Transcriptome profiling of Diachