A chromosome-level genome assembly of the spider mite Tetranychus piercei McGregor

Abstract

Despite the rapid advances in sequencing technology, limited genomic resources are currently available for phytophagous spider mites, which include many important agricultural pests. One of these pests is Tetranychus piercei (McGregor), a serious banana pest in East Asia exhibiting remarkable tolerance to high temperature. In this study, we assembled a high-quality genome of T. piercei using a combination of PacBio long reads and Illumina short reads sequencing. With the assistance of chromatin conformation capture technology, 99.9% of the contigs were anchored into three pseudochromosomes with a total size of 86.02 Mb. Repetitive elements, accounting for 14.16% of this genome (12.20 Mb), are predominantly composed of long-terminal repeats (30.7%). By combining evidence of ab initio prediction, transcripts, and homologous proteins, we annotated 11,881 protein-coding genes. Both the genome and proteins have high BUSCO completeness scores (>94%). This high-quality genome, along with reliable annotation, provides a valuable resource for investigating the high-temperature tolerance of this species and exploring the genomic basis that underlies the host range evolution of spider mites.

Full text

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A chromosome-level genome
assembly of the spider mite
Tetranychus piercei McGregor
Lei Chen
1,2, Xin-Yue Yu1,2, Feng Zhang
1, Hua-Meng Zhang1, Li-Xue Guo1, Lu Ren1,
Xiao-Yue Hong1 & Jing-Tao Sun1 ✉
Despite the rapid advances in sequencing technology, limited genomic resources are currently available
for phytophagous spider mites, which include many important agricultural pests. One of these pests is
Tetranychus piercei (McGregor), a serious banana pest in East Asia exhibiting remarkable tolerance to
high temperature. In this study, we assembled a high-quality genome of T. piercei using a combination
of PacBio long reads and Illumina short reads sequencing. With the assistance of chromatin
conformation capture technology, 99.9% of the contigs were anchored into three pseudochromosomes
with a total size of 86.02 Mb. Repetitive elements, accounting for 14.16% of this genome (12.20 Mb),
are predominantly composed of long-terminal repeats (30.7%). By combining evidence of ab initio
prediction, transcripts, and homologous proteins, we annotated 11,881 protein-coding genes. Both the
genome and proteins have high BUSCO completeness scores (>94%). This high-quality genome, along
with reliable annotation, provides a valuable resource for investigating the high-temperature tolerance
of this species and exploring the genomic basis that underlies the host range evolution of spider mites.
Background & Summary
Phytophagous spider mites in Tetranychidae comprise more than 1,300 species, many of which are serious
agricultural pests, such as Tetranychus urticae Koch, Panonychus citri McGregor1. Spider mites are notorious
for developing rapid resistance to pesticides, causing signicant economic losses since the widespread use of
synthetic insecticides and fungicides aer World War II2. e global acaricide market was valued at up to 400
million dollars annually3. In addition to their economic importance, spider mites exhibit diverse host ranges,
from monophagous (e.g. Tetranychus lintearius) to extremely polyphagous (e.g. T. urticae)4, making them an
ideal system for exploring the mechanisms underlying the evolution of host range. e two-spotted spider mite
(T. urticae) is the rst species of Chelicerate to have its whole genome sequenced, which was obtained by Sanger
sequencing and assembled at the scaold level5. Subsequently, in 2019, the genome was further rened and
anchored into three pseudochromosomes6. is genome provides important insights into the host adaptation
and pesticide resistance evolution of T. urticae and suggests a possible link between its rapid development of
pesticide resistance and its strong adaptive ability to host plants5,7.
Tetranychus piercei (McGregor) is a major pest on banana (Musa spp.), papaya (Carica papaya) and other
crops in East Asia8–10. It can also feed on plants such as mulberry (Morus alba), rose (Rosa sp.), peach (Prunus
persica), sweetsop (Annona squamosa), cassava (Manihot esculenta) and g (Ficus carica)4. Being the phyloge-
netically older sister species of T. urticae, T. piercei has a narrower host range and distinct host plant prefer-
ences11,12. Notably, T. piercei exhibits greater tolerance to high temperature compared to T. urticae, positioning
it as a potential replacement for T. urticae as a major pest in the context of global warming13. However, limited
information on its genetic resources hinders our understanding of its strong tolerance to high temperature and
the evolution of the detoxication system in Tetranychinae.
