Background & Summary

Odontothrips loti Haliday (Thysanoptera: Thripidae) is a destructive, oligophagous pest that mainly feeds on leguminous crops, particularly alfalfa Medicago sativa L.1,2. As the predominant pest of alfalfa, in North China, the major alfalfa-growing region, O. loti can cause damage to 70%~100% of plants on average3,4. Thrips attack the entire life cycle of the host plants, causing the plants to wilt or stop growing and the leaves to turn dry (Fig. 1), which not only leads to severe yield and forage quality reductions but also exacerbates the spread of plant viruses5,6,7. Several features of thrips such as small body size, cryptic behavior, and high fecundity make them difficult to control.

Fig. 1
figure 1

Odontothrips loti (a), alfalfa with O. loti damage (b) and without O. loti damage (c).

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Taking advantages of the low-cost of next generation sequencing (NGS) technology, researchers could identify functional genes related to virus transmission or pesticide resistance from the whole genome level through the construction of genome map, understand the evolution of pesticide resistance and virus transmission mechanisms, and control pest by gene regulation, making it possible to develop new pest management strategies8,9,10,11,12,13,14,15. As the genetic information of O. loti is still largely unknown currently, we aimed to disclose it for the development of novel O. loti control strategies.

In this study, we present a high-quality chromosome-level genome of O. loti, which was obtained using a combination of ONT long-read sequencing, Illumina short-read sequencing and chromosome conformation capture (Hi-C) technologies. Comparative genomic analysis was also performed on O. loti and another fourteen insect species to explore their phylogenetic relationship and genomic features. We provide the first genome assembly for a thrip in the Odontothrips genus to facilitate better understanding the genome evolution of thrips and developing novel control strategies for this important alfalfa pest.

Methods

Sample preparation

Odontothrips loti individuals were initially collected from the alfalfa field at Shangzhuang Experimental Station at the China Agricultural University (40°8’15”N, 116°11’18”E), and the colony was established and maintained for approximately 10 generations in the laboratory using the ‘Zhongmu No.1’ alfalfa at the temperature of 25 ± 1 °C, the relative humidity of 65 ± 5%, and the light: dark cycle of 16 h:8 h. The developmental stages of the thrips were examined under a light microscope. Individuals were collected, flash frozen in liquid nitrogen, and stored at −80 °C until use. Detailed information for O. loti sampling was shown in Table 1.

Table 1 Sample information of Odontothrips loti in this study.
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Genomic DNA sequencing

For Illumina short-read sequencing, the genomic DNA was isolated from of a single female adult following Chen’s protocol16, briefly, using sodium dodecyl sulfate (SDS) and proteinase K digestion, followed by phenol-chloroform extraction. The library (150 bp inserts) was constructed with Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA), and sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA), generating 43.66 Gb of raw data with 150 bp pair-end reads. Adapters and low-quality short reads were removed by Fastp (v0.21.0)17 with default parameters, resulting in a total of 42.05 Gb (~123 × coverage) of clean data (Table 2). The short-read data was used for genome survey and assembly polish.

Table 2 Library sequencing data and methods used in this study to assemble the Odontothrips loti genome.
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For long-read genomic DNA sequencing, we used approximately 800 mixed-sex adult thrips. Genomic DNA was extracted using the SDS method16, and the DNA fragment size and the degree of degradation were checked on a 0.7% agarose gel. The purity and concentration of extracted DNA were determined with NanoDrop One (Thermo Fisher Scientific). The library was constructed with SQK-LSK109 kit (Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions and sequenced on the Oxford Nanopore PromethION platform (Oxford Nanopore Technologies, Oxford, UK). We obtained 41.19 Gb (~120 × coverage) of raw long-read data with mean length of 6,182.26 bp (N50 = 16,150 bp). We then used Oxford Nanopore GUPPY (v0.3.0, https://timkahlke.github.io/LongRead_tutorials/BS_G.html) to filter reads with quality score < 7 and obtained 39.63 Gb (~116 × coverage) of clean reads. The cleaned long-read data were used for contig-level genome assembly (Table 2).

Hi-C library preparation and sequencing

The Hi-C sequencing library was prepared with 800 mixed-sex adult thrips. Samples were cross-linked with a 2% formaldehyde isolation buffer and then treated with DpnII (New England Biolabs, Beijing, CN) to digest nuclei. Biotinylated nucleotides were used to repair tails, and the ligated DNA was split into fragments of 300–700 bp in length. The resulting Hi-C library was sequenced in Illumina Novoseq 6000 for 150 bp paired-end reads. After applying the same filter criteria for short reads, a total of 31.78 Gb (~93 × coverage) of clean data was generated to assist the chromosome-level assembly (Table 2).

