De novo Transcriptome Analysis of Chinese Citrus Fly, Bactrocera minax (Diptera: Tephritidae), by High-Throughput Illumina Sequencing.

Jia Wang,Ke-Cai Xiong,Ying-Hong Liu

doi:10.1371/journal.pone.0157656

Abstract

The Chinese citrus fly, Bactrocera minax (Enderlein), is one of the most devastating pests of citrus in the temperate areas of Asia. So far, studies involving molecular biology and physiology of B. minax are still scarce, partly because of the lack of genomic information and inability to rear this insect in laboratory. In this study, de novo assembly of a transcriptome was performed using Illumina sequencing technology. A total of 20,928,907 clean reads were obtained and assembled into 33,324 unigenes, with an average length of 908.44 bp. Unigenes were annotated by alignment against NCBI non-redundant protein (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. Genes potentially involved in stress tolerance, including 20 heat shock protein (Hsps) genes, 26 glutathione S-transferases (GSTs) genes, and 2 ferritin subunit genes, were identified. These genes may play roles in stress tolerance in B. minax diapause stage. It has previously been found that 20E application on B. minax pupae could avert diapause, but the underlying mechanisms remain unknown. Thus, genes encoding enzymes in 20E biosynthesis pathway, including Neverland, Spook, Phantom, Disembodied, Shadow, Shade, and Cyp18a1, and genes encoding 20E receptor proteins, ecdysone receptor (EcR) and ultraspiracle (USP), were identified. The expression patterns of 20E-related genes among developmental stages and between 20E-treated and untreated pupae demonstrated their roles in diapause program. In addition, 1,909 simple sequence repeats (SSRs) were detected, which will contribute to molecular marker development. The findings in this study greatly improve our genetic understanding of B. minax, and lay the foundation for future studies on this species.

Highlights

The high-throughput sequencing technologies, referred to as the next-generation sequencing, such as Solexa/Illumina, SOLID/ABI, 454/Roche platform, has widely been used to PLOS ONE | DOI:10.1371/journal.pone.0157656 June 22, 2016Transcriptome Analysis of Bactrocera minax had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
The contigs were further assembled into 33,324 unigenes with a mean sequencing Raw data (G) Total number of raw reads Low quality data percentage (%) rRNA percentage (%) Clean data (G) Total number of clean reads Q20 percentage (%) Q30 percentage (%) GC percentage (%) N percentage (%) Number of contigs Length of contigs N50 length of contigs Mean length of contigs Number of unigenes Length of unigenes N50 length of unigenes Mean length of unigenes
8,357 (25.08%) and 3,795 (11.39%) unigenes are longer than 1,000 and 2,000bp, respectively, and only 9960 (29.89%) unigenes are shorter than 300bp (S2 Fig), demonstrating the effectiveness of Illumina sequencing technology in rapidly capturing a large portion of the transcriptome and providing a sequence basis for future studies

Summary

Introduction

The high-throughput sequencing technologies, referred to as the next-generation sequencing, such as Solexa/Illumina, SOLID/ABI, 454/Roche platform, has widely been used to PLOS ONE | DOI:10.1371/journal.pone.0157656 June 22, 2016Transcriptome Analysis of Bactrocera minax had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Transcriptome Analysis of Bactrocera minax database, Cluster of Orthologous Groups (COG), and gene ontology (GO). The genes encoding Hsps, GSTs, Ferritins, enzymes in ecdysone biosynthesis pathway, and ecdysone receptors were identified. Unigenes putatively encoding Hsps, GSTs, enzymes in 20E biosynthesis pathway, and 20E receptors were identified by alignment against databases with a cut-off E-value < 10−5.

Results

Conclusion