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. 2019 Feb 5;19(4):2537–2550. doi: 10.3892/mmr.2019.9938

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida (Savigny, 1826)

Chang Liu 1,*, Xuan Wang 1,*, Jiu-Bo Tian 1, Pei-Yuan Ma 1, Fan-Xiu Meng 1, Qi Zhang 1, Bao-Feng Yu 1,✉, Rui Guo 1, Zhi-Zhen Liu 1, Hai-Long Wang 1, Jun Xie 1, Niu-Liang Cheng 1, Jian-Hua Wang 2, Bo Niu 1,2,✉, Gong-Qin Sun 1,3
PMCID: PMC6423580  PMID: 30720084

Abstract

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full-length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5′End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high-quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P-value <1×10−8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein-protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high-quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

Keywords: Earthworm Eisenia fetida (Savigny, 1826); cDNA library; expressed sequence tags; GO annotation; KEGG pathway

Introduction

Earthworms are terrestrial annelids in the oligochaeta subclass, with a generally preferred habitat of damp and loose soil. They include ~3,000 species worldwide, with 229 species in China (1,2). In a wide variety of soil types, earthworms serve vital roles in converting large pieces of organic matter into rich humus to enrich soil fertility. The earthworms are the highest evolutionary species capable of regenerating an anterior portion containing the central nerve system, heart and clitellum (3). The anterior regeneration is a unique developmental process that requires cell proliferation, re-differentiation and sophisticated cell-cell communication. This process can serve as a useful model for investigating normal development and differentiation (4).

Over the past several years, cDNA library construction and analysis have become established as indispensable methods for functional genome analysis since they provide detailed information about the genomic mechanisms underlying the diverse processes of an organism (5). However, conventionally generated cDNA libraries contain a high percentage of 5′-truncated clones, limiting the utility of such libraries. The Switching Mechanism at 5′End of RNA Template (SMART) technique (6) amplifies and enriches the full-length mRNA, and thus generates cDNA libraries with a significantly improved ratio of full-length to partial cDNA sequences. In the present study, the SMART technique was adopted to construct a high quality library of full-length cDNAs representative of adult earthworms, namely of the earthworm Eisenia fetida (Savigny, 1826).

Unlike other model organisms, none of the oligochaete genomes have been sequenced to the best of our knowledge, and genomic research on earthworms lags behind that of other model species such as Mus musculus. In the absence of the full genome sequences, expressed sequence tags (ESTs) aid the rapid detection of expressed genes via sequence analysis, and are a significant resource for comparative and functional genomic studies (2). In addition, among the biological techniques for transcriptome analysis, the determination of ESTs is considered the simplest method for profiling the transcriptome, which is also particularly useful in the development of cDNA microarrays for systematic identification of differentially expressed genes (7). Analysis of ESTs is an effective method for rapidly analyzing gene expression, characterizing gene functions and discovering new genes that are important for specific developmental and physiological processes (8). The present study established 593 ESTs, representing 168 genes and 425 unknown tags, providing a gene expression profile of earthworm development. This collection of ESTs may provide a valuable basis for future research on the physiology of earthworms.

Materials and methods

Isolation of total RNA and mRNA

Eisenia fetida earthworms were purchased from Beijing Shuangqiao Farm (Beijing, China). Fully developed adult Eisenia fetida earthworms weighing 0.3–0.6 g (live weight) were selected for all experiments. All earthworm tissues were harvested and total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The integrity of total RNA was analyzed by electrophoresis using 1% agarose gels. Isolation of poly(A) mRNA from total RNA was carried out using an MN-NucleoTrap®mRNA kit (Machery-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's protocol. Oligo(dT) beads suspension was applied to total RNA and incubated at 68°C for 5 min before eluting mRNA. Then ethidium bromide (EB) staining was applied and 1% agarose gel was used to visualize the result. The isolated mRNA was further vacuum concentrated using Concentrator plus™ (Eppendorf, Hamburg, Germany). The quantity and integrity of isolated mRNA were determined using a nanodrop spectrophotometer and agarose gel electrophoresis, respectively.

cDNA library construction

A total of ~8,048.4 ng mRNA was used for single-stranded cDNA synthesis. The purified mRNA was used as a template, Oligod(T)18 with XhoI cleavage site was used as the primer, and first strand cDNA was transcribed at 42°C using SuperScript™ II RnaseH-Reverse (Thermo Fisher Scientific, Inc.). Then the mRNA was digested using RNaseH, and the resultant mRNA fragments were used as further primers. The first cDNA chain was used as a template for double-stranded cDNA synthesis, using DNA Polymerase I (Takara Biotechnology Co., Ltd., Beijing, China). The ends of the double-stranded cDNA were ligated by T4 DNA polymerase and the ligation products were purified by phenol/chloroform/isoamyl alcohol to remove excess impurities such as protein. Subsequently, the double-stranded DNA fragments were ligated into EcoRI Adaptor using T4 DNA ligase at 4°C overnight. Then the double-stranded DNA fragments were phosphorylated with T4 Polynucleotide Kinase and digested with XhoI. Following XhoI digestion of the double-stranded cDNA, producing XhoI sticky ends, a QIAEXII Gel Extraction kit (Beijing BioDev-Tech, Beijing, China) was used to recycle 0.5–4 Kb fragments. The recycled cDNA was preserved at −20°C. Then the cDNA was ligated into the pBluescript II SK(+) XR vector (Promega Corporation, Madison, WI, USA) in a 3:1 molar ratio with T4 DNA ligase at 4°C overnight. To reduce the redundancy and avoid the underrepresentation of different transcript species, cDNA fragments with different fractionated sizes were balanced and subjected to library construction (9,10). Prior to transformation, mixing of all ligated products with microporous membranes was performed to remove salt ions. Subsequently the products were transformed into 5×107/ml DH10B competent cells (Thermo Fisher Scientific, Inc.), plated on agar plates (10 cm diameter) by pipetting the cells onto the middle of the plate and spreading, and monoclonal colonies were selected for PCR amplification. The inserted sequences in the plasmids were amplified by PCR using T3 primers (5′-ATTAACCCTCACTAAAGGGA-3′) and T7 primers (5′-TAATACGACTCACTATAGGG-3′). The total volume of PCR reaction mixture was 20 µl, containing 1 µl template, 10 µl 2XTaq MasterMix (CWBIO, Beijing, China), 1 µl T3 primers (10 pmol), 1 µl T7 primers (10 pmol) and 7 µl ddH2O (CWBIO). Cycling conditions were: 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 40 sec and 72°C for 60 sec, followed by 72°C for 5 min.