ONT-Transcriptome sequencing

For ONT-transcriptome sequencing, approximately 240 thrips including nymph and adult were mixed for RNA extraction with the RNA Easy Fast Tissue/Cell Kit (Tiangen). NanoDrop (Thermo Fisher Scientific) and Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) were used to evaluate the quality of extracted RNA. SQK-PCS109 and SQK-PBK004 kit (Oxford Nanopore Technologies) were used for reverse transcript and construction of cDNA library, and sequencing was proceeded on the PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK). A total of 10.24 Gb of clean reads were generated with mean length of 1,034.61 bp (N50 = 1,238 bp), used to assist genome annotation (Table 2).

Estimation of genomic characteristics

Genomic characteristics were estimated based on 42.05 Gb of short-read data using a K-mer-based statistical analysis in Jellyfish (v2.3.0)18 and GenomeScope219 (p = 2, k = 19). Based on 19-mer depth analysis, the genome size and heterozygosity were estimated to be 341.3 Mb and 1.49%, respectively, therefore, this genome is considered highly heterozygous (Fig. 2).

Fig. 2
figure 2

Characteristics of the Illumina short-read sequencing of the Odontothrips loti genome.

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Genome assembly

Contig level assembly

We first used NextDenovo (v2.5.0)20 to generate a draft assembly, and conducted two rounds of polish with ONT long reads on Racon (v1.4.11, https://github.com/lbcb-sci/racon). Illumina reads were mapped to the assembly using BWA v0.7.17 and another two rounds of contig polishing were performed with Pilon (v1.23)21. Owing to its highly heterozygous feature, Purge_haplotigs (v1.0.4, https://github.com/skingan/purge_haplotigs_multiBAM) was applied to de-heterozygosis the draft genome to generate the final contig-level genome, which was 346.58-Mb long and similar to the estimated size, with the N50 contig length of 8.59 Mb (Table 3).

Table 3 Major indicators of the Odontothrips loti genome.
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Hi-C scaffolding

Low-quality raw reads (quality score <20,length shorter than 30 bp) and adaptors were removed using Fastp (v0.21.0)17. The clean reads were then mapped to the contig assembly using HICUP (v0.8.0)22 to filter unmapped reads, invalid pairs, dangling end and repeats resulting from PCR amplification. The valid paired-end pairs were used for contig cluster, order and orient by ALLHIC (v0.9.8)23. The interaction between contig pairs were converted into binary files by 3D-DNA24 and Juicer (v1.6)25. The HiCExplorer (v3.6)26 was used to generate the heat maps of contig interaction intensity and location. The Juicebox (v1.11.08)27 was subsequently employed to review assembly manually. In summary, the resulting chromosome-level genome length was 346.59 Mb with a scaffold N50 of 18.52 Mb (Table 3), around 86.93% (301.28 Mb) of the genome bases were anchored onto 16 chromosomes (Fig. 3a), and most syntenic blocks of genome presents in the low GC content region (Fig. 3b).

Fig. 3
figure 3

Heatmap of genome-wide Hi-C data and circular representation of the chromosomes of Odontothrips loti. (a) The heatmap of chromosome interactions in O. loti. The frequency of Hi-C interaction links is represented by colors, which ranges from yellow (low) to red (high). (b) Circos plot of distribution of the genomic elements in O. loti. The tracks indicate (i) length of the chromosome, (ii) gene density, (iii) distribution of transposable element (TE) density, and (iv) GC density. Center: intra-genomic syntenic blocks of O. loti. The densities of genes, TEs, and GC were calculated in 500 kb windows.

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Predicting repeats

We used ReaptModeler (v.1.0.11, https://github.com/Dfam-consortium/RepeatModeler) to predict repeat sequence. LTR_FINDER (vOfficial, -size 1000000 -time 300)28 and LTR_retriever (v2.9.0)29 were used to find and de-redundant the LTR sequence. These two de novo library were combined with RepBase30 for further prediction by RepeatMasker (v4.0.9,-nolow -no_is -norna)31. RepeatProteinMask (-noLowSimple -pvalue 0.0001) was used for homo-prediction. All results were de-redundant and merged to the final repeat sequence. In summary, 115.26 Mb repeat sequences were identified, accounting for 33.26% of the O. loti genome (Table 4). Among these repeat sequences, most (18.85%) are DNA transposon, followed by 10.13% of long terminal repeats (LTRs), 3.45% of long interspersed nuclear elements (LINEs) and only 0.40% of short interspersed nuclear elements (SINEs) (Table 4).

