Complete Mitogenome of Cruznema tripartitum Confirms Highly Conserved Gene Arrangement within Family Rhabditidae

The specimen of C. tripartitum were originally extracted from soil in a chestnut orchard in Guangzhou, China (GPS coordinates: 23°56¢44²N, 114°42¢15²E). This species is a free-living bacterivore, and the population was established through pure culture using nematode growth medium (NGM) supplemented with Escherichia coli OP50 and maintained at 24°C.

An approximate number of 8,000 living individual nematodes were used for genomic DNA extraction using Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s protocol. The DNA concentration was examined by a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) using the Qubit 1X dsDNA HS Kit (Yeasen Biotech, Shanghai, China). A total of 200 ng DNA extraction was used for sequencing.

For library preparation, the TruSeq DNA Sample Prep Kit (Illumina Inc. San Diego, CA, USA) was used according to the manufacturer’s instructions. Library concentration and fragment size distribution were determined using the Qubit fluorometer and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was conducted by Personalbio, Ltd. (Shanghai, China) using an Illumina NovaSeq platform.

For quality control and trimming, reads less than 50 bp, Q-score less than 20, or number of n > 3 were removed by FASTP (Chen et al., 2018). The resultant reads were mapped to the reference mitogenomes of 32 nematode species and 11 insect species using NextGenMap program (Sedlazeck et al., 2013) with a similarity threshold of 0.3. The candidate raw reads that matching at least one direction in the alignment results are extracted by SAMtools (Li et al., 2009). Reads were assembled in NOVOPlasty (Dierckxsens et al., 2017) using seed-extend algorithm. Since no cox1 gene was available in GenBank for the genus Cruznema, a partial cox1 belonging to the closely related Pelodera strongyloides (GenBank accession number LR700242) was used as a “seed” sequence. A total of 554,098 reads were used for the final assembly that generated a circular DNA molecule.

After mitogenome assembly, protein-coding genes (PCGs) and non-coding region were predicted using the invertebrate mitochondrial code (genetic code 5) on the MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt et al., 2013). Secondary structures of transfer RNA (tRNA) genes were predicted using MiTFi (Jühling et al., 2012) as implemented in MITOS and depicted using the web server FORNA (http://rna.tbi.univie.ac.at/forna/) (Kerpedjiev et al., 2015).

Multiple alignments of amino acid sequences of 12 PCGs were carried out individually using TranslatorX (Abascal et al., 2010) under invertebrate mitochondrial genetic code and MAFFT approach. The concatenate sequence matrix was generated using Geneious (Biomatters, Auckland, New Zealand) and 12 PCG alignments were arranged in following order: nad6-nad5-nad4-nad4L-nad3-nad2-nad1-cytb-cox3-cox2-cox1-atp6. The phylogeny placement of the newly sequenced C. tripartitum was inferred by maximum likelihood (ML) method together with the full mitogenomes of 36 other nematode species. An isopod parasitic mermithid nematode Thaumamermis cosgrovei was selected as the outgroup. The phylogeny tree was constructed using RAxML 8.2.12 (Stamatakis et al., 2014) with MtZoa protein substitution model and under a total of 1,000 bootstrap replicates. The phylogeny analysis was performed in the CIPRES Science Gateway (Miller et al., 2010).

A total of ca.105 million reads (paired-end 150 bp) were generated and 99.04% of these reads were of high-quality (Q-score > 30). Approximately 10 GB raw data were mapped to the references, resulting in 0.53% mitochondrial reads; and these reads were subsequently used for mitogenome assembly.

The complete mitogenome of C. tripartitum (GenBank accession OM234676) is a 14,067 bp single circular DNA that contains 12 PCGs (atp6, cob, cox1-3, nad1-6, and nad4L), two ribosomal RNA genes, and 22 tRNA genes (Fig. 1). The atp8 gene was missing in the assembled mitogenome. All genes of C. tripartitum mitogenome are encoded in the same direction, which is a result similar to all other known chromadorean nematodes. The nucleotide composition is strongly biased toward A + T (78.7%), with 48.1% for T, 7.5% for C, 30.6% for A, and 13.8% for G. An intergenic non-coding region was found between trnA and trnP in a length of 696 bp. This region has much higher A + T content (87.1%) in comparison with any other part of mitogenome.

Figure 1

For PCGs, eight genes (cox1, cox2, cox3, nad2, nad3, nad5, nad6, and nad4L) use ATT as the start codon, while another two (atp6 and cob) start with ATA, and two (nad1 and nad4) start with TTG (Table 1). All PCGs are preferred to use TAA as their termination codon and have T-rich codons peference. A total of 22 tRNAs were recovered, ranging in size from 54 bp (trnK, trnC, trnP, trnS2, and trnV) to 62 bp (trnK) (Table 1 and Fig. 2). Two ribosomal RNA genes are located between trnE and trnS2 (rrnS, 697 bp) and between trnH and nad3 (rrnL, 957 bp), respectively (Table 1). The A + T contents of rrnS and rrnL are 76.8% (36.6% A and 40.2% T) and 81.1% (37.9% A and 43.2% T), respectively.

Figure 2

Maximum likelihood phylogenetic analysis of C. tripartitum was conducted using the translated amino acid data set of 12 PCGs (3,893 alignment characters) together with those of 36 other nematodes (32 chromadoreans and 4 enopleans; available in NCBI GenBank) as references (Fig. 3). In general, the phylogeny we revealed was congruent with previously published phylogeny inferred by rRNA genes (Blaxter et al., 1998; De Ley and Blaxter, 2004; Holterman et al., 2006; Meldal et al., 2007) or mitogenome (Kim et al., 2015, 2017). The family Rhabditidae is paraphyletic, containing a moderately supported major clade (BS = 87) and a separated Diploscapter coronatus sistering to several genera in Rhabditina, Tylenchina, and Spirurina. In the major clade of Rhabditidae, the newly sequenced C. tripartitum is sister to Oscheius chongmingensis, Litoditis marina, and species in the genus Caenorhabditis.

Figure 3

The analyses of gene arrangement suggested Rhabditidae representing by C. tripartitum, O. chongmingensis, L. marina, Diplocapter coronatus and genus Caenorhabditis are highly conserved in arrangement pattern, except for C. briggsae which alanine was not annotated (Fig. 4). A similar arrangement pattern was also found in some of Diplogasteridae species, i.e., Pristionchus pacificus shares exactly the same pattern, while Koerneria sudhausi only slightly differs in the placement of a few tRNAs (trnM, trnD, and trnA). Interestingly, such conserved arrangement may also extend to the suborder Tylenchina, e.g., the superfamily Aphelenchoidea represented by Aphelenchoides medicagus, A. besseyi, Bursaphelenchus xylophilus, and B. mucronatus share identical arrangement of PCGs to those species from Rhabditidae, although translocations can apply to some of the tRNAs.

Figure 4