Background & Summary

Bean flower thrips Megalurothrips usitatus is a highly harmful pest of leguminous crops in the genera Glycine, Arachis, and Vigna1,2,3,4. The insect lays eggs in plant tissue and feeds on leaves, flowers and pods, causing economic losses worldwide, particularly in southern China, India, Japan, the Philippines, and Australia1,3,5,6. Its small body size, cryptic behavior, and fast transmission present difficulties in pest control6,7.

Attempts to mitigate agricultural damage have largely involved chemical insecticides8,9,10,11,12. However, excessive pesticide usage leaves residues that risk consumer health and also induce resistance in pest insects. Understanding the evolution of pesticide resistance is necessary for developing novel management strategies, but the genetics of M. usitatus remains poorly understood. Filling this knowledge gap will benefit our efforts at pest control.

In this study, we assembled a chromosome-level genome of M. usitatus using a combination of PacBio long read, Illumina short-read sequencing, and chromosome conformation capture (Hi-C) technologies. We compared the genomic features of M. usitatus with those of other insects to explore the genomic signatures of resistance. The high-quality reference genome of the bean flower thrips obtained in this study will lay the foundation for future investigations on the ecology of thrips and provide valuable genetic information for its management.

Methods

Sample preparation and genomic DNA sequencing

Megalurothrips usitatus samples were collected from Wanning, Hainan province, and reared for approximately 100 generations in the laboratory. Adults were fed Lablab purpureus and kept at 25 ± 1 °C, 70 ± 5% relative humidity, and 14:10 light:dark cycle. Stages were confirmed under a light microscope and verified with pictorial keys13. Individuals were then quickly placed into collection tubes, flash-frozen in liquid nitrogen, and stored at −80 °C until use.

We prepared approximately 2,000 mixed-sex M. usitatus individuals for genome sequencing. Genomic DNA was extracted using the CTAB method, followed by purification using a Blood and Cell Culture DNA Midi Kit (QIAGEN, Germany). The purity and concentration of extracted DNA were determined with 0.75% agarose gel electrophoresis and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA), respectively. The library constructed from the extracted DNA was approximately 10–20 Kb in size. A PacBio Sequel sequencer (Pacific Biosciences, Menlo Park, USA) was used for long DNA fragments, and Illumina Novoseq 6000 was used to generate 150 bp paired-end short reads. The sequencing yielded 98.30 Gb (412.78 × coverage) of long-reads with an N50 length of 14,475 bp and an average length of 10,352.68 ± 2.46 bp (mean ± S.E.). The Illumina platform sequenced 58.80 Gb raw data, of which adapters and low-quality short reads were removed using Fastp version 0.21.014 with default parameters, resulting in a total of 55.86 Gb (234.57 × coverage) clean data (Table 1).

Table 1 Library sequencing data and methods used in this study to assemble the Megalurothrips usitatus genome.
Full size table

Hi-C library preparation and sequencing

Chromosome conformation capture (Hi-C) sequencing used fresh tissues from 1,500 mixed-sex M. usitatus individuals. The samples were cross-linked with a 2% formaldehyde isolation buffer and then treated with DpnII (NEB) to digest nuclei. Biotinylated nucleotides were used to repair the tails, and the ligated DNA was split into fragments of 350 bp in length. The resulting Hi-C library was sequenced in Illumina Novoseq. 6000 with paired-end 150 bp. After applying the same filter criteria for short reads, a total of 53.90 Gb (226.34 × coverage) of clean data was generated (Table 1).

Transcriptome sequencing

A pooled M. usitatus sample was prepared using 30 eggs, 20 pseudo-pupae, 10 females, and 10 males. Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, USA). A paired-end library was constructed using the TruSeq RNA Library Preparation Kit (Illumina, USA) and sequenced on an Illumina Novoseq 6000 platform. It resulted in a total of 5.61 Gb RNA-seq clean data (Table 1). Additionally, total RNA (1 µg) was used to construct a full-length transcript isoform library using the SMRT bell Express Template Prep Kit 2.0 (Pacific Biosciences, USA). Target-size sequences were generated using the PacBio sequel II platform. A total of 47.67 Gb full-length transcriptome data was obtained (Table 1).

