Genome sequence of the root-knot nematode Meloidogyne luci

Root-knot nematodes (RKN) from the genus Meloidogyne parasitize a wide range of host plants and have a global distribution. They are considered the most important group of plant-parasitic nematodes (Jones et al., 2013). Field infestations result in economic damage due to reduction or loss of crop yield with estimated global annual losses of $110bn (Danchin et al., 2013; Bebber et al., 2014). Among RKN, the tropical species belonging to Meloidogyne Clade I reproduce asexually by mitotic parthenogenesis (except M. floridensis) and parasitize a broader range of hosts than their sexual relatives (Castagnone-Sereno and Danchin, 2014). Several genomes of Clade I tropical Meloidogyne spp. have been sequenced (Abad et al., 2008; Lunt et al., 2014; Blanc-Mathieu et al., 2017; Szitenberg et al., 2017) and have revealed them to be complex allopolyploids with heterozygous duplicated genome regions and abundant transposable elements (Blanc-Mathieu et al., 2017; Szitenberg et al., 2017). Previous genome assemblies largely relied on short-read next-generation sequencing which limited the contiguity of the assemblies. Sato et al. (2018) found that applying long-read sequencing technologies such as Pacific Biosciences single-molecule real-time (SMRT) significantly improved the contiguity of their Meloidogyne arenaria assembly.

The species belonging to the Meloidogyne ethiopica group include the closely related species M. ethiopica, M. inornata and M. luci (Gerič Stare et al., 2019). The phylogenetic positions of different populations of M. ethiopica group species within Clade I Meloidogyne are incompletely resolved. Isolated specimens of M. luci in Europe were previously misidentified as M. ethiopica due to their high similarity (Gerič Stare et al., 2017). We used long-read Pacific Biosciences Sequel and short-read Illumina HiSeqX sequencing data to produce a high-quality Meloidogyne luci genome assembly. The M. luci population SI-Smartno was isolated from tomato plants grown in a commercial production greenhouse in Šmartno, Slovenia (Gerič Stare et al., 2018). A line (V13) was reared from the progeny of a single female and multiplied on tomato (Solanum lycopersicum “Val”). Nematode eggs were obtained by hypochlorite extraction (Hussey and Barker, 1973) and cleaned by sucrose flotation (McClure et al., 1973). Genomic DNA (gDNA) was obtained by phenol-chloroform extraction from the nematode eggs ground in liquid nitrogen. Following fluorometric quantification (Qubit; Thermo Fisher Scientific), a total of 6.64 µg of gDNA was used for Illumina whole-genome sequencing (WGS) on HiSeqX platform. 150 bp paired-end reads were generated from 350 bp insert TruSeq DNA PCR-Free libraries, yielding 206,071,630 reads (30.9 Gb). Reads were quality checked with FastQC v0.11.8 (Andrews, 2018) and trimmed with Trimmomatic v0.36 (Bolger et al., 2014) using the Phred quality score cutoff at 20. Prior to Pacific Biosciences SMRT sequencing on the Sequel, gDNA was assessed using the Femto Pulse system (Agilent) and a total of 10 µg of gDNA was used. The SMRTbell Express template prep kit (Pacific Biosciences) was used to prepare PacBio library >20 kb using standard protocol without shearing (Procedure & Checklist – Preparing >15 kb Libraries Using SMRTbell® Express Template Preparation Kit). Blue Pippin (Sage Science, MA, USA) was used for size selection (25 kb cutoff). Libraries were sequenced on the Sequel using v2.1 Sequencing and Binding kits generating 3,617,847 reads (42.4 Gb). We generated approximately 150-fold and 200-fold genome coverage using Illumina and PacBio data, respectively. Adapter and barcode sequences were filtered out within the Sequel instrument and assembled with HGAP4 pipeline (SMRT Link suite v5.1.0.26412, Pacific Biosciences) and polished with Pilon (Walker et al., 2014) using trimmed Illumina data.

The assembled M. luci SI-Smartno genome consists of 327 contigs with a minimum contig length of 10,147 bp and N50 contig length of 1,711,905 bp. The total length of assembly is 209.16 Mb. Smudgeplot v0.1.3 (Ranallo-Benavidez et al., in press) and Jellyfish v1.0 (Marçais and Kingsford, 2011) were used to estimate genome ploidy based on the counting of k-mers (k = 21) on short-read data. The genome is estimated to be triploid (AAB). Blobtools (Laetsch and Blaxter, 2017) was used to assess contaminant DNA presence (Fig. 1B). The assembly is currently the most contiguous RKN assembly (Table 1) available with an estimated coverage of 95.16% of the coding space based on Core Eukaryotic Genes Mapping Approach (CEGMA) analysis (Parra et al., 2007) and the average number of CEGs at 2.88 supports the triploid genome model (Fig. 1A). The polished assembly was 88.1% complete based on the eukaryote set (n = 303) of Benchmarking Universal Single-Copy Orthologs (Simão et al., 2015). The assembly of M. luci SI-Smartno can now be used to determine the correct phylogenetic position of the clade, identification of genetic changes related to the origins of virulence, and in the study of evolutionary history of this organism.

Figure 1: