De novo assembly and analysis of the Heortia vitessoides transcriptome via high-throughput Illumina sequencing
Graphical abstract
Introduction
Aquilaria sinensis (Loureiro) Sprenger, a tree belonging to Thymelaeaceae, is an economically significant tree species. It produces valuable agarwood that is widely used in traditional medicine and the incense industry (Qiao et al., 2016). A. sinensis, distributed in the southern coastal areas of China, belongs to the list of rare and endangered Chinese plants (Chen et al., 2011a). In recent years, Heortia vitessoides Moore outbreaks have seriously threatened the large- and small-scale planting of A. sinensis (Wen et al., 2016). H. vitessoides is a defoliating pest affecting A. sinensis forests and H. vitessoides larvae feed solely on the leaves of A. sinensis in southern China (Qiao et al., 2013). The biological characteristics (Cao et al., 2013), pesticide control methods (Chen et al., 2011b, Lu et al., 2014, Zhou et al., 2016), and behavior (X.F. Jin et al., 2016) of H. vitessoides have been extensively studied for application in insect control, but to our knowledge, the molecular biology of H. vitessoides has not been studied.
Next-generation sequencing (NGS) technologies, including the Illumina Solexa, Roche 454, and ABI SOLiD platforms, can provide genomic and transcriptomic data inexpensively and rapidly (Schuster, 2008, Ansorge, 2009, Metzker, 2010). In recent years, NGS has been applied to large population studies and to non-model species without reference genomic resources (Song et al., 2016). Application of NGS to entomological research has greatly promoted its progress. Illumina transcriptome sequencing of organisms with complete genomes has confirmed that the relatively short reads produced can be effectively assembled and used for gene discovery and gene expression analysis (Marioni et al., 2008, Hegedus et al., 2009, Wang et al., 2011). Over the past several years, Illumina de novo transcriptome sequencing has been used for the assembly of de novo transcriptomes for many insects, including Aedes aegypti (Gibbons et al., 2009), whitefly (Wang et al., 2010), brown planthopper (Xue et al., 2010), oriental fruit fly (Shen et al., 2011), and mountain pine beetle (Christopher et al., 2012).
In this study, a cDNA library was constructed from the total RNA of H. vitessoides adults. Using Illumina HiSeq high-throughput sequencing, we are the first to obtain de novo transcriptome data for H. vitessoides, a non-model species with no reference genomic resources. Bioinformatic methodologies were used to classify unigenes by function and SSR loci were studied using the transcriptome data. Additionally, unigenes coding for proteins related to insecticide resistance were retrieved from the assembled sequences. We believe that obtaining genetic information on H. vitessoides by employing our transcriptome data would allow for further investigations into the functions of important genes and for a better understanding of its molecular biology. Future development of management strategies directed toward this insect would greatly benefit from the present study and from ongoing research such as RNA interference (RNAi) control of this insect.
Section snippets
Insects
The H. vitessoides adults used in this study were obtained from an A. sinensis plantation (22°01′N, 110°25′E) in Huazhou, Guangdong, China, on January 7, 2016. No chemical treatment was applied before or during collection. Ten adults were collected, anesthetized with ice, washed with sterile water, disinfected in 75% alcohol, and snap frozen in liquid nitrogen; the 10 adults were considered one sample.
Extraction and testing of RNA
Total RNA was extracted from H. vitessoides using Total RNA Kit II (OMEGA), following the
Sequencing and de novo assembly
A library of H. vitessoides adults was constructed and sequenced using Illumina HiSeq 2000 high-throughput sequencing technology. This sample yielded a total of 60,587,900 clean reads (7,572,046,316 clean bases); 97.84% of the clean reads had a Phred quality score of ≥ Q30 (with an error probability of 0.1%). The GC content of the transcriptome was 44.00% (Table 1). Clean reads were assembled using Trinity and TGICL software. Finally, we obtained 66,673 unigenes with 63,789,080 bp. The lengths of
Conclusion
In this study, a de novo transcriptome was assembled for H. vitessoides adults. A total of 60,587,900 clean read fragments containing 7,572,046,316 bases were generated. We obtained 66,673 unigenes with an average length of 957 bp. Using five databases (NR, Swiss-Prot, KEGG, KOG, and GO), we obtained 28,202, 27,339, 9172, 24,278, and 24,861 unigenes, respectively. In total, 24,861 (37.29%) unigenes were assigned to 64 subcategories belonging to three main categories, i.e., biological process,
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (No. 31470653) and the Natural Science Foundation of Guangdong Province (No. 2015A030313416).
References (45)
Next-generation DNA sequencing techniques
New Biotechnol.
(2009)- et al.
Azadirachtin ingestion is lethal and inhibits expression of ferritin and thioredoxin peroxidase genes of the sweetpotato whitefly Bemisia tabaci
J. Asia Pac. Entomol.
(2016) - et al.
De novo sequencing and characterization of the Bradysia odoriphaga (Diptera: Sciaridae) larval transcriptome
Comp. Biochem. Physiol. Part D
(2015) - et al.
Transcriptome analysis of the Japanese pine sawyer beetle, Monochamus alternatus (Coleoptera: Cerambycidae) by high-throughput Illumina sequencing
J. Asia Pac. Entomol.
(2015) - et al.