In this study, we employed a combination of PacBio continuous long reads, accurate Illumina short reads and
chromosomal conformation capture (Hi-C) data to assemble a chromosomal-level genome of T. piercei, which
includes 3 chromosomes and 11,881 protein-coding genes. Synteny analysis revealed dramatic chromosomal rear-
rangement between T. piercei and T. urticae. is high-quality genome will facilitate in-depth biological studies of
T. piercei and enable exploration of the genomic basis underlying the host range evolution of spider mites.
1Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China. 2These authors
contributed equally: Lei Chen, Xin-Yue Yu. ✉e-mail: jtsun@njau.edu.cn
DATA DESCRIPTOR
OPEN
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Methods
Raw material collection. At least 100 wild spider mites, including larvae, nymphs, and adults, were col-
lected from Trachycarpus fortunei in Sanya Hainan province (18.29°N, 109.47°E), a tropical region in China. We
amplied the nuclear ribosomal internal transcribed spacer (ITS) sequence to conrm the species identication
of T. piercei14. By backcrossing male sons with a virgin female, an isofemale line was constructed to minimize
heterozygosity. e isofemale line was reared on potted soybeans at a population size of thousands for at least 20
generations.
DNA and RNA sequencing. Total genomic DNA was extracted from more than 5,000 adult females using
MagAttract® HMW DNA Kit. e PacBio 30 kb SMRTbell library was prepared with more than 5 μg gDNA
using the SMRTbellTM Prep Kit 2.0 (Pacic Biosciences). e mode of Continuous Long Read (CLR) was run
on the Sequel IIe system, and generated 6.44 Gbp raw data (75-fold depth). Illumina whole-genome sequencing
was prepared using a 350 bp-insert fragment library (150 bp paired-end) by Truseq DNA PCR-free Kit, which
was further sequenced on an Illumina NovaSeq 6000 platform. High-throughput chromosome conformation
capture (Hi-C) included cross-linking, HindIII restriction enzyme digestion, end repair, DNA cyclization, puri-
cation and capture. e Hi-C library with 300–700 bp insert size library was sequenced on the NovaSeq 6000
platform, and 8.45 Gbp reads were generated to scaold chromosomes. Total RNA was extracted from 100 adult
females feeding on common beans using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s
instructions. RNA library was constructed using the VAHTS mRNA-seq v2 Library Prep Kit (Vazyme, Nanjing,
China) and sequenced on an Illumina NovaSeq 6000 platform. Finally, we generated 6.44 Gb (75×) PacBio long
reads, 13.10 Gb (152×) Illumina short reads, 8.45 Gb Hi-C (98×) reads, and 12.62 Gb transcriptome reads for our
genome assembly.
Genome survey. Duplicate and low-quality Illumina raw reads (base quality < Q20, length < 15 bp, polymer
A/G/C > 10 bp) were trimmed and removed using BBtools package v38.8215. e 21-mer depth distribution was
counted using script khist.sh of BBtools v38.82. GenomeScope v2.016 was used to estimate the genome size and
heterozygosity of T. piercei with the maximum kmer coverage at 1,000×. Based on the distribution of kmer cov-
erage and frequency, the estimated genome size of T. piercei was 86.45 Mb, with a heterozygosity rate of around
0.001% and a repeat content proportion of approximately 14.6% (Fig.1).
Chromosome staining. Unfertilized (haploid) and fertilized (diploid) eggs of T. piercei, laid within 8 hours,
were collected in a centrifuge tube containing phosphate buered saline (PBS; 0.85% NaCl, 1.4 mM KH2PO4,
8 mM Na2HPO4, PH 7.1). e PBS was then discarded, and 500 μL 50% sodium hypochlorite solution was added,
allowing it to stand for 2–3 minutes. Aer discarding the sodium hypochlorite solution, a mixture of hexane and
methanol (1:1) was added to the centrifuge tube, and the contents were vigorously shaken for 3 min to remove the
chorion. e eggs were rehydrated through an ethanol series (95, 70, 50 and 35%) and then washed in PBT (PBS
containing 0.1% Triton X-100) for 15 min. Aer being washed ve times for 1 min each in PBS, the eggs were
incubated with a uorescence quenching agent containing DAPI at room temperature for 5 min. Subsequently,
the eggs were mounted on slides and covered with coverslips for further microscopic investigation using a Leica
Fig. 1 Genome survey at 21-mer of T. piercei estimated by GenomeScope. e vertical dotted lines represent
the peaks of dierent coverages for the heterozygous, the homozygous, and the duplicated sequences (the last
two peaks) separately.
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TCS SP8 confocal microscope. e egg DNA staining of diploid females and haploid males (Fig.2a,b) consistently
indicates that the genome of T. piercei consists of three chromosomes.
