF 623 kb)

Comparison of distance to neighbouring gene for effectors and non-effectors. A. The log nucleotide distance to next gene 5′ and 3′ for a random subset of non-effectors (n = 138). B. The log nucleotide distance to next gene 5′ and 3′ for high-confidence effectors (n = 138). C. Comparing the distance of each gene to its neighbour either upstream or downstream suggests that despite being located in gene dense regions of the genome, effectors have a skewed distribution of gene density compared to an identically sized subset of non-effectors (lower case letters indicate homogenous subsets, Student’s t-test correcting for multiple comparisons p

Comparison of gene expression between parasitic G. pallida life stages. The key transitions to be captured in terms of gene expression of all genes, and in particular for effectors, is from outside the plant (J2) compared to inside the plant (sedentary female). There is almost no difference in global gene expression between the early sedentary time points [22]. A. 84 % of the variation in expression at 7 days post infection (dpi) is explained by variation in expression at 14 dpi. B. This correlation is even more profound if the analysis is restricted to only the effectors (89 %). Similar correlations are possible between 14 and 21 dpi, albeit of lesser magnitude but an identical trend (60 % correlation for all genes, and 64 % correlations for specifically effectors). This is not the case, however, when comparing J2 and 7 dpi (44 % for all genes, and zero correlation for all effectors). Fourteen dpi provides an ideal intermediate for the sedentary stages. (PDF 4379 kb)

Supplementary methods and results. (DOCX 28 kb)

Synteny clusters. Network representation of i-ADHoRe results. Scaffolds are represented as nodes (blue = G. rostochiensis, ochre = G. pallida) whose diameter is scaled by scaffold length and vertices are drawn between them if five or more anchorpoints (i.e. syntenic proteins, numbers on vertices indicate count of anchorpoints) have been found. The biggest cluster (dashed black line) is composed of 26 G. rostochiensis scaffolds (4.99 Mb) and 35 G. pallida scaffolds (5.54 Mb). Part of this cluster (grey dotted line) is shown in Fig. 6. (PDF 570 kb)

Differential expression super-clusters. Ninety-four percent of all differentially expressed genes are manually grouped into 25 biologically relevant expression super-clusters. For each super-cluster, individual cluster graphs are shown where for all expression displayed as centred log fold-change is in the order, Cyst, Cyst, Egg, Egg, J2, J2, 14 dpi, 14 dpi. (PDF 1669 kb)

Phylum wide analysis of GC/AG splice sites in nematodes. The percentage of GC/AG splices sites with associated consensus sequences are shown for 17 species against a schematic phylogeny of the phylum Nematoda (adapted from [89]). Red numbers indicate those which likely represent under reporting due to over-strict parameter settings during gene prediction. (PDF 1371 kb)

Blob plots. A. Blob plot of the initial G. rostochiensis assembly, displaying some minor contamination from Actinobacteria and Ascomycota. Each scaffold is drawn as a circle based on its GC content (X-axis) and log-coverage (Y-axis), with a diameter proportional to its length and coloured by its taxonomic annotation at the phylum-level. In the legend, colours of phyla are listed together with scaffold-count, scaffold span and scaffold N50. The histograms above and to the right of the main scatter plot sum contig spans for GC proportion bins and log-coverage bins, respectively. B. Blob plot of the G. rostochiensis assembly after removal of contaminant scaffolds. (PDF 8314 kb)

Comparison of gene expression between parasitic G. pallida life stages. The key transitions to be captured in terms of gene expression of all genes, and in particular for effectors, is from outside the plant (J2) compared to inside the plant (sedentary female). There is almost no difference in global gene expression between the early sedentary time points [22]. A. 84 % of the variation in expression at 7 days post infection (dpi) is explained by variation in expression at 14 dpi. B. This correlation is even more profound if the analysis is restricted to only the effectors (89 %). Similar correlations are possible between 14 and 21 dpi, albeit of lesser magnitude but an identical trend (60 % correlation for all genes, and 64 % correlations for specifically effectors). This is not the case, however, when comparing J2 and 7 dpi (44 % for all genes, and zero correlation for all effectors). Fourteen dpi provides an ideal intermediate for the sedentary stages. (PDF 4379 kb)

Differential expression super-clusters. Ninety-four percent of all differentially expressed genes are manually grouped into 25 biologically relevant expression super-clusters. For each super-cluster, individual cluster graphs are shown where for all expression displayed as centred log fold-change is in the order, Cyst, Cyst, Egg, Egg, J2, J2, 14 dpi, 14 dpi. (PDF 1669 kb)