Estimation of genomic characteristics

Genomic characteristics were determined based on 55.86 Gb of short-read data using a K-mer-based statistical analysis in JELLYFISH version 2.1.315 with the following parameters: ‘count -m 17 -C -c 7 -s 1 G -F 2’. Genome heterozygosity and genome size were estimated in GenomeScope version 2.016 with default parameters. Based on 17-mer depth analysis, genome size and heterozygosity were estimated to be 255.81 Mb and 0.85%, respectively (Fig. 1).

Fig. 1: Genomic characteristics of Megalurothrips usitatus based on Illumina short-read data obtained in GenomeScope version 2.0 with 17 K-mer.
figure 1

The K-mer distributions showed double peaks: the first peak with a coverage of 100 indicates genome duplication and the highest peak with a coverage of 200 represents a genome-size peak. Genome size was calculated to be 255.81 Mb with a heterozygous rate of 0.85%.

Full size image

Genome assembly

We assembled a draft genome using wtdbg2 version 2.5 with default parameters17. We then had it polished using RACON version 1.4.1318 with parameters ‘-m 8 -x −6 -g −8 -w 500 -u’ and Pilon version 1.1419 with default parameters based on 98.30 Gb long reads and 55.86 Gb short reads.

A scaffolding pipeline based on Durand (2016)20 was used to generate a high-quality chromosome-scale genome. Initially, Hi-C data were mapped to the contig assembly using BWA-MEM version 0.7.1721 with the following parameters: ‘mem -SP5M’. Next, the DpnII sites were generated using the ‘generate_site_positions.py’ script in Juicer version 1.520. The 3D-DNA pipeline (-r 2) was subsequently employed to order, orient, and cluster the contig22. After viewing Hi-C contact maps, the chromosome-scale genome was assembled in Juicebox version 1.11.08 (https://github.com/aidenlab/Juicebox). The genome assembly was screened for contaminant sequences by using the “Contamination in Sequence Databases” in NCBI. A total of 33 sequences were labeled as contaminant and removed (available in Figshare). To identify the mitochondrial genome, we amplified the cytochrome oxidase subunit 1 (COI) gene fragment with primer pairs LCO1490 and HCO2198, and obtained a DNA barcode sequence of approximately 610 bp23. We then used BLAST version 2.2.2824 (-evalue 1e-5) to find assembly sequences of a high similarity to the COI fragment (>98%), and identified one unplaced sequence (scaffold46) as mitochondrial sequence. The resulting chromosome-level genome was 238.14 Mb with a scaffold N50 of 13.85 Mb, maximum length of 20.88 Mb, and GC rate of 55.90% (Table 2). 91.89% of the genome was anchored to 16 pseudo-chromosomes (Table 2), which were well-distinguished from each other based on the chromatin interaction heatmap (Fig. 2).

Table 2 Statistics for the chromosomal-level genome of the Megalurothrips usitatus.
Full size table
Fig. 2: Genome-wide contact matrix of Megalurothrips usitatus generated using Hi-C data.
figure 2

Each black square represents a pseudo-chromosome. The color bar indicates the interaction intensity of Hi-C contacts.

Full size image

Predicting repeats

Repeat sequences were annotated in Extensive de novo TE Annotator (EDTA) version 1.9.425. In brief, LTR retrotransposons were identified in LTR FINDER version 1.0726, LTRharvest27, and LTR retriever version 2.9.028 with default parameters. Next, TIR Learner29 and HelitronScanner30 were used to classify DNA transposons with default parameters. RepeatMasker version 4.0.7 (-gff -xsmall -no_is)31 and RepeatProteinMasker version 4.0.7 (-engine wublast) were utilized to identify repeat sequences based on RepBase edition 2017012732. Repeats were masked with de novo predictions using RepeatModeler version 2.0.1 with parameters ‘-engine ncbi -pa 4’. Additionally, Tandem Repeats Finder33 was used to annotate tandem repeats with parameters ‘2 7 7 80 10 50 500 -f -d -m’. Overall, 20.20% of the assembled genome was classified as repetitive sequences in the M. usitatus genome (Table 3). Tandem repeat elements were found to be the most abundant (8.42%), followed by the terminal inverted repeat category (5.39%) (Table 3).