Bioinformatic analysis

cDNA clones were selected randomly from the cDNA library and the vector sequences were trimmed from the raw sequence data using Vecscreen tool (www.ncbi.nlm.nih.gov/tools/vecscreen/) from the National Center for Biotechnology Information. The sequence of each EST was also edited, mainly to remove ambiguous bases and poor-quality sequences (nucleotide sequences <100 bp). All edited sequences were assembled into groups using SeqMan software version 8.0 (DNASTAR, Madison, WI, USA). The processed cDNA sequences were used to perform a BLAST search in the GenBank database to compare all available ESTs and genes to date (11). The Basic Local Alignment Search Tool (BLAST; blast.ncbi.nlm.nih.gov/Blast.cgi) results with P-values <1×10−8 were generally regarded as a significant match (12,13). A large-scale Unigene assembly of the ESTs was initiated to identify and functionally annotate as many unique transcripts as possible. BLAST analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and protein and nucleic acid databases was conducted for examination of biological functions. The ESTs homologous to known proteins were further annotated for Gene Ontology (GO; www.geneontology.org) terms and the GO analysis was carried out using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt; www.webgestalt.org/option.php) (14,15).

Results

Construction of cDNA library

Obtaining an adequate quantity of high quality mRNA initially is the key to yielding a sufficient quantity of first-strand full-length cDNA by reverse transcription. In the present study, total RNA was extracted from the tissues of earthworms. As shown in Fig. 1, 28s and 18s bands were clearly visible in the electrophoresis gel of total RNA, indicating that the total RNA was obtained. The optical density (OD)260/OD280 ratio for the total RNA was 2.04, well within the range of 1.8–2.1, indicating that the isolated total RNA was suitable for cDNA library construction.

Figure 1.

Figure 1.

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Result of the electrophoresis of total RNA. A total of 1,056 µg RNA was obtained from the tissues of earthworms. The optical density values were A260=0.880 and A280=0.430; A260/A280=2.04.

Once the double-stranded cDNA was synthesized as described in the Materials and methods, the present study determined the size distribution of the products. Diffuse strips between 0.5–4.0 kb could be detected by 1% agarose gel electrophoresis, which indicated that double-stranded cDNAs were successfully synthesized. A cDNA library of 4.12×105 clones was obtained and half of the bacteria were cultured for amplification, which produced a total of 1.4×1011 clones. Several colonies were selected, and the inserted sequences in the plasmids were amplified by PCR using T3 and T7 primers. The PCR products were detected using 1% agarose gel electrophoresis as clear bands. No nonspecific bands were identified and the recombinant rate was 97% (Fig. 2).

Figure 2.

Figure 2.

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Amplified inserts of cDNA clones from the constructed cDNA library. M, DNA marker; Lanes 1–32, insert cDNA (>500 bp) clones from phage plaques.

To investigate the quality of the full-length cDNA library, the lengths of the cDNA inserts were assessed. Sequence outputs were manually edited to remove vector and ambiguous sequences. Then, the sequence data of the cDNA clones obtained by random partial sequencing were searched in the NCBI GenBank using BLAST to identify similarities with sequences in the nucleic acid databases. An evaluation of cDNA insert size and its distribution revealed a low level of insert size bias in the final cDNA library. The majority of the cDNA inserts were larger than 500 bp.

EST analysis

Instead of the amplified library, the primary cDNA library was used to generate ESTs to reduce the redundancy of cDNA clones. Following the removal of the redundant sequences and low-quality sequences (<100 bp), 1,148 effective sequences (>100 bp) from the total cDNA sequences were obtained. As shown in Fig. 3, 53 ESTs were 100–300 bp, 261 ESTs were 300–500 bp, 828 ESTs were 500–700 bp and 6 ESTs were larger than 700 bp. Taken together, 1,148 ESTs were larger than 100 bp. Among them, the shortest sequence was 100 bp, the longest was 718 bp and the average length was ~452 bp. Following sequencing, a homology BLAST search and assembling of the data, 368 singletons and 225 contigs were obtained out of the 1,140 high-quality ESTs, as shown in Table I. Additionally, a total of 593 individual ESTs were analyzed and 168 ESTs annotated in GenBank with nematode homology (Table I).

Figure 3.

Figure 3.

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Different groups of ESTs, separated by length. ESTs, expressed sequence tags.

Table I.

Summary of ESTs obtained from the cDNA library of earthworms.

ESTs Number
Total number of ESTs 1,256
Total length of ESTs (bp) 568,140
Average length of ESTs (bp) 452.34
Unique genes 593
Contigs 225
Singletons 368
Annotation 168
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ESTs, expressed sequence tags.

GO annotation and bioinformatic analysis

The cDNA functions were classified using the GO database into the three main categories of molecular functions, cell components and biological processes.

GO annotation of genes associated with molecular functions indicated that among the 168 ESTs, 46% (77/168 ESTs) were associated with growth and metabolic pathways, with the distribution of the 77 ESTs shown in Fig. 4A. Out of the 77 ESTs, 21% (16 ESTs) were associated with ‘proteolytic enzymes’, 16% (12 ESTs) with ‘protein ligases’, 14% (11 ESTs) with ‘oxido-reductases’, 13% (10 ESTs) with ‘energy release’, 10% (8 ESTs) with ‘signal transduction and cell communication’, 5% (4 ESTs) with ‘transport’ and only 3% (2 ESTs) with ‘post-translational modification’, ‘protein turnover’ and ‘chaperones’ (Fig. 4B).

Figure 4.

Figure 4.

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Distribution of GO molecular functions. (A) The distribution of 77 expressed sequence tags with molecular functions. (B) The classification of identified genes based on the relevant molecular functions. GO, Gene Oncology.

Cellular components associated with the cDNAs included ‘myosin’, the ‘citrate lyase compound’, the ‘mitochondrial inner membrane translocase compound’, ‘microtubules’, the ‘mitochondrial inner membrane’ and ‘nucleosomes’. The proportions of cellular components are presented in Fig. 5. It can be observed that the proportion of ‘myosin’ among cellular components was the largest. Myosin, an actin-dependent molecular motor, is involved in a number of important functions in earthworms. In particular, the myosin network can drive movement and support different moving speeds of earthworms. This specific feature is closely related to the free moving ability of earthworms (16).

Figure 5.

Figure 5.

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Distribution of GO cellular components. GO, Gene Oncology.

Regarding biological processes, known genes were determined as those presenting significant matches to protein sequences with known functions in non-redundant nucleotide databases. According to these biological functions, the biological processes component was divided into different functions including ‘larval development’ (46%), ‘changes of cell morphogenesis’ (6%), ‘the process of cytokinesis’ (12%), ‘post-translational protein modification’ (15%), ‘stress response’ (5%), ‘cell redox homeostasis’ (2%), ‘protein polymerization’ (2%) and ‘protein catabolism’ (12%), as shown in Fig. 6. It was concluded from the above data that promoting growth was considered to be an important biological function of genes associated with biological processes.

Figure 6.

Figure 6.

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Classification of identified genes based on relevant biological processes.