De Novo assembly of the transcriptome for oriental armyworm Mythimna separata (Lepidoptera: Noctuidae) and analysis on insecticide resistance-related genes
J. Insect Sci.
(2016) - et al.
De novo transcriptome sequencing and analysis of Rapana venosa from six different developmental stages using Hi-seq 2500
Comp. Biochem. Physiol., Part D
(2016) - et al.
Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines
Free Radic. Biol. Med.
(2001) - et al.
Chlorantraniliprole induces up-regulated expression of cytochrome P450 genes in Spodoptera exigua (Lepidoptera: Noctuidae)
Acta Entomol. Sin.
(2015) - et al.
Molecular cloning, expression and oxidative stress response of a mitochondrial thioredoxin peroxidase gene (AccTpx-3) from Apis cerana cerana
J. Insect Physiol.
(2013) - et al.
Identification of a thioredoxin peroxidase gene involved in resistance to nucleopolyhedrovirus infection in Helicoverpa armigera with RNA interference
J. Insect Physiol.
(2015)
Identification of permethrin-inducible cytochrome P450s from the diamondback moth, Plutella xylostella (L.) and the possibility of involvement in permethrin resistance
Pestic. Biochem. Physiol.
A method used for distinguishing the pupae sexuality of Heortia vitessoides
Chin. For. Sci. Technol.
Correlation of catalase and peroxidase with pesticide tolerance in massonpine caterpillar
Acta Entomol. Sin.
Studies on biological characteristics of Heortia vitessoides Moore on Aquilaris sinensis
Chin. Plant Prot.
Studies on the Synergistic Effects of Chlorantraniliprole and Beta-cypermethrin on Heoritia vitessoides Moore
J. Shandong For. Sci. Technol.
Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests
Insect Biochem. Mol. Biol.
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research
Bioinformatics
Benchmarking next-generation transcriptome sequencing for functional and evolutionary genomics
Mol. Biol. Evol.
Full-length transcriptome assembly from RNA-Seq data without a reference genome
Nat. Biotechnol.
Cloning and tissue-specific expression of a chitin deacetylase gene from Helicoverpa armigera (Lepidoptera: Noctuidae) and its response to Bacillus thuringiensis
J. Insect Sci.
Deepn sequencing of the zebrafish transcriptome response to mycobacterium infection
Mol. Immunol.
Effect of the host plant on the antioxidative defence in the midgut of Lymantria dispar L. caterpillars of different population origins
J. Insect Physiol.
Cited by (15)
Fatty acid-binding protein gene is indispensable for molting process in Heortia vitessoides (Lepidoptera: Crambidae)
2023, Journal of Integrative AgricultureChromosomal-level reference genome of the moth Heortia vitessoides (Lepidoptera: Crambidae), a major pest of agarwood-producing trees
2022, GenomicsCitation Excerpt :These include gene families related to detoxification, metabolism, and development including UDP-glucuronosyltransferases (UGTs, Pfam: PF00201), abhydrolipases (Pfam: PF04083), short-chain dehydrogenases/reductases (SDRs, Pfam: PF00106), cytochromes P450 (CYPs, Pfam: PF00067), C-type lectins (Pfam: PF00059), and lepidopteran-specific chorion gene family (Pfam: PF01723), and cytosolic sulfotransferases (SULTs) (Table 1; Supplementary Table 3). A total of 67 CYPs could be identified in the H. vitessoides genome assembly, which contains an additional 21 CYPs to those identified in a previous transcriptomic study [13] (Supplementary Table 3). Moreover, copy number expansion was identified in two CYP families (CYP6AB and CYP9G) that function in xenobiotic metabolism and plant allelochemical detoxification [9] (Supplementary Fig. 2).
Chitin deacetylase 1 and 2 are indispensable for larval–pupal and pupal–adult molts in Heortia vitessoides (Lepidoptera: Crambidae)
2019, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyCitation Excerpt :At 4, 8, 12, and 24 h after the administration of 20E and 1 × PBS, six larvae from each group were collected and stored at −80 °C. Based on the H. vitessoides transcriptome (SRX3035102; Cheng et al., 2017), cDNA unigenes encoding putative CDA1 and CDA2 were obtained. ORF finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to acquire the cDNA sequences of CDA1 and CDA2 ORF.
Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera: Crambidae) by antennal transcriptome analysis
2019, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsCitation Excerpt :The distribution of H. vitessoides ranges from India, Nepal, China, Sri Lanka, through South-East Asia and the East Indies to Queensland, the New Hebrides, and Fiji (Qiao et al., 2012). The biological characteristics, pesticide control methods, and behavior of H. vitessoides have been extensively studied for applications in insect control (Cheng et al., 2017). However, there have been no reports detailing an effective method of controlling this pest.
Identification and characterization of the catalase gene involved in resistance to thermal stress in Heortia vitessoides using RNA interference
2018, Journal of Thermal BiologyCitation Excerpt :At least three independent biological replications were performed for each treatment. From the transcriptome of adult H. vitessoides (accession number: SRX3035102; Cheng et al., 2017), a candidate CAT gene was identified by keyword searching. The sequence of CAT was confirmed by comparison with other sequences available in GenBank using the nucleotide BLAST and BLAST-X tools available on the NCBI website (https://www.ncbi.nlm.nih.gov/).