Table 3 Classification of repeat annotation in the Megalurothrips usitatus genome.
Full size table

Gene and functional predictions

Genes in the assembled genome were predicted using a combination of homology-based, transcriptome-based, and ab initio methods. Homology-based predictions involved downloaded sequences of peptides and transcripts from Aptinothrips rufus (http://v2.insect-genome.com/Organism/87), Frankliniella occidentalis (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/697/945/GCF_000697945.3_Focc_3.1), and Thrips palmi (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/012/932/325/GCF_012932325.1_TpBJ-2018v1). The IsoSeq version 3.4.0 workflow was utilized to generate 28,608 high-quality transcripts from the full-length transcriptome data, with quality parameters of 0.99 (https://github.com/PacificBiosciences/IsoSeq). Next, RNA-seq short data were mapped to the reference genome using HISAT2 version 2.2.134 with the parameter ‘-k 2’. The mapped reads were then assembled into transcripts using StringTie version 2.4.035 with default parameters. Homologous proteins and transcripts were aligned using Exonerate version 2.4.0 with default parameters to train the gene sets. Meanwhile, a sorted and mapped bam file of RNA-seq data was transferred to a hints file using the bam2hints program in AUGUSTUS version 3.2.336 with the parameter ‘–intronsonly’. The trained gene sets and hint files were combined as inputs for AUGUSTUS version 3.2.336, which predicted coding genes from the assembled genome with default parameters. Finally, homology-based, de novo-derived, and transcript genes were merged in MAKER version 2.31.10 to generate a high-confidence gene set37. It resulted in the annotation of 14,000 M. usitatus genes. The average transcript length was 2,243.30 bp with an average length of coding sequence (CDS) of 1,588.94 bp. The average exon number per gene was 7.38, and the average exon length was 303.85 bp (Table 4).

Table 4 Gene annotation statistics of the Megalurothrips usitatus genome.
Full size table

Gene structure and annotations were determined through several methods, including eggnog-mapper38 (-m diamond–tax_scope auto–go_evidence experimental–target_orthologs all–seed_ortholog_evalue 0.001–seed_ortholog_score 60–query-cover 20–subject-cover 0 –override), InterProscan version 5.039 (-iprlookup -goterms -appl Pfam -f TSV), BLAST version 2.2.2824 (-evalue 1e-5), and HMMER version 3.3.240 (–noali–cut_ga Pfam-A.hmm). These methods were used to search against multiple public databases, including NCBI non-redundant protein (Nr), Gene Ontology (GO), Clusters of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and Pfam. Most genes (91.74%) were successfully annotated with at least one public database (Table 5).

Table 5 Functional annotation of the Megalurothrips usitatus genome.
Full size table

Comparative genomic analysis

To identify single-copy orthologous genes, we utilized the longest protein sequence of each gene from M. usitatus and multiple other species (Table 6), including F. occidentalis41, T. palmi42, Acyrthosiphon pisum43, Triatoma rubrofasciata44, Columbicola columbae45, Aedes aegypti46, Danaus plexippus47, Tribolium castaneum48, Apis mellifera49 and Daphnia galeata50. We performed all-to-all single-copy ortholog BLAST comparisons in OrthoFinder version 2.5.451 with the parameters ‘-a blast -M msa’. We aligned the resulting single-copy orthologous genes using MAFFT version 7.487 (–auto)52 and further trimmed the poorly aligned regions using Gblocks version 0.91b53 (-t = p -b4 = 5). We maintained the genes that met the stationary, reversible and homogeneous (SRH) assumptions54 using IQ-TREE version 2.2.055 with a p-value cut-off of 0.05. We finally obtained 1,573 single-copy genes under these criteria. Next, We used FASconCAT-G version 1.05.156 to concatenate the genes to form a supermatrix, which was used for subsequent phylogenetic analysis.

Table 6 Genome datasets were used for comparative genomic analysis in the study.
Full size table

We performed a maximum likelihood analysis of concatenated sequences in IQ-TREE version 2.2.055 with 1,000 UFBoot replicates (–bb 1,000 –model JTT + I + G4). The minimum correlation coefficient for the convergence criterion was set at 0.99 (-bcor 0.99). The age of each node was estimated using a correlated rates clock in MCMCTREE of PAML version 4.457. To estimate the divergence times, we selected fossil records listed in Table 7.