KEGG pathway annotation

KEGG is a collection of online databases describing pathways associated with biochemical, genomic and enzymatic processes. Furthermore, it provides annotations of biochemical pathways for the species in which the genome has been sequenced (17). In this analysis, proteins are not viewed as individual gene products but are organized into pathways and networks according to their biological function(s). In the present study, from the data in Table II, 15 of the 168 ESTs were revealed to be involved in metabolism. Notably, 4 of the 15 ESTs (27%) were involved in the ubiquitin-mediated proteolysis pathway, which was the most represented. The glutathione metabolism (3 ESTs) and arachidonic acid metabolism (2 ESTs) pathways were the second and third most represented pathways, respectively. Additionally, chondroitin sulfate biosynthesis, heparan sulfate biosynthesis, riboflavin metabolism, selenoamino acid metabolism, γ-hexachlorocyclohexane degradation, and the fructose and mannose metabolism pathways were also represented.

Table II.

Pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database.

Pathway Count P-value Q-value Protein Input symbol
Ubiquitin-mediated proteolysis 4 1.12×10−5 1.73×10−4 ubc-14;ubc-13; ubc-18;let-70 Y87G2A.9;Y54G2A.31; R01H2.6;M7.1
Glutathione metabolism 3 2.55×10−5 2.63×10−4 W07G4.4;F26E4.12; C11E4.1 W07G4.4;F26E4.12; C11E4.1
Arachidonic acid metabolism 2 2.01×10−4 1.25×10−3 F26E4.12; C11E4.1 F26E4.12; C11E4.1
Chondroitin sulfate biosynthesis 1 6.54×10−3 2.03×10−2 sqv-8 ZK1307.5
Heparan sulfate biosynthesis 1 1.14×10−2 2.75×10−2 sqv-8 ZK1307.5
Riboflavin metabolism 1 1.14×10−2 2.75×10−2 F02E9.7 F02E9.7
Selenoamino acid metabolism 1 3.39×10−2 4.62×10−2 seld-1 Y45F10A.4
γ-hexachlorocyclohexane degradation 1 4.02×10−2 4.62×10−2 F02E9.7 F02E9.7
Fructose and mannose metabolism 1 4.18×10−2 4.62×10−2 R04B5.5 R04B5.5
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Mutual relationship between 287 genes

Relationships among the 168 ESTs were analyzed using the STRING database, and a functional association network was determined with 287 nodes, as shown in Table III. Each node corresponds to a gene and each (weighted) edge represents the evidence of a functional association between the gene pair. Predicted potential regulators are presented in Fig. 7. It can be seen from the STRING results that the most associated nodes (blue) included the 19 genes: Ribosomal protein L (rpl)-1, ribosomal protein S (rps)-0, rpl-4, rpl-5, rps-13, rps-2, acidic ribosomal protein (rla)-1, translocon-associated protein-4, transcription factor BTF3 homolog, rpl-7A, iff-1, rps-17, elongation factor 1α3, polyadenylate-binding protein-1, rps-28, translationally-controlled tumor protein homolog-1, rpl-18, rps-4 and adenosine triphosphate synthase subunit β mitochondrial precursor-2. Of note, the genes rpl-4, rps-13, rpl-5, rpl-1, rps-0, rla-1 and rpl-7A served critical roles in overall gene expression networks.

Table III.

Relationships between the 287 associated genes, including several important parameters.

