Full-Length SMRT Transcriptome Sequencing and SSR Analysis of Bactrocera dorsalis (Hendel).

Huili Ouyang,Fengping Qin,Xiaoyun Wang,Wen Lu,Chao Chen,Xialin Zheng

doi:10.3390/insects12100938

Abstract

Simple SummaryIn this study, a full-length transcriptome was analyzed with single-molecule real-time (SMRT) sequencing, which was first used to discover simple sequence repeat (SSR) genetic markers from B. dorsalis. Moreover, SSR markers from isoforms were screened for the identification of species diversity. These results could provide molecular biology methods for further population research.Bactrocera dorsalis (Hendel), as one of the most notorious and destructive invasive agricultural pests in the world, causes damage to over 250 different types of fruits and vegetables throughout tropical and subtropical areas. PacBio single-molecule real-time (SMRT) sequencing was used to generate the full-length transcriptome data of B. dorsalis. A total of 40,319,890 subreads (76.6 Gb, clean reads) were generated, including 535,241 circular consensus sequences (CCSs) and 386,916 full-length non-concatemer reads (FLNCs). Transcript cluster analysis of the FLNC reads revealed 22,780 high-quality reads (HQs). In total, 12,274 transcripts were functionally annotated based on four different databases. A total of 1978 SSR loci were distributed throughout 1714 HQ transcripts, of which 1926 were complete SSRs and 52 were complex SSRs. Among the total SSR loci, 2–3 nucleotide repeats were dominant, occupying 83.62%, of which di- and tri- nucleotide repeats were 39.38% and 44.24%, respectively. We detected 105 repeat motifs, of which AT/AT (50.19%), AC/GT (39.15%), CAA/TTG (32.46%), and ACA/TGT (10.86%) were the most common in di- and tri-nucleotide repeats. The repeat SSR motifs were 12–190 bp in length, and 1638 (88.02%) were shorter than 20 bp. According to the randomly selected microsatellite sequence, 80 pairs of primers were designed, and 174 individuals were randomly amplified by PCR using primers. The number of primers that had amplification products with clear bands and showed good polymorphism came to 41, indicating that this was a feasible way to explore SSR markers from the transcriptomic data of B. dorsalis. These results lay a foundation for developing highly polymorphic microsatellites for researching the functional genomics, population genetic structure, and genetic diversity of B. dorsalis.

Highlights

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), known as the oriental fruit fly, is one of the most devastating and highly invasive agricultural pests in the world, causing severe damage to over 250 species of commercial fruits and vegetables [1–5]
Larvae were subjected to a banana diet, whereas adult flies were fed with an artificial diet made from yeast extract and sugar (4:1)
The PacBio Sequel system generated a total of 40,319,890 subreads with an average read length of 1009 bp

Summary

Introduction

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), known as the oriental fruit fly, is one of the most devastating and highly invasive agricultural pests in the world, causing severe damage to over 250 species of commercial fruits and vegetables [1–5]. The broad range of distribution, the large number of host plants, and the complex interactions between B. dorsalis and diverse environments may be due to its high genetic variation, which makes it challenging to manage this pest. SSR markers commonly present high levels of intra- and inter-specific variations. Because of their characteristics of assay technique, reproducibility, multi-allelic nature, codominant inheritance, abundance, and genome-wide coverage, they have been extensively applied to genetic diversity, genetic structure analysis, and paternity testing in arthropods [6–9]. There have been 28,201 insect microsatellite sequences included in the National Center for Biotechnology Information (NCBI), of which 8539 belong to Order

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