Ma et al. Parasites Vectors (2020) 13:321 https://doi.org/10.1186/s13071-020-04187-y Parasites & Vectors RESEARCH Open Access The complete mitochondrial genome of the Columbia lance nematode, Hoplolaimus columbus, a major agricultural pathogen in North America Xinyuan Ma1, Paula Agudelo1, Vincent P. Richards2 and J. Antonio Baeza2,3,4* Abstract Background: The plant-parasitic nematode Hoplolaimus columbus is a pathogen that uses a wide range of hosts and causes substantial yield loss in agricultural felds in North America. This study describes, for the frst time, the complete mitochondrial genome of H. columbus from South Carolina, USA. Methods: The mitogenome of H. columbus was assembled from Illumina 300 bp pair-end reads. It was annotated and compared to other published mitogenomes of plant-parasitic nematodes in the superfamily Tylenchoidea. The phylogenetic relationships between H. columbus and other 6 genera of plant-parasitic nematodes were examined using protein-coding genes (PCGs). Results: The mitogenome of H. columbus is a circular AT-rich DNA molecule 25,228 bp in length. The annotation result comprises 12 PCGs, 2 ribosomal RNA genes, and 19 transfer RNA genes. No atp8 gene was found in the mitog- enome of H. columbus but long non-coding regions were observed in agreement to that reported for other plant- parasitic nematodes. The mitogenomic phylogeny of plant-parasitic nematodes in the superfamily Tylenchoidea agreed with previous molecular phylogenies. Mitochondrial gene synteny in H. columbus was unique but similar to that reported for other closely related species. Conclusions: The mitogenome of H. columbus is unique within the superfamily Tylenchoidea but exhibits similarities in both gene content and synteny to other closely related nematodes. Among others, this new resource will facilitate population genomic studies in lance nematodes from North America and beyond. Keywords: Hoplolaimus, Lance nematode, Ecdysozoa, Mitochondrial genome, Phylogeny, de novo assembly Background plant-parasitic nematodes are currently recognized as In the phylum Nematoda, plant-parasitic species can be major pathogens of agricultural crops worldwide, which distinguished from animal parasites as well as non-par- leads to more than 150 billion USD losses annually in the asitic relatives because their mouthparts and stylet are USA [3]. In a recent USA survey of agricultural patho- well developed allowing them to penetrate sturdy plant gens, six main genera of plant-parasitic nematodes were cell walls while digging and feeding [1, 2]. A number of recognized as serious crop threats [1]: cyst nematodes (Heterodera spp.); lance nematodes (Hoplolaimus spp.); root-knot nematodes (Meloidogyne spp.); lesion nema- *Correspondence: [email protected] todes (Pratylenchus spp.); reniform nematodes (Rotylen- 2 Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634, USA chulus spp.); and dagger nematodes (Xiphinema spp.). Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ma et al. Parasites Vectors (2020) 13:321 Page 2 of 10 Moreover, some of the above pathogens, like lance nema- noticed that short nuclear and/or mitochondrial gene todes, can damage horticultural felds, golf courses, and markers are sometimes uninformative for revealing fne turfgrasses. Tese plant-parasitic nematodes can also to moderate population genetic structure within a spe- cause serious indirect environmental problems by favor- cies [22]. Tis shortcoming can be addressed by devel- ing chemical overuse during nematode management [4]. oping genomic resources in this relevant group of lance Lance nematodes are all species of migratory ecto-endo nematodes. Although the genomes of a number of plant- parasites with a distinct cephalic region and a massive parasitic nematodes have been sequenced and analyzed well-developed stylet [4]. According to the current taxo- before [23–28], no genomic resources exist for lance nomical view that relies on a combination of molecular nematodes, yet. and morphological characters, lance nematodes belong In this study, we de novo sequenced and assembled the to the class Chromadorea, infraorder Tylenchomorpha complete mitochondrial genome of the Columbia lance [2, 4–6]. Tey