node1 node2 node1_string_id node2_string_id node1_external_id node2_external_id coexpression combined_score
rpl-5 atp-2 502,561 496,479 F54C9.5.1 C34E10.6.3 0.866 0.871
rpl-18 snr-2 510,406 508,903 Y45F10D.12.2 W08E3.1 0.426 0.468
trap-4 rps-2 511,330 497,379 Y56A3A.21.2 C49H3.11.1 0.243 0.919
sod-2 daf-21 498,735 497,248 F10D11.1.1 C47E8.5.1 0 0.572
rla-2 rps-4 511,588 510,280 Y62E10A.1.1 Y43B11AR.4.2 0.981 0.998
rpl-1 drs-1 511,889 493,869 Y71F9AL.13a.1 B0464.1.1 0.492 0.523
skr-1 sdhd-1 501,798 500,588 F46A9.5.3 F33A8.5.2 0 0.587
prdx-6 sod-2 509,858 498,735 Y38C1AA.11 F10D11.1.1 0.233 0.726
act-4 rps-0 505,013 493,798 M03F4.2a B0393.1.1 0.426 0.425
daf-21 rps-13 497,248 495,308 C47E8.5.1 C16A3.9.1 0.402 0.474
rpl-1 rpl-4 511,889 493,528 Y71F9AL.13a.1 B0041.4.1 0.8 0.999
cyc-1 atp-2 497,719 496,479 C54G4.8.1 C34E10.6.3 0.849 0.873
rla-1 act-4 509,817 505,013 Y37E3.7.2 M03F4.2a 0.538 0.538
rpl-7A rps-17 509,604 506,860 Y24D9A.4a T08B2.10.1 0.872 0.923
rps-28 rpl-5 510,228 502,561 Y41D4B.5.2 F54C9.5.1 0.77 0.887
rpl-18 cyc-1 510,406 497,719 Y45F10D.12.2 C54G4.8.1 0.387 0.411
tpi-1 rps-0 509,457 493,798 Y17G7B.7.2 B0393.1.1 0.477 0.49
daf-21 drs-1 497,248 493,869 C47E8.5.1 B0464.1.1 0.281 0.543
lsm-3 rla-2 511,591 511,588 Y62E10A.12.2 Y62E10A.1.1 0 0.408
rpl-1 rps-4 511,889 510,280 Y71F9AL.13a.1 Y43B11AR.4.2 0.819 0.989
tpi-1 atp-2 509,457 496,479 Y17G7B.7.2 C34E10.6.3 0.448 0.877
lev-11 unc-60 509,147 496,717 Y105E8B.1d C38C3.5b.1 0 0.431
rpl-5 icd-1 502,561 497,806 F54C9.5.1 C56C10.8.1 0.864 0.87
rla-2 pab-1 511,588 509,182 Y62E10A.1.1 Y106G6H.2a.4 0.102 0.904
unc-54 mlc-3 498,853 498,666 F11C3.3.1 F09F7.2a.2 0.452 0.452
alp-1 cyc-1 507,123 497,719 T11B7.4d C54G4.8.1 0 0.409
mca-3 cmd-1 511,739 507,723 Y67D8C.10b T21H3.3.1 0 0.581
cyc-1 rps-2 497,719 497,379 C54G4.8.1 C49H3.11.1 0.579 0.579
rpl-1 daf-21 511,889 497,248 Y71F9AL.13a.1 C47E8.5.1 0.251 0.415
rla-2 iff-1 511,588 506,602 Y62E10A.1.1 T05G5.10 0.436 0.521
rpl-1 rla-1 511,889 509,817 Y71F9AL.13a.1 Y37E3.7.2 0.794 0.997
tpi-1 vha-4 509,457 506,234 Y17G7B.7.2 T01H3.1.1 0.483 0.483
vha-4 atp-2 506,234 496,479 T01H3.1.1 C34E10.6.3 0.304 0.724
tpi-1 sod-2 509,457 498,735 Y17G7B.7.2 F10D11.1.1 0.213 0.84
rps-4 act-4 510,280 505,013 Y43B11AR.4.2 M03F4.2a 0.429 0.469
rpl-18 rps-2 510,406 497,379 Y45F10D.12.2 C49H3.11.1 0.999 0.999
rpl-18 rps-13 510,406 495,308 Y45F10D.12.2 C16A3.9.1 0.999 0.999
rps-17 tct-1 506,860 499,979 T08B2.10.1 F25H2.11.2 0.874 0.884
nmy-1 mlc-4 502,225 497,828 F52B10.1 C56G7.1.2 0 0.698
drs-1 rpl-4 493,869 493,528 B0464.1.1 B0041.4.1 0.581 0.74
rpl-5 rps-0 502,561 493,798 F54C9.5.1 B0393.1.1 0.999 0.999
sdhd-1 cey-1 500,588 500,586 F33A8.5.2 F33A8.3.2 0.224 0.624
rpl-18 rps-28 510,406 510,228 Y45F10D.12.2 Y41D4B.5.2 0.78 0.928
rps-28 rps-13 510,228 495,308 Y41D4B.5.2 C16A3.9.1 0.794 0.979
pab-1 rpl-5 509,182 502,561 Y106G6H.2a.4 F54C9.5.1 0.762 0.978
rps-4 icd-1 510,280 497,806 Y43B11AR.4.2 C56C10.8.1 0.826 0.833
eft-3 atp-2 505,324 496,479 R03G5.1a.2 C34E10.6.3 0.478 0.602
iff-1 rps-0 506,602 493,798 T05G5.10 B0393.1.1 0.604 0.623
rpl-18 pab-1 510,406 509,182 Y45F10D.12.2 Y106G6H.2a.4 0.522 0.95
iff-1 rpl-4 506,602 493,528 T05G5.10 B0041.4.1 0.603 0.656
rla-2 rpl-18 511,588 510,406 Y62E10A.1.1 Y45F10D.12.2 0.967 0.997
crt-1 rpl-5 509,852 502,561 Y38A10A.5.1 F54C9.5.1 0.452 0.465
rps-28 tpi-1 510,228 509,457 Y41D4B.5.2 Y17G7B.7.2 0.278 0.451
rps-4 iff-1 510,280 506,602 Y43B11AR.4.2 T05G5.10 0.64 0.888
icd-1 rps-13 497,806 495,308 C56C10.8.1 C16A3.9.1 0.856 0.856
trap-4 rla-1 511,330 509,817 Y56A3A.21.2 Y37E3.7.2 0.176 0.923
nmy-1 unc-54 502,225 498,853 F52B10.1 F11C3.3.1 0.102 0.565
trap-4 rps-13 511,330 495,308 Y56A3A.21.2 C16A3.9.1 0.148 0.942
iff-1 rps-13 506,602 495,308 T05G5.10 C16A3.9.1 0.614 0.614
maoc-1 ftn-2 498,218 497,902 E04F6.3 D1037.3.4 0.997 0.996
rpl-18 eft-3 510,406 505,324 Y45F10D.12.2 R03G5.1a.2 0.428 0.938
exos-2 cpf-1 512,052 500,247 Y73B6BL.3 F28C6.3 0.403 0.403
rla-2 rla-1 511,588 509,817 Y62E10A.1.1 Y37E3.7.2 0.999 0.999
rla-2 eft-3 511,588 505,324 Y62E10A.1.1 R03G5.1a.2 0.104 0.91
iff-1 rps-2 506,602 497,379 T05G5.10 C49H3.11.1 0.696 0.903
rpl-7A rpl-4 509,604 493,528 Y24D9A.4a B0041.4.1 0.955 0.996
tpi-1 daf-21 509,457 497,248 Y17G7B.7.2 C47E8.5.1 0.313 0.404
crt-1 rps-0 509,852 493,798 Y38A10A.5.1 B0393.1.1 0.402 0.462
rpl-7A iff-1 509,604 506,602 Y24D9A.4a T05G5.10 0.477 0.536
pab-1 rps-17 509,182 506,860 Y106G6H.2a.4 T08B2.10.1 0.217 0.936
tpi-1 rps-17 509,457 506,860 Y17G7B.7.2 T08B2.10.1 0.341 0.412