Table 7 Fossils were used for estimating divergence times and calibration point prior settings in the analysis.
Full size table

Gene-family expansion and contraction were estimated using CAFÉ version 4.2 with parameters ‘lambda -s -t’, based on maximum likelihood and reduction methods58. Phylogenetic tree topology and branch lengths were considered when inferring the significance of changes to gene-family size in each branch. The results revealed 684 expanded gene families and 1,639 contracted gene families in M. usitatus (Fig. 3). Next, functional enrichment analysis (GO enrichment and KEGG pathway) was performed in KOBAS version 3.059. Significantly enriched GO terms were those with an adjusted p < 0.05 under Fisher’s exact test. Expanded gene families were enriched in cAMP signaling pathway, fatty acid metabolism, detoxification metabolism (ABC transporters) and the ionotropic glutamate receptor pathway (Fig. 4a, available in Figshare). Contracted gene families were enriched in chitin-based cuticle development, sensory perception of taste and NADP + activity (Fig. 4b, available in Figshare).

Fig. 3: Genome evolution of Megalurothrips usitatus.
figure 3

A time-calibrated phylogenetic tree inferred from 1,573 single-copy orthologs using IQ-TREE version 2.2.0 was shown. The upper panel in wheat represents Paraneoptera insects and the lower panel in light-blue represents Holometabola. The divergence between M. usitatus and F. occidentalis diverged 73.93 Mya (Million years ago). Bootstrap support values based on 1,000 replicates are equal to 100 (orange dot). The number of expanded (+red) and contracted (−blue) gene families are shown for each lineage.

Full size image
Fig. 4: Functional annotation of expanded and contracted gene families.
figure 4

(a) Expanded genes. (b) Contracted genes. Each row represents an enriched function, and the bar length represents the enrichment ratio (input gene number/background gene number). Bar colors represent different clusters. If any cluster has more than five terms, the top five with the highest enrichment ratio are displayed.

Full size image

Data Records

Genomic PacBio sequencing data were deposited in the Sequence Read Archive at NCBI under accession number SRR2213748560.

Genomic Illumina sequencing data were deposited in the Sequence Read Archive at NCBI under accession SRR2213748261.

RNA-seq data were deposited in the Sequence Read Archive at NCBI under accession number SRR2213748462.

Full-length transcript isomer sequencing data were deposited in the Sequence Read Archive at NCBI under accession number SRR2213748363.

Hi-C sequencing data were deposited in the Sequence Read Archive at NCBI under accession number SRR2213748164.

The final chromosome assembly was deposited in GenBank at NCBI under accession number JAPTSV00000000065.

The contaminant file, single-copy orthologous genes, gene-family expansion and contraction, gene function annotation, and repeat annotation are available in Figshare66.

Technical Validation

DNA integrity

The integrity of extracted genomic DNA was determined using 0.75% agarose gel electrophoresis and analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). DNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit 2.0 (Thermo Fisher Scientific, USA). Absorbance at 260/280 nm was approximately 1.8.

Assessment of genome assemblies

We assessed the accuracy of the final genome assembly by mapping Illumina short reads to the M. usitatus genome with BWA-MEM version 0.7.1721. The analysis showed that 96.52% of short reads were successfully mapped to the M. usitatus genome (Table 8). We further assessed the base quality of genome assembly by estimating the quality value score (QVS) using Merqury version 1.167, which showed a high QVS of 32.65 (Table 8). These findings indicate that the quality of our assembled genome is high.

Table 8 Assessment metrics for the final genome assembly of Megalurothrips usitatus.
Full size table

Furthermore, we evaluated the completeness of the final genome assembly using Benchmarking Universal Single-Copy Orthologs (BUSCO version 3.0.2) insecta_odb1068, which includes 1,367 orthologous genes. The analysis revealed a high completeness of 97.40% for the M. usitatus genome with only 0.60% of BUSCO genes being fragmented, 2.00% being missing, and 0.40% being duplicated (Table 8). These BUSCO results were comparable to the completeness for other thrips genomes, such as T. palmi (97.20%), F. occidentalis (98.50%), and A. rufus (95.00%) (Table 9).

Table 9 Comparisons of genome assemblies of different thrips.
Full size table