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

Abstract

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.

Transcriptional Analysis of Microsatellites in Velvetbean Caterpillar Anticarsia gemmatalis Hübner, 1818

“…3). This slight change in nucleotide composition in SUSBt and RESBt libraries compared to the others may be associated with the participation of TEs, since under stress these elements are more active [Pappalardo et al, 2021], and it is already known that in Lepidoptera microsatellites can be associated with TEs [Tay et al, 2010]. Ding et al [2017] observed that in the mono-and trinucleotide repeat types the composition was predominantly A/T, exactly as found in our study for A. gemmatalis.…”

“…Ding et al [2017], in a vast study of SSRs in more than 130 insect genomes (deposited in a database), reported that the most abundant repeat motifs in insect genomes were di-(27.2%) and trinucleotide (22%) types. The trinucleotide-type repeats were also prominent in the transcriptomes of B. dorsalis (44.24%) [Ouyang et al, 2021] and D. citri, where 42.95% of the identified SSRs were of this type [Chen et al, 2014]. One of the explanations for this is that trinucleotide-type repeats would have possible stability in the protein-coding regions when compared to other types of repeat unit, rarely producing sliding mutations of the coding frame [Ouyang et al, 2021].…”

“…Other works with microsatellites in transcriptomes have already been performed in other insect orders. Ouyang et al [2021] evidenced 1,978 loci of SSRs in the transcriptome of Bactrocera dorsalis (Diptera), and Duan et al [2017] identified 7,936 SSR loci for Rhopalosiphum padi (Hemiptera). Studies with locust species showed 5,696 microsatellites in Epacromius coerulipes [Jin et al, 2015] and 2,093 for Gampsocleis gratiosa [Zhou et al, 2016].…”

“…It is already known that the quantity of microsatellites found in insect genomes and transcriptomes can vary greatly depending on the organism and methodology used [Ouyang et al, 2021], and that these are not so abundant in some species [Fagerberg et al, 2001]. Thus, in Dialeurodes citri (Hemiptera) only 149 SSRs were characterized [Chen et al, 2014] or, for example, in Euphydryas editha (Lepidoptera) only 92 SSRs were identified [Mikheyev et al, 2010].…”

A full-length transcriptome and gene expression analysis of three detoxification gene families in a predatory stink bug, Picromerus lewisi

“…SMRT sequencing can provide a new comprehension of full-length sequences, greatly facilitating the transcriptome research of species lacking high-quality reference genomes and improving genome annotation. To date, only a few full-length transcriptome analyses have been reported in insects, such as Dendroctonus ponderosae ( Keeling et al, 2012 ), Chouioia cunea ( Pan et al, 2019 ), Sogatella furcifera ( Chen et al, 2020 ), Aphis aurantii ( Hong et al, 2020 ), Rhynchophorus ferrugineus ( Yang H et al, 2020 ), Bactrocera dorsalis ( Ouyang et al, 2021 ), Spodoptera frugiperda ( Yang et al, 2021 ), Diabrotica virgifera ( Zhao et al, 2021 ). There are still no published reference genomes for predatory stink bugs.…”