act-4 rpl-4 505,013 493,528 M03F4.2a B0041.4.1 0.471 0.572
crt-1 rps-2 509,852 497,379 Y38A10A.5.1 C49H3.11.1 0.507 0.523
rpl-7A tct-1 509,604 499,979 Y24D9A.4a F25H2.11.2 0.793 0.794
exos-2 lsm-3 512,052 511,591 Y73B6BL.3 Y62E10A.12.2 0.164 0.449
rla-1 icd-1 509,817 497,806 Y37E3.7.2 C56C10.8.1 0.835 0.835
crt-1 cmd-1 509,852 507,723 Y38A10A.5.1 T21H3.3.1 0.116 0.609
pab-1 atp-2 509,182 496,479 Y106G6H.2a.4 C34E10.6.3 0.432 0.432
inf-1 rpl-5 503,072 502,561 F57B9.6a.3 F54C9.5.1 0.409 0.409
trap-4 rps-0 511,330 493,798 Y56A3A.21.2 B0393.1.1 0.174 0.911
rpl-18 atp-2 510,406 496,479 Y45F10D.12.2 C34E10.6.3 0.476 0.659
rps-2 drs-1 497,379 493,869 C49H3.11.1 B0464.1.1 0.664 0.663
rpl-5 drs-1 502,561 493,869 F54C9.5.1 B0464.1.1 0.332 0.403
rpl-5 daf-21 502,561 497,248 F54C9.5.1 C47E8.5.1 0.559 0.676
rpl-18 rpl-5 510,406 502,561 Y45F10D.12.2 F54C9.5.1 0.999 0.999
trap-4 rpl-5 511,330 502,561 Y56A3A.21.2 F54C9.5.1 0.273 0.949
snr-2 rps-0 508,903 493,798 W08E3.1 B0393.1.1 0.35 0.414
unc-54 daf-21 498,853 497,248 F11C3.3.1 C47E8.5.1 0 0.674
rpl-18 rps-0 510,406 493,798 Y45F10D.12.2 B0393.1.1 0.999 0.999
rpl-7A icd-1 509,604 497,806 Y24D9A.4a C56C10.8.1 0.737 0.756
tpi-1 rps-2 509,457 497,379 Y17G7B.7.2 C49H3.11.1 0.484 0.521
qua-1 nas-4 506,538 494,442 T05C12.10 C05D11.6 0.42 0.419
try-1 cpn-1 513,205 501,580 ZK546.15 F43G9.9.1 0.408 0.408
rla-1 iff-1 509,817 506,602 Y37E3.7.2 T05G5.10 0.551 0.691
rpl-18 iff-1 510,406 506,602 Y45F10D.12.2 T05G5.10 0.564 0.577
pab-1 eft-3 509,182 505,324 Y106G6H.2a.4 R03G5.1a.2 0.398 0.708
abcf-2 rps-2 508,270 497,379 T27E9.7.1 C49H3.11.1 0.268 0.452
tct-1 rps-0 499,979 493,798 F25H2.11.2 B0393.1.1 0.987 0.987
alp-1 daf-21 507,123 497,248 T11B7.4d C47E8.5.1 0 0.998
daf-21 rps-0 497,248 493,798 C47E8.5.1 B0393.1.1 0.44 0.445
lev-11 act-4 509,147 505,013 Y105E8B.1d M03F4.2a 0.244 0.441
daf-21 atp-2 497,248 496,479 C47E8.5.1 C34E10.6.3 0.415 0.745
rla-1 tct-1 509,817 499,979 Y37E3.7.2 F25H2.11.2 0.998 0.999
rps-17 iff-1 506,860 506,602 T08B2.10.1 T05G5.10 0.569 0.678
rps-28 rla-1 510,228 509,817 Y41D4B.5.2 Y37E3.7.2 0.785 0.963
try-1 cyp-31A2 513,205 503,484 ZK546.15 H02I12.8 0.543 0.543
rpl-1 rpl-5 511,889 502,561 Y71F9AL.13a.1 F54C9.5.1 0.799 0.995
rps-28 rps-0 510,228 493,798 Y41D4B.5.2 B0393.1.1 0.76 0.966
act-4 unc-54 505,013 498,853 M03F4.2a F11C3.3.1 0.168 0.474
rpl-5 tct-1 502,561 499,979 F54C9.5.1 F25H2.11.2 0.996 0.996
rla-1 rps-13 509817 495,308 Y37E3.7.2 C16A3.9.1 0.999 0.999
rpl-1 rps-17 511,889 5068,60 Y71F9AL.13a.1 T08B2.10.1 0.799 0.993
rpl-1 rla-2 511,889 511,588 Y71F9AL.13a.1 Y62E10A.1.1 0.772 0.996
cey-1 cpf-1 500,586 500,247 F33A8.3.2 F28C6.3 0 0.904
pab-1 rps-13 509,182 495,308 Y106G6H.2a.4 C16A3.9.1 0.327 0.928
sqv-8 ret-1 512,932 508,785 ZK1307.5 W06A7.3f 0 0.579
rps-28 rpl-7A 510,228 509,604 Y41D4B.5.2 Y24D9A.4a 0.752 0.838
icd-1 rps-0 497,806 493,798 C56C10.8.1 B0393.1.1 0.875 0.88
rla-1 tpi-1 509,817 509,457 Y37E3.7.2 Y17G7B.7.2 0.501 0.501
icd-1 rps-2 497,806 497,379 C56C10.8.1 C49H3.11.1 0.913 0.913
sod-2 atp-2 498,735 496,479 F10D11.1.1 C34E10.6.3 0.271 0.595
hum-5 arx-6 506,256 496,567 T02C12.1 C35D10.16 0 0.421
rla-1 eft-3 509,817 505,324 Y37E3.7.2 R03G5.1a.2 0.189 0.92
iff-1 rpl-5 506,602 502,561 T05G5.10 F54C9.5.1 0.58 0.61
atp-2 rpl-4 496,479 493,528 C34E10.6.3 B0041.4.1 0.828 0.835
rpl-5 rps-13 502,561 495,308 F54C9.5.1 C16A3.9.1 0.999 0.999
unc-87 unc-60 498,495 496,717 F08B6.4a C38C3.5b.1 0 0.407
mlc-3 unc-87 498,666 498,495 F09F7.2a.2 F08B6.4a 0.516 0.516
gpd-3 atp-2 504,646 496,479 K10B3.7.2 C34E10.6.3 0.214 0.523
rpl-5 rps-2 502,561 497,379 F54C9.5.1 C49H3.11.1 0.999 0.999
tct-1 icd-1 499,979 497,806 F25H2.11.2 C56C10.8.1 0.746 0.751
rpl-1 rps-2 511,889 497,379 Y71F9AL.13a.1 C49H3.11.1 0.798 0.995
pab-1 icd-1 509,182 497,806 Y106G6H.2a.4 C56C10.8.1 0.458 0.458
rps-4 rps-13 510,280 495,308 Y43B11AR.4.2 C16A3.9.1 0.999 0.999
rps-4 rpl-7A 510,280 509,604 Y43B11AR.4.2 Y24D9A.4a 0.949 0.989
pab-1 rpl-4 509,182 493,528 Y106G6H.2a.4 B0041.4.1 0.69 0.966
rps-17 rps-0 506,860 493,798 T08B2.10.1 B0393.1.1 0.999 0.999
rps-17 icd-1 506,860 497,806 T08B2.10.1 C56C10.8.1 0.856 0.861
rpl-7A drs-1 509,604 493,869 Y24D9A.4a B0464.1.1 0.451 0.519
rpl-18 rla-1 510,406 509,817 Y45F10D.12.2 Y37E3.7.2 0.996 0.999
rpl-1 rpl-18 511,889 510,406 Y71F9AL.13a.1 Y45F10D.12.2 0.832 0.993
daf-21 unc-60 497,248 496,717 C47E8.5.1 C38C3.5b.1 0 0.911
sqv-8 cyp-31A2 512,932 503,484 ZK1307.5 H02I12.8 0.411 0.411
