Life stage-specific (eggs, infective J2, J3, J4, virgin females) cDNA libraries of H. glycines, the soybean cyst nematode, were generated to provide templates for EST sequencing, totaling 20,100 5' ESTs or almost 10 million nucleotides (GC content 48.9%). Sequences from all five developmental stages were represented in approximately equal proportions (Table 1). In addition to these stage-specific libraries, 1,858 H. glycines sequences previously deposited in GenBank were included in the dataset for this study, bringing the total number of sequences analyzed here to 21,958. This dataset was used by Affymetrix (Santa Clara, CA, USA) to form 6,860 unique contigs (average length 552 nucleotides, average size 3 ESTs), which then were represented by 7,530 probesets on the Affymetrix Soybean Genome Array GeneChip (gene discovery rate 31%; 6,860/21,958). Of the 6,860 unique contigs, 3,499 consisted of only one EST, so-called singletons (16% of all ESTs analyzed). On the other extreme, contig number HgAffx.13905.2 was formed by 599 ESTs. Furthermore, the 40 contigs that contained the largest number of ESTs represented 8.3% of all ESTs studied (Table 2).

In order to determine sequence similarities of our contigs and in particular to identify genes that are conserved between different nematode species, we BLAST searched the 6,860 H. glycines contigs versus three databases (Figure 1). About half of the contigs (44%) matched sequences in at least one of these three databases at a threshold value of E = 1e^-20. Examination of the BLAST match distribution revealed that 19% of the contigs that matched all three databases are most likely representing highly conserved genes involved in fundamental housekeeping processes in metazoans, while the 31% of contigs exclusively matching sequences in the cyst nematode database contained genes that likely are important for specific host adaptations of Heterodera spp.

When assessing BLAST hit identities, the cluster that contained the most ESTs (HgAffx.13905.2; 599 ESTs) belonged to a gene coding for a putative cuticular collagen. Identities of other highly represented contigs were actin, tropomyosin and myosin, as well as additional house-keeping genes like ribosomal components, ubiquitin, arginine kinase, synaptobrevin and heat shock proteins. Interestingly, four putative parasitism gene sequences from the esophageal gland cells were among the 40 contigs with the highest EST constituents: three of unknown function (AAO33474.1, AAL78212.1, AAP30835.1) 78 and a β-1,4-endoglucanase-2 precursor.

We further determined which H. glycines genes showed the highest degree of conservation when compared to C. elegans. BLASTX searches of all 6,860 soybean cyst nematode contigs against the Wormpep database revealed that 34.9% matched C. elegans entries at a threshold value of E = 1e^-20. The products of the 25 most conserved genes were heat shock proteins, proteins related to transcription and translation (for example, elongation and splicing factors and RNA polymerase II) and structural proteins, including tubulin and actin, as well as enzymes, including guanylate cyclase (Additional data file 3).

In order to identify H. glycines genes that are developmentally regulated and to document their expression profiles, we designed a microarray experiment using three complete and independent biological replications (that is, three independent sample series representing three complete life cycles). We identified 6,695 probesets (Additional data file 4) as described in Materials and methods that were differentially expressed with a false discovery rate (FDR) of 5% when observed over the entire life cycle of H. glycines. This group of probesets equals 89% of all H. glycines probesets on the microarray. In other words, the vast majority of H. glycines genes represented on the GeneChip significantly changed expression during the nematode life cycle. We then grouped these 6,695 probesets into 10 clusters based on their expression profiles (Figure 2). As an exemplary gene family, we analyzed the expression pattern of FMRF (Phe-Met-Arg-Phe-NH₂)-related neuropeptide (FaRP)-encoding genes. This group encodes a specific class of neuronally expressed tetrapeptides that are potent myoactive transmitters in nematode neuromusculature 5253545556, which are expressed in motor neurons that act on body wall muscle cells 575859. Based on our BLAST searches against various databases as detailed above, we identified five probesets for genes encoding FaRPs (HgAffx.23446.1.S1_at, HgAffx.23636.1.S1_at, HgAffx63.1.S1_at, HgAffx20469.1.S1_at, HgAffx.24161.1.S1_at). All five probesets were co-expressed with each other and showed an expression peak in the infective J2 stage (Additional data file 1). These FaRP probesets were differentially expressed when observed over the entire life cycle of the nematode, and, with the exception of HgAffx.20469.1.S1_at, which was found in cluster 4, all probesets were grouped in cluster 7 (Figure 2). The general profile of cluster 4 showed an expression peak in infective J2 and fell steadily in later life stages, while cluster 7 demonstrated the same overall pattern but showed a more pronounced increase from egg to infective J2.