Table 4 Statistics of the repeat sequences annotation in Odontothrips loti genome.
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Protein-coding genes and functional predictions

We utilized a pipeline include three strategies: transcriptome-based prediction, homology-based prediction, and ab initio prediction to annotate protein coding genes. For transcriptome-based prediction, we use NanoFilt (v2.8.0, -q 7 -l 100 -headcrop 30 -minGC 0.3)32, Pychopper (v2.7.2, https://github.com/epi2me-labs/pychopper), racon (v1.4.11, https://github.com/lbcb-sci/racon), minimap2 (v2.17-r941)33, stringtie (v2.1.4)34 and TransDecoder (v5.1.0, https://github.com/TransDecoder/TransDecoder) for ONT-transcriptome reads to predicted protein-coding gene. For homology-based prediction, tblastn (v2.7.1)35 with an E-value cutoff of 1e-5 and Exonerate (v2.4)36 were used to predict gene structure by comparing with 3 closely related species (Megalurothrips usitatus, Thrips palmi, Frankliniella occidentalis) and model species Drosophila melanogaster. Before ab initio prediction, repetitive elements from the whole genome were soft-masked. Augustus (v3.3.2)37, GenScan (v1.0)38 and GlimmerHMM (v3.0.4)39 were used for de novo prediction. Finally, MAKER (v2.31.10)40 integrated the above three strategies, resulting in a non-redundant gene set, with weighting as default. Overall, 20,128 protein coding genes were obtained (Table 5).

Table 5 Statistics for the Odontothrips loti functionally annotated protein-coding genes.
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For functional annotation, protein sequences were aligned to Non-Redundant protein (NR), Universal Protein (Uniprot), Protein Families Analysis and Modeling (Pfam), Clusters of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database. Gene Ontology (GO) terms was obtained from Uniport. InterProScan (v5.52-86.0)41 was used to search the conserved sequences, motifs and domains. There were 12,229 (60.76%) and 8,527 (42.36%) genes annotated to GO terms and KEGG pathways respectively. A total of 18,837 genes (93.59%) were annotated using at least one public database (Table 5).

Data Records

The assembly genome sequence and annotation data were deposited in Figshare42 and GenBank43. Raw data from Nanopore (CRR997575)44, Illumina (CRR997573)45 and Hi-C (CRR997574)46 genome sequencing and RNA-seq (CRR997576)47 were deposited in the Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa)48, and were related to the BioProject PRJCA022165.

Technical Validation

Genome quality assessment

We assessed the quality of chromosome-level genome from the three aspects: continuity, consistency, and completeness. First, the scaffold N50 of O. loti is 18.52 Mb (Table 3), representing the continuity of genome. Second, we evaluated the consistency of the genome by calculating the comparison rate and coverage of Illumina reads through BWA (v0.7.17)49, resulting 91.11% short reads were aligned to and covered 94.68% of the ref-genome. Third, we used BUSCO (v4.1.4)50 to estimate the completeness of chromosome-level genome by searching the 1367 BUSCO genes in insecta_odb10 (https://busco-data.ezlab.org/v5/data/lineages/). The results showed a high completeness level with 99.2%, 99.2%, 95.6%, 94.4% complete genes found in the contig-level genome, chromosome-level genome, annotated gene sets and protein-coding gene sets, respectively (Fig. 4).

Fig. 4
figure 4

Benchmarking of genome completeness of Odontothrips loti genome assembly and annotation, evaluated by BUSCO based on insect_odb10 database which includes 1,367 genes. C: the number of complete genes, S: the number of complete and single-copy genes, D: the number of complete and duplicated genes, F: the number of incomplete genes, M: the number of missing genes.

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Evaluation of gene prediction

To verify the accuracy and reliability of the gene prediction, we determined the distribution of gene length, CDS length, exon length and intron length in O. loti, D. melanogaster51 and other four related species (M. usitatus8, T. palmi12, F. occidentalis14, S. biformis13). The consistent tendency among the thrips supported an ideal annotated gene dataset in O. loti (Fig. 5).

Fig. 5
figure 5

Annotated genes comparison of the distribution of (a) gene length (b) CDS length (c) exon length (d) intron length in Odontothrips loti with Drosophila melanogaster and four closely related species. The x-axis represents the length, and the y-axis represents the density of genes.

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