act-4 rps-13 505,013 495,308 M03F4.2a C16A3.9.1 0.53 0.53
tct-1 rps-13 499,979 495,308 F25H2.11.2 C16A3.9.1 0.996 0.996
crt-1 eft-3 509,852 505,324 Y38A10A.5.1 R03G5.1a.2 0.573 0.573
rps-2 daf-21 497,379 497,248 C49H3.11.1 C47E8.5.1 0.658 0.721
let-70 cyp-31A2 505,189 503,484 M7.1 H02I12.8 0 0.522
rps-13 drs-1 495,308 493,869 C16A3.9.1 B0464.1.1 0.528 0.528
crt-1 pdi-3 509,852 503,539 Y38A10A.5.1 H06O01.1.3 0.726 0.739
rpl-18 rpl-7A 510,406 509,604 Y45F10D.12.2 Y24D9A.4a 0.925 0.984
rps-13 rps-0 495,308 493,798 C16A3.9.1 B0393.1.1 0.999 0.999
rps-4 rpl-4 510,280 493,528 Y43B11AR.4.2 B0041.4.1 0.999 0.999
rps-4 rps-2 510,280 497,379 Y43B11AR.4.2 C49H3.11.1 0.999 0.999
rla-1 rpl-7A 509,817 509,604 Y37E3.7.2 Y24D9A.4a 0.872 0.912
rps-28 rps-17 510,228 506,860 Y41D4B.5.2 T08B2.10.1 0.805 0.989
rpl-18 tct-1 510,406 499,979 Y45F10D.12.2 F25H2.11.2 0.963 0.966
rla-2 rps-28 511,588 510,228 Y62E10A.1.1 Y41D4B.5.2 0.799 0.879
eft-3 rpl-4 505,324 493,528 R03G5.1a.2 B0041.4.1 0.713 0.97
tpi-1 rpl-4 509,457 493,528 Y17G7B.7.2 B0041.4.1 0.489 0.498
tpi-1 gpd-3 509,457 504,646 Y17G7B.7.2 K10B3.7.2 0.608 0.998
unc-54 unc-60 498,853 496,717 F11C3.3.1 C38C3.5b.1 0 0.587
tpi-1 cey-1 509,457 500,586 Y17G7B.7.2 F33A8.3.2 0 0.403
hum-5 unc-54 506,256 498,853 T02C12.1 F11C3.3.1 0 0.54
rps-4 pab-1 510,280 509,182 Y43B11AR.4.2 Y106G6H.2a.4 0.344 0.955
rla-2 icd-1 511,588 497,806 Y62E10A.1.1 C56C10.8.1 0.809 0.81
rpl-7A rpl-5 509,604 502,561 Y24D9A.4a F54C9.5.1 0.934 0.994
rps-17 eft-3 506,860 505,324 T08B2.10.1 R03G5.1a.2 0.187 0.916
rpl-18 rpl-4 510,406 493,528 Y45F10D.12.2 B0041.4.1 0.999 0.999
rla-2 daf-21 511,588 497,248 Y62E10A.1.1 C47E8.5.1 0.38 0.548
rps-4 tct-1 510,280 499,979 Y43B11AR.4.2 F25H2.11.2 0.941 0.946
rps-4 rps-28 510,280 510,228 Y43B11AR.4.2 Y41D4B.5.2 0.76 0.971
rla-2 rpl-5 511,588 502,561 Y62E10A.1.1 F54C9.5.1 0.967 0.999
rps-4 rpl-5 510,280 502,561 Y43B11AR.4.2 F54C9.5.1 0.999 0.999
rla-1 pab-1 509,817 509,182 Y37E3.7.2 Y106G6H.2a.4 0.29 0.929
rla-2 rps-2 511,588 497,379 Y62E10A.1.1 C49H3.11.1 0.991 0.999
iff-1 tct-1 506,602 499,979 T05G5.10 F25H2.11.2 0.483 0.483
rla-1 rps-2 509,817 497,379 Y37E3.7.2 C49H3.11.1 0.997 0.999
rpl-1 pab-1 511,889 509,182 Y71F9AL.13a.1 Y106G6H.2a.4 0.237 0.918
rps-28 rps-2 510,228 497,379 Y41D4B.5.2 C49H3.11.1 0.732 0.981
rpl-5 rpl-4 502,561 493,528 F54C9.5.1 B0041.4.1 0.999 0.999
rps-4 rps-17 510,280 506,860 Y43B11AR.4.2 T08B2.10.1 0.995 0.999
rpl-18 rps-17 510,406 506,860 Y45F10D.12.2 T08B2.10.1 0.956 0.996
rpl-1 rpl-7A 511,889 509,604 Y71F9AL.13a.1 Y24D9A.4a 0.796 0.96
inf-1 rps-0 503,072 493,798 F57B9.6a.3 B0393.1.1 0.414 0.433
alp-1 unc-87 507,123 498,495 T11B7.4d F08B6.4a 0.413 0.421
rla-1 rpl-4 509,817 493,528 Y37E3.7.2 B0041.4.1 0.997 0.999
rps-2 rps-13 497,379 495,308 C49H3.11.1 C16A3.9.1 0.998 0.999
pdi-3 rps-2 503,539 497,379 H06O01.1.3 C49H3.11.1 0.457 0.457
cpl-1 cpf-1 506,335 500,247 T03E6.7.2 F28C6.3 0 0.408
gyg-1 cey-1 502,889 500,586 F56B6.4a F33A8.3.2 0 0.55
hum-5 nmy-1 506,256 502,225 T02C12.1 F52B10.1 0 0.544
gpd-3 sod-2 504,646 498,735 K10B3.7.2 F10D11.1.1 0 0.588
rps-4 tpi-1 510,280 509,457 Y43B11AR.4.2 Y17G7B.7.2 0.444 0.444
rla-2 trap-4 511,588 511,330 Y62E10A.1.1 Y56A3A.21.2 0.076 0.916
rps-28 rpl-4 510,228 493,528 Y41D4B.5.2 B0041.4.1 0.756 0.847
drs-1 rps-0 493,869 493,798 B0464.1.1 B0393.1.1 0.464 0.494
sdhd-1 cyc-1 500,588 497,719 F33A8.5.2 C54G4.8.1 0.382 0.54
rpl-1 icd-1 511,889 497,806 Y71F9AL.13a.1 C56C10.8.1 0.777 0.787
rps-17 rps-13 506,860 495,308 T08B2.10.1 C16A3.9.1 0.997 0.999
rla-1 rps-0 509,817 493,798 Y37E3.7.2 B0393.1.1 0.999 0.999
daf-21 rpl-4 497,248 493,528 C47E8.5.1 B0041.4.1 0.59 0.634
tpi-1 cyc-1 509,457 497,719 Y17G7B.7.2 C54G4.8.1 0.442 0.444
trap-4 rpl-4 511,330 493,528 Y56A3A.21.2 B0041.4.1 0.308 0.951
rpl-1 rps-28 511,889 510,228 Y71F9AL.13a.1 Y41D4B.5.2 0.793 0.939
rps-28 tct-1 510,228 499,979 Y41D4B.5.2 F25H2.11.2 0.683 0.683
eft-3 rpl-5 505,324 502,561 R03G5.1a.2 F54C9.5.1 0.701 0.974
sod-2 cyc-1 498,735 497,719 F10D11.1.1 C54G4.8.1 0.456 0.604
pdi-3 phy-2 503,539 500,798 H06O01.1.3 F35G2.4.1 0 0.4
rpl-18 daf-21 510,406 497,248 Y45F10D.12.2 C47E8.5.1 0.387 0.401
rpl-1 tct-1 511,889 499,979 Y71F9AL.13a.1 F25H2.11.2 0.751 0.757
rpl-1 iff-1 511,889 506,602 Y71F9AL.13a.1 T05G5.10 0.612 0.624
rpl-1 tpi-1 511,889 509,457 Y71F9AL.13a.1 Y17G7B.7.2 0.396 0.614
rps-0 rpl-4 493,798 493,528 B0393.1.1 B0041.4.1 0.999 0.999
eft-3 rps-13 505,324 495,308 R03G5.1a.2 C16A3.9.1 0.362 0.956
act-4 unc-60 505,013 496,717 M03F4.2a C38C3.5b.1 0 0.863