Phylogenetic analysis of Worm-specific Argonauts (WAGOs). A. A total of 604 nematode and non-nematode argonaute proteins drawn as an unrooted phylogram. It is assumed that each subtree (Bâ F) is effectively rooted by the other subtrees; however, the extreme divergence between these proteins yields low support for some subtrees. Bâ F Subtrees containing G. rostochiensis WAGOs (GrWAGOs). Branch widths of subtrees are drawn proportional to branch support. Coloured boxes indicate membership of taxa to phylogenetic groups (nematode Clade I, Clade III, Clade IV and Clade V, non-nematode taxa) and coloured stars indicate clades composed entirely of Heteroderidae-WAGOs (Globodera spp., Meloidogyne spp., Heterodera glycines). Orange dots indicate GrWAGOs in differential expression super-clusters 19, 20 or 21. GrWAGOs are placed within Nematode WAGO-subclades ALG1/ALG2 (1), RDE1/ERGO1/PRG1/2 (2), WAGO1/2/4/5 (7), CSR/WAGO-III/WAGO-IV (8) and NRDE/WAGO-10/11 (5), sensu Buck and Blaxter, 2013 [30]. As expected, no GrWAGOs were observed in WAGO-subclades SAGO2/PPW, WAGO-III/-V and ALG3/ALG4. Subclades NRDE/WAGO-10/11, WAGO1/2/4/5 and CSR/WAGO-III/WAGO-IV show increased numbers of paralogous expansion of these gene families within Clade IV in general and Heteroderidae in particular. B All Globodera spp. and Meloidogyne spp. exhibit one ALG1/ALG2 orthologue each, which form a clade. C The RDE1/ERGO1/PRG1/2 subtree contains another Heteroderidae-specific clade; however, one M. incognita sequence is sister to a subclade of Clade III taxa. This is surprising since the Clade III parasites A. suum and B. pahangi are thought to have lost the piRNA pathway [30] and may be an artefact. D The NRDE/WAGO-10/WAGO-11 subtree shows an expansion of paralogous Heteroderidae-WAGOs, with three GrWAGOs expressed at 14 dpi. E The WAGO1/2/4/5 subtree depicts two Heterodera-specific expansions, of which the larger subclade contains four GrWAGOs expressed at 14 dpi. F The CSR/WAGO-III/WAGO-IV subtree contains another two Heteroderidae-specific expansion, of which one also includes a sequence from the Clade V nematode Dictyocaulus viviparus (ochre star). (PDF 64 kb)

Gene duplication in the G. rostochiensis and related genomes. Comparing the identity of each gene to the next most similar gene in the genome gives insights into potential duplication within the genome sequence. In a diploid species, with a good assembly and gene prediction, we expect no overrepresentation of duplicates at any particular divergence, as is seen in the genome of M. hapla. The G. rostochiensis protein set has a very similar distribution to that of M. hapla, but in G. pallida there is an overrepresentation of genes that are >97Â % identical to each other. As reported previously, the protein set from M. incognita has a distinct excess of duplicates at ~96Â % identity, thought to derive from a hybrid origin, and subsequent aneuploidy changes, of this species [23, 88]. G. pallida is not believed to derive from a hybridisation event [26] and so this is probably a reflection of duplication at the assembly stage (i.e. retention of allelic copies of loci because of the high level of heterozygosity in UK populations). (PDF 1365 kb)

Phylum wide analysis of GC/AG splice sites in nematodes. The percentage of GC/AG splices sites with associated consensus sequences are shown for 17 species against a schematic phylogeny of the phylum Nematoda (adapted from [89]). Red numbers indicate those which likely represent under reporting due to over-strict parameter settings during gene prediction. (PDF 1371 kb)

Phylogenetic analysis of Worm-specific Argonauts (WAGOs). A. A total of 604 nematode and non-nematode argonaute proteins drawn as an unrooted phylogram. It is assumed that each subtree (Bâ F) is effectively rooted by the other subtrees; however, the extreme divergence between these proteins yields low support for some subtrees. Bâ F Subtrees containing G. rostochiensis WAGOs (GrWAGOs). Branch widths of subtrees are drawn proportional to branch support. Coloured boxes indicate membership of taxa to phylogenetic groups (nematode Clade I, Clade III, Clade IV and Clade V, non-nematode taxa) and coloured stars indicate clades composed entirely of Heteroderidae-WAGOs (Globodera spp., Meloidogyne spp., Heterodera glycines). Orange dots indicate GrWAGOs in differential expression super-clusters 19, 20 or 21. GrWAGOs are placed within Nematode WAGO-subclades ALG1/ALG2 (1), RDE1/ERGO1/PRG1/2 (2), WAGO1/2/4/5 (7), CSR/WAGO-III/WAGO-IV (8) and NRDE/WAGO-10/11 (5), sensu Buck and Blaxter, 2013 [30]. As expected, no GrWAGOs were observed in WAGO-subclades SAGO2/PPW, WAGO-III/-V and ALG3/ALG4. Subclades NRDE/WAGO-10/11, WAGO1/2/4/5 and CSR/WAGO-III/WAGO-IV show increased numbers of paralogous expansion of these gene families within Clade IV in general and Heteroderidae in particular. B All Globodera spp. and Meloidogyne spp. exhibit one ALG1/ALG2 orthologue each, which form a clade. C The RDE1/ERGO1/PRG1/2 subtree contains another Heteroderidae-specific clade; however, one M. incognita sequence is sister to a subclade of Clade III taxa. This is surprising since the Clade III parasites A. suum and B. pahangi are thought to have lost the piRNA pathway [30] and may be an artefact. D The NRDE/WAGO-10/WAGO-11 subtree shows an expansion of paralogous Heteroderidae-WAGOs, with three GrWAGOs expressed at 14 dpi. E The WAGO1/2/4/5 subtree depicts two Heterodera-specific expansions, of which the larger subclade contains four GrWAGOs expressed at 14 dpi. F The CSR/WAGO-III/WAGO-IV subtree contains another two Heteroderidae-specific expansion, of which one also includes a sequence from the Clade V nematode Dictyocaulus viviparus (ochre star). (PDF 64 kb)

Additional file 5. Overview of primers used in qRT-PCR and for confirmation of T-DNA insert.

Additional file 5. Overview of primers used in qRT-PCR and for confirmation of T-DNA insert.