Furthermore, we formed expression clusters for all 15 possible pairwise comparisons of all six life stages under study, as well as for comparisons of groups of life stages, that is: all pre-penetration (egg, infective J2) versus all post-penetration (parasitic J2, J3, J4, virgin females) life stages; and motile (pre-penetration J2) versus all non-motile parasitic (parasitic J2, J3, J4, virgin females) life stages. A summary displaying the number of probesets showing differential expression (FDR 5%) in these comparisons is given in Table 3.

We used InterProScan 60 to conduct functional classification for all 6,860 contigs in all expression clusters of each comparison. The relative abundance of the 25 InterPro domains with the highest representation in each of the 10 clusters for contigs that showed differential expression (FDR 5%) throughout the entire life cycle is summarized in Additional data file 5. While most clusters contained a wide range of genes represented by diverse InterPro domains, collagen domains stood out, in that they accumulated in cluster 2 at a high frequency relative to other InterPro domains. Since it has been suggested that down-regulation of collagens might be a common feature in dauer and infective stages of nematodes 27, we analyzed the expression profiles of H. glycines collagens in more detail. Using a reciprocal BLAST strategy as described in Materials and methods, we identified eight H. glycines probesets representing seven unique contigs orthologous to C. elegans collagens (Table 4). The temporal expression pattern of these seven orthologs was very similar (Figure 3) and congruent with observations in other nematode species 1451, which supports the hypothesis that down-regulation of collagen transcription is a conserved characteristic of non-molting infective and dauer-stage nematodes 27.

In addition to providing a comprehensive gene characterization and expression resource, we wished to demonstrate the applicability and power of our data by addressing the question of whether the infective J2 stage of H. glycines is biochemically analogous to the C. elegans dauer larva stage. We compiled a list of 1,839 C. elegans genes that were identified by Wang and Kim 61 as so-called dauer-regulated genes by conducting microarray experiments comparing gene expression in C. elegans larvae that were in transition from dauer to non-dauer with that of freshly fed L1 larvae that had been starved. Dauer-regulated genes showed significant expression changes during a dauer exit time course that were not related to the introduction of food 61. Reciprocal BLAST searches resulted in the identification of 438 H. glycines probesets that could be categorized unambiguously as orthologs of these C. elegans dauer-regulated genes (Additional data file 6). Because of the deliberate redundancy of the Affymetrix GeneChip, these 438 probesets corresponded to 396 unique H. glycines gene predictions. In other words, we identified H. glycines orthologs for 22% of the 1,839 C. elegans dauer-regulated genes (396/1,839).

We also compiled a list of H. glycines genes that are orthologous to the 488 C. elegans gene subset of the C. elegans dauer-regulated genes that Wang and Kim 61 determined to be up-regulated during the dauer stage and down-regulated upon dauer exit, a group that was called dauer-enriched. These genes presumably define dauer-specific properties, including stress resistance and longevity. These dauer-enriched genes were of particular interest to us because up-regulation of orthologous genes in the H. glycines infective J2 stage would suggest involvement in developmental arrest of these genes not only in C. elegans, but also in H. glycines. Using the same reciprocal BLAST search strategy, we identified 74 H. glycines probesets corresponding to 69 unique H. glycines contigs or genes that are orthologous to 57 unique C. elegans dauer-enriched genes (Table 5), which represent 14%.