crt-1 pab-1 509,852 509,182 Y38A10A.5.1 Y106G6H.2a.4 0.425 0.453
tct-1 atp-2 499,979 496,479 F25H2.11.2 C34E10.6.3 0.575 0.583
rps-13 rpl-4 495,308 493,528 C16A3.9.1 B0041.4.1 0.998 0.999
rps-2 rpl-4 497,379 493,528 C49H3.11.1 B0041.4.1 0.999 0.999
rla-2 rps-17 511,588 506,860 Y62E10A.1.1 T08B2.10.1 0.997 0.999
rla-1 rps-17 509,817 506,860 Y37E3.7.2 T08B2.10.1 0.966 0.997
rps-17 rps-2 506,860 497,379 T08B2.10.1 C49H3.11.1 0.969 0.999
cyc-1 eat-6 497,719 493,780 C54G4.8.1 B0365.3.2 0.295 0.425
trap-4 rpl-18 511,330 510,406 Y56A3A.21.2 Y45F10D.12.2 0.37 0.956
tpi-1 rps-13 509,457 495,308 Y17G7B.7.2 C16A3.9.1 0.51 0.556
rla-2 rps-0 511,588 493,798 Y62E10A.1.1 B0393.1.1 0.994 0.999
icd-1 drs-1 497,806 493,869 C56C10.8.1 B0464.1.1 0.403 0.509
sod-2 ftn-2 498,735 497,902 F10D11.1.1 D1037.3.4 0 0.494
pab-1 cey-1 509,182 500,586 Y106G6H.2a.4 F33A8.3.2 0.317 0.447
pab-1 rps-0 509,182 493,798 Y106G6H.2a.4 B0393.1.1 0.546 0.958
rla-1 rpl-5 509,817 502,561 Y37E3.7.2 F54C9.5.1 0.996 0.999
rps-17 rpl-4 506,860 493,528 T08B2.10.1 B0041.4.1 0.995 0.999
lsm-3 rpl-4 511,591 493,528 Y62E10A.12.2 B0041.4.1 0.126 0.622
tct-1 rpl-4 499,979 493,528 F25H2.11.2 B0041.4.1 0.919 0.926
icd-1 daf-21 497,806 497,248 C56C10.8.1 C47E8.5.1 0.439 0.478
rpl-1 rps-0 511,889 493,798 Y71F9AL.13a.1 B0393.1.1 0.808 0.992
rpl-1 trap-4 511,889 511,330 Y71F9AL.13a.1 Y56A3A.21.2 0.258 0.92
rps-4 eft-3 510,280 505,324 Y43B11AR.4.2 R03G5.1a.2 0.402 0.939
rps-4 rla-1 510,280 509,817 Y43B11AR.4.2 Y37E3.7.2 0.997 0.999
atp-2 rps-0 496,479 493,798 C34E10.6.3 B0393.1.1 0.432 0.763
rpl-7A rps-2 509,604 497,379 Y24D9A.4a C49H3.11.1 0.914 0.978
rla-2 rpl-7A 511,588 509,604 Y62E10A.1.1 Y24D9A.4a 0.936 0.958
rps-28 icd-1 510,228 497,806 Y41D4B.5.2 C56C10.8.1 0.772 0.772
rpl-18 icd-1 510,406 497,806 Y45F10D.12.2 C56C10.8.1 0.857 0.863
inf-1 rps-2 503,072 497,379 F57B9.6a.3 C49H3.11.1 0.395 0.417
tpi-1 tct-1 509,457 499,979 Y17G7B.7.2 F25H2.11.2 0.428 0.476
rps-17 rpl-5 506,860 502,561 T08B2.10.1 F54C9.5.1 0.972 0.998
snr-2 cpf-1 508,903 500,247 W08E3.1 F28C6.3 0.245 0.919
pab-1 rps-2 509,182 497,379 Y106G6H.2a.4 C49H3.11.1 0.704 0.968
skr-1 pas-2 501,798 497,931 F46A9.5.3 D1054.2.1 0 0.899
rla-2 act-4 511,588 505,013 Y62E10A.1.1 M03F4.2a 0.508 0.508
icd-1 rpl-4 497,806 493,528 C56C10.8.1 B0041.4.1 0.826 0.858
eft-3 rps-2 505,324 497,379 R03G5.1a.2 C49H3.11.1 0.66 0.971
rpl-1 rps-13 511,889 495,308 Y71F9AL.13a.1 C16A3.9.1 0.8 0.997
rps-2 atp-2 497,379 496,479 C49H3.11.1 C34E10.6.3 0.943 0.947
unc-54 unc-87 498,853 498,495 F11C3.3.1 F08B6.4a 0.566 0.776
tct-1 rps-2 499,979 497,379 F25H2.11.2 C49H3.11.1 0.999 0.999
trap-4 rps-4 511,330 510,280 Y56A3A.21.2 Y43B11AR.4.2 0.245 0.921
rps-28 iff-1 510,228 506,602 Y41D4B.5.2 T05G5.10 0.432 0.635
rla-1 daf-21 509,817 497,248 Y37E3.7.2 C47E8.5.1 0.394 0.543
rps-4 crt-1 510,280 509,852 Y43B11AR.4.2 Y38A10A.5.1 0.491 0.5
rpl-18 crt-1 510,406 509,852 Y45F10D.12.2 Y38A10A.5.1 0.595 0.597
rps-2 rps-0 497,379 493,798 C49H3.11.1 B0393.1.1 0.999 0.999
lev-11 unc-87 509,147 498,495 Y105E8B.1d F08B6.4a 0.272 0.454
trap-4 rps-17 511,330 506,860 Y56A3A.21.2 T08B2.10.1 0.308 0.929
tct-1 daf-21 499,979 497,248 F25H2.11.2 C47E8.5.1 0.648 0.713
crt-1 rpl-4 509,852 493,528 Y38A10A.5.1 B0041.4.1 0.472 0.604
rpl-18 rps-4 510,406 510,280 Y45F10D.12.2 Y43B11AR.4.2 0.999 0.999
eft-3 rps-0 505,324 493,798 R03G5.1a.2 B0393.1.1 0.523 0.959
rpl-1 eft-3 511,889 505,324 Y71F9AL.13a.1 R03G5.1a.2 0.348 0.948
iff-1 icd-1 506,602 497,806 T05G5.10 C56C10.8.1 0.47 0.47
rpl-7A rps-13 509,604 495,308 Y24D9A.4a C16A3.9.1 0.944 0.979
eft-3 ftn-2 505,324 497,902 R03G5.1a.2 D1037.3.4 0 0.999
rps-4 drs-1 510,280 493,869 Y43B11AR.4.2 B0464.1.1 0.422 0.434
snr-2 cey-1 508,903 500,586 W08E3.1 F33A8.3.2 0 0.899
rpl-1 atp-2 511,889 496,479 Y71F9AL.13a.1 C34E10.6.3 0.144 0.403
rla-2 rpl-4 511,588 493,528 Y62E10A.1.1 B0041.4.1 0.995 0.999
act-2 act-4 506,426 505,013 T04C12.5 M03F4.2a 0 0.481
cyn-7 daf-21 512,151 497,248 Y75B12B.2.2 C47E8.5.1 0.359 0.408
rla-2 tct-1 511,588 499,979 Y62E10A.1.1 F25H2.11.2 0.96 0.963
rps-4 rps-0 510,280 493,798 Y43B11AR.4.2 B0393.1.1 0.999 0.999
rpl-18 drs-1 510,406 493,869 Y45F10D.12.2 B0464.1.1 0.408 0.474
atp-2 eat-6 496,479 493,780 C34E10.6.3 B0365.3.2 0.666 0.707
rpl-7A rps-0 509,604 493,798 Y24D9A.4a B0393.1.1 0.942 0.947
lsm-3 snr-2 511,591 508,903 Y62E10A.12.2 W08E3.1 0.323 0.625
rla-2 rps-13 511,588 495,308 Y62E10A.1.1 C16A3.9.1 0.996 0.999
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Figure 7.