exhibit a wide range of hosts, including, nematode H. columbus. Other than annotating and pro- among others, turf grasses, cereals, soybean, corn, cot- viding a detailed description of the mitochondrial chro- ton, sugar cane, and some trees [1, 4, 7]. Tey live in soil, mosome in this crop pathogen, we used protein-coding feed on plant roots, move inside or around plant tissue, genes to explore phylogenetic relationships among plant- and destroy cortex cells, that can result in root necrotic parasitic nematodes belonging to the class Chromadorea, lesions [8]. Hoplolaimus columbus, also known as the superfamily Tylenchoidea. ‘Columbia lance nematode’, is considered among the most economically important species in the world [1]. Methods Tis nematode was described as a new species from sam- Collection of specimens, DNA extraction ples collected in Columbia, South Carolina, USA. Later, and whole‑genome amplifcation the same species was reported in the states of North Soil samples containing specimens of H. columbus were Carolina, Georgia, Alabama, and Louisiana [8–10]. In collected from the Edisto Research Center in Black- the feld, H. columbus is parasitic on cotton and soybean, ville, South Carolina (33°21’56.2”N, 81°19’46.9”W) and on which pathogenicity has been demonstrated; produc- transported to Clemson University for further study. In tion losses for cotton are typically 10–25%, and losses for the laboratory, nematodes were frst extracted from soil soybean can be as high as 70% in the southeastern USA samples using the sugar centrifugal fotation method [11–14]. Although H. columbus has been found in some [29]. A few fxed specimens were then identifed using Asian countries [2], there are no reports yet of crop dam- diagnostic key characters under an optical microscope age in the region. Hoplolaimus columbus belongs to the [30]. Next, live nematodes (n = 9) were submerged into subgenus Basirolaimus together with 17 other nematode distilled water, starved for two weeks, and placed in a species [2]. Species in the subgenus Basirolaimus have 3% hydrogen peroxide solution (Aaron Industry, Clin- been reported in Asian countries, including China, India ton, SC, USA) for 5 min before washing them in distilled and Japan [2]. Nonetheless, the only species in the sub- water three times to eliminate potential microorganisms genus Basirolaimus so far recognized as a major agricul- inhabiting their surface. Ten, the same nematodes were tural pathogen is H. columbus (i.e. in the USA) [2, 15, 16]. placed separately in DNA Away solution (Molecular Bio- Considering its wide distribution and damage to crops, Products Inc., San Diego, CA, USA) to eliminate poten- a better genomic understanding of H. columbus would tial DNA and DNase contamination and washed three prove helpful to understand its population genetic struc- times using PCR-grade water. Total DNA from each H. ture and efects, or the lack thereof, on commercially rel- columbus specimen was extracted using a Sigma-Aldrich evant crops. extract-N-Amp kit (XNAT2) (Sigma-Aldrich, St. Louis, Morphological characteristics alone have limited MO, USA). Te whole genome size of H. columbus was function to distinguish among closely related species in estimated to be ~300 million bp using fow cytometry the genus Hoplolaimus given the remarkable similarity [31, 32]. Whole-genome amplifcation (WGA) of each of internal and external organs and body parts among individual nematode was then performed using an Illus- closely related species [5, 7]. Molecular markers have tra Ready-To-Go GenomiPhi V3 DNA amplifcation kit been shown to be useful for species identifcation and for (GE Healthcare, Chicago, IL, USA) following the manu- understanding phylogenetic relationships and popula- facturer’s instructions. Tree WGA replicates per nema- tion genetics in diferent species of lance nematodes [5, tode were performed, and the one with the highest DNA 15–17]. Although previous work has provided valuable concentration tested using a Qubit fuorometer (Invitro- insights for nematode phylogeny [18–21], it has been gen, Carlsbad, CA, USA) was selected for the next gen- eration sequencing library preparation. Ma et al. Parasites Vectors (2020)