Genome assembly. e CLR reads were set as input to Flye v2.9 and Raven v1.6.0 to assemble continuous
long reads17,18. e better assembly from Flye, which had a greater N50 length and completeness, was retained as
the following primary assembly. One round of built-in long reads polishing was performed by Flye v2.9. en,
two rounds of short reads were used to polish and ll in gaps of the primary assembly with NextPolish v1.4.019.
Haplotigs and duplication caused by haplotype divergence were eliminated by Purge_dups v1.2.5 using the align-
ment program Minimap2 v2.2320,21. Hi-C reads were aligned to the purged genome using BWA v0.7.1722 to anchor,
order and orient contigs into chromosomal assembly following 3D-DNA pipeline23. en, we manually reviewed
and corrected assembled errors using Juicebox v1.11.0824. Vector contaminants were checked against the UniVec
database using BLAST + v2.11.0 with the VecScreen parameters25. Bacterial and human being contaminants were
detected in the assembly against the Nt database using MMseqs v13-4511126. e completeness of genome assem-
bly was evaluated by BUSCO version 5.2.227 using the arachnida_odb10 dataset (creation date 2020-08-05). e
reads from the whole genome sequencing were aligned back to the genome assembly to access the mapping rate.
Aer de novo assembly, polishing and purging, 56 contigs (N50 of 3.33 Mb) with a total length of 86.13 Mb
were generated, accounting for 99.63% of the estimated genome. By combining Hi-C with high-throughput
sequencing and manual adjustment, we anchored these contigs into 4 scaolds, including 3 megascaolds and
1 unplaced scaold (Fig.2c). is unplaced scaold was identied as bacterial contamination by NCBI and
was subsequently excluded. Finally, a total length of 86.02 Mb contigs was assigned to 3 pseudochromosomes
(Fig.3), with scaold N50 of 29.25 Mb (Table1). e GC content of the T. piercei genome was 32.17%, which is
similar to that of T. urticae (32.25%).
Genome annotation. e repetitive elements were identied using RepeatModeler v2.0.2, which discovered
the complete long terminal repeats (LTR) with the integration of LTRharvest and LTR_retriever28. RepeatMasker
v4.1.2p1 and RMBlast v2.11.0 were searched against the custom repeat library of Dfam 3.5 and Repbase
v20181026 to so mask repeats of the genome assembly29–31. Two ab initio gene prediction soware, BRAKER2
v2.1.6 and GeMoMa v1.832,33, were used to nd protein-coding gene structure based on the masked genome.
Transcriptome mapping conducted by HISAT2 v2.2.0 and homologous proteins from ve species (Daphnia
magna, Dermacentor silvarum, Drosophila melanogaster, T. urticae, and Varroa destructor) were provided to
assist gene prediction of BRAKER2/GeMoMa. Genome-guided transcript assembly was performed by StringTie
v2.1.6, and the results were used as mRNA evidence for MAKER2 v3.01.0334,35. e same homologous proteins
Fig. 2 Chromosome staining, Hi-C heatmap and synteny. (a) DAPI staining of chromosomes in diploid female
egg of T. piercei. e blue signals represent condensed chromosomal regions, while the red dashed lines in the
model panel represent simulated chromosome boundaries. (b) DAPI staining of chromosomes in haploid
male egg. (c) Genome-wide chromosomal interaction heatmap generated in Hi-C interaction analysis with
each chromosome in the blue box. e frequency of Hi-C interaction links is represented by the color, which
ranges from white (low) to red (high). (d) Synteny dot plot based on protein homologous between T. piercei and
T. urticae.
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were also fed to MAKER2 as the protein evidence. Finally, MAKER2 combined ab initio prediction, mRNA and
homology-protein evidence to generate gene models with direct predictions not allowed for transcripts and
proteins. Proteins with lengths shorter than 30 aa were discarded. e functional annotation (TableS1) of pre-
dicted protein sequences was searched against UniProt, InterProScan and eggNOG databases. Diamond v2.0.1136
under the ‘very sensitive’ mode was used to assign gene function of the best hits in the UniProt database. Protein
domains, Gene Ontology terms and pathways were supported by Pfam, SMART, Superfamily and CDD using
InterProScan537. Complementary annotation of ko, KEGG categories were provided using eggNOG-mapper
v2.1.5 against EggNOG 5.0 database38,39.
To explore chromosomal synteny between T. piercer and T. uritcae6, we conducted a homologous search
between their respective protein sequences using Diamond v2.0.1136 with default parameters. e resulting
homologous dot plot was visualized to detect collinearity, inversions and translocations using WGDI v0.6.540.