Figure 7.

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Connectivity between the predicted regulators and the clusters in the STRING network: Experimentally-derived interactions (green), co-expression (navy blue), and co-occurrences (red) in the genomes are shown. Colored circles represent input genes.

Discussion

The present study reported on the construction of a high-quality cDNA library from tissues of the earthworm Eisenia fetida (Savigny, 1826), following preliminary analysis of ESTs, putative functional analysis of the ESTs and the gene expression pattern associated with the physiological functions of this organism. cDNA libraries are widely used to identify genes and splice variants and are considered to be a physical resource for the construction of full-length clones (18,19). In the present study, a cDNA library was utilized to provide a molecular resource for the analysis of genes involved in the specific biology of earthworms in terms of their development, survival, pathogenicity and virulence. There are two main factors to consider when assessing the quality of a cDNA library: Representation and cDNA lengths. According to Clarke-Carbon's formula (20), a cDNA library should contain at least 1.7×105 independent clones to ensure that 99% of low-abundance mRNAs will be represented in the library (21). Furthermore, the average length of the inserted cDNAs should be no less than 1.0 kb to ensure the integrity of cDNAs, indicating that in the present study the fragment sizes were effective for ensuring full-length cDNAs in the cDNA library. Since selection bias could favor the smaller cDNAs, the present study used fewer PCR cycles to minimize such bias as previously suggested (10). In addition, up to 25 PCR amplification cycles were used to generate an adequate amount of cDNA for cloning.

The generation of ESTs is an effective and unique approach in molecular studies as it allows for the analysis and measurement of gene expression, as well as simultaneous discovery of new genes. As each EST represents a copy of the functional part of a genome, the study of ESTs is believed to be a more effective way to discover functional genes (22). Furthermore, analysis of the expression of a large number of genes combined with the knowledge of their functions enables insight into the overall situation in terms of biological processes (23), for the current purposes in earthworms. In the present study, ~91% of the ESTs generated were sequences with known or putative functions, while the remainder represented unknown proteins or sequences with no similarities to those in the databases. Although close to 600 ESTs were reported, this is actually far from what could be considered as a ‘complete’ transcriptome (which usually includes between 15,000–20,000 to >100,000 ESTs). Therefore, the present characterization of this seemingly partial transcriptome may far from reflect the full transcriptomic profile of tissues in earthworms.

A comparison of the classification of ESTs with a C. elegans cDNA library based on their putative functions was conducted. Based on identification of clusters via GO analysis, 168 ESTs were matched to C. elegans genes by BLASTx. It is well known that earthworms serve significant roles in organic matter decomposition and mineral cycling, and thus are considered to be important contributors to soil fertility and humification processes (24). In the present study, hydrolytic enzyme activity, conjugating protein ligase activity, oxidation reduction and energy release activity of metabolic enzymes accounted for a large proportion of the molecular functions component. These molecular functions are considered to be a key part of the physiological functions of earthworms, which allow earthworms to survive in different soil environments.

Cell components, as part of GO annotation, are mainly categorized based on subcellular location (including cell cytoplasm, mitochondria, lysosomes, nucleus, microtubules, plasma membrane and myosin), which is highly important for the study of protein functions (25). The results of subcellular localization analysis can provide significant clues to aid the understanding of protein functions. In the present study, several cell components were determined through GO analysis of the 168 ESTs (annotated genes). It was evident that these cell components had strong associations with the regulation of gene expression during the biological development of earthworms, enabling the regeneration of the anterior portion, alterations in movement ability and tissue differentiation.

Due to the temporal specificity of gene expression and interactions with other gene products, the specific pathway undertaken, sequence of gene expression and expression pattern may ultimately change the effects of multiple pathways in earthworms (2). Therefore, the cDNA library and transcription profile of genes representative of fully developed adult earthworms may differ markedly to those of juvenile earthworms. In the present study, gene expression profiles representative of adult earthworm development were generated; however, gene expression profiling of juvenile earthworms was not performed, nor were analyses of the expected differential gene expression between juveniles and adults. In the present study, 168 individual ESTs of earthworms were analyzed in terms of KEGG pathway annotation, which identified 9 corresponding categories. Among them, glutathione metabolism is involved in antioxidant defense systems in Eisenia Andrei, and the associated enzymes are mainly identified in cytosolic fractions (26). Chondroitin sulfate and heparan sulfate biosynthesis are involved in biosynthesis pathways, which have important effects on growth and regeneration. In addition, heparan sulfate is also considered to be a type of anticoagulant, which may also be a function of arachidonic acid (27). Therefore, understanding the molecular function of earthworms may provide some basis for the treatment of thrombotic disorders.

Information on functional annotation and relevant biological interactions associated with a particular gene is available from many online resources. The gene network comprises a collection of genes that cooperate with each other to control the main biological processes. The STRING database suggested a functional context for earthworm lumbrokinase with unknown specificity. A previous study revealed that lumbrokinase and dilong administration can efficiently reduce the incidence of cardiac disease among nonsmokers exposed to second-hand smoke (28). In this regard, the discovery of genes and protein interactions in earthworms has provided a basis for further investigation into human diseases.

Acknowledgements

Not applicable.

Glossary

Abbreviations

EST

expressed sequence tags

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

Funding

The present study was supported by the National Natural Science Foundation of China (grant nos. 30472251, 30901821 and 81370312), the Natural Science Foundation for Young Scientists of Shanxi Province, China (grant no. 2010021035-2), Natural Science Foundation of Shanxi Province, China (grant no. 2015011113), International Scientific and Technological Cooperative Foundation of Shanxi Province, China (grant no. 201703D421023), the Fund for Shanxi ‘1331 Project’ Key Subjects Construction and the Fund for Shanxi Key Subjects Construction.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

BFY, BN and JX conceived and designed the experiments and bioinformatics analysis. CL and FXM performed bioinformatics analysis, and wrote the manuscript. XW, PYM, QZ and JBT performed the experiments. RG, ZZL, HLW and NLC contributed to designing the present study and revising the manuscript. JHW and GQS analyzed and interpreted the data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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