Data Records
The raw reads and genome assembly have been deposited in the NCBI databases under BioProject
PRJNA833563. e PacBio, Illumina, Hi-C, and transcriptome data are available under identication num-
bers SRR23622209-SRR2362221241. e nal chromosome assembly has been deposited at GenBank under the
accession number GCA_036759885.142. e nal chromosome-level genome assembly, annotation, and protein
sequences are available at the Figshare database (https://doi.org/10.6084/m9.gshare.22215145)43.
Technical Validation
Evaluation of the genome assembly. Compared to T. urticae6, our genome assembly exhibits greater
contiguity and completeness attributed to the utilization of long reads and Hi-C sequencing. e contigs num-
ber, contigs N50 of T. piercei are much better than those of the two-spotted spider mite (56 vs. 1,996; 3.33 Mb vs.
0.21 Mb; Table1). We mapped whole-genome resequencing reads to the T. piercei genome and found that 92.5%
20
10
0
30
20
10
0
20
10
0
GC Content
Gene Density
LTR/Gypsy
LTR/Copia
DNA Transposons
250 μm
Fig. 3 Circular karyotype representation of the chromosomes in non-overlapping windows of 100 kb. Tracks
from inside to outside are GC content, gene density, Gypsy density, Copia density and DNA transposons density.
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of PacBio long reads and 96.51% of Illumina short reads could be well aligned. e complete benchmarking
universal single-copy orthologs (BUSCOs) under genome mode were used to assess the genome completeness.
A total of 94.6% (2,775/2,934) complete BUSCOs were identied, including 89.0% (2,612) single-copy BUSCOs,
5.6% (163) duplicated BUSCOs, 0.7% (22) fragmented BUSCOs, and 4.7% (137) missing BUSCOs.
Repeat elements and protein-coding genes. A total of 12.20 Mb repetitive elements were identied,
accounting for 14.16% of the genome (Fig.3). Besides unclassied repeats (4.67%), long-terminal repeat (LTR)
represented the most common repeat element (4.36%), followed by simple repeats (2.04%), DNA transposons
(1.67%), long-interspersed elements (LINE, 0.68%), and others (0.75%).
Using multiple lines of evidence, we annotated 11,881 protein-coding genes for T. piercei (Table1). Most
genes (>97%) had ‘annotation edit distance’ (AED) scores smaller than 0.5, indicating strong support from
the evidence of transcript and homologous protein. Under the protein model, the complete BUSCOs for our
genome annotation were 2,773 (94.5%). Although T. urticae had more coding genes than T. piercei (19,102 vs.
11,881), the results of BUSCOs suggested that T. piercei had higher completeness (90.4% vs. 94.5%; Table1).
Based on the synteny of homologous proteins, we found that the chromosomes of the two species underwent
dramatic inversion and translocation events (Fig.2d). More than half of Chr3 in T. piercer is syntenic to frag-
ments of Chr1 and Chr2 in T. urticae.
Code availability
All commands and pipelines were executed following the manuals and protocols of the corresponding
bioinformatic soware. e versions and parameters of the soware have been detailed in the Methods section.
Received: 6 December 2023; Accepted: 25 March 2024;
Published: xx xx xxxx
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Assembly
Length of contigs 86.13 Mb 90.83 Mb
Length of chromosome 86.02 Mb 85.75 Mb
Contig N50 3.33 Mb 0.21 Mb
Scaold N50 29.25 Mb 29.22 Mb
Contigs number 56 1996
Scaolds number 4 601
Pseudochromosomes number 3 3
Length of Chr1 26,633,653 bp 32,654,540 bp
Length of Chr2 30,138,663 bp 29,215,314 bp
Length of Chr3 29,245,017 bp 23,884,949 bp
GC% 32.17 32.25
BUSCO completeness 94.6% 94.6%
Annotation
No. of protein genes 11,881 19,102
Mean protein length 494.7 aa 363 aa
Exons per gene 3.35 3.53
Mean exon length 459.6 bp 362.5 bp
Introns per gene 2.46 2.87
Mean intron length 603.0 bp 501.7 bp
BUSCO completeness 94.5% 90.4%
Tab le 1. Statistics for the assembly and annotation of Tetrany chu s genome.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. U2003112,
32020103011, 32202290) and the Natural Science Foundation of Jiangsu Province (Grant No. BK20221003).
Author contributions
J.T.S., F.Z. and L.C. designed the research. X.Y.Y. contributed to the sampling and sequencing. F.Z. assembled and
annotated the genome. L.C., X.Y.Y., H.M.Z. and L.G.X. analyzed the data and drew the pictures. L.R. performed
chromosome staining. L.C. and J.T.S. wrote the dra manuscript, and X.Y.H. improved the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information e online version contains supplementary material available at https://doi.
org/10.1038/s41597-024-03189-0.
Correspondence and requests for materials should be addressed to J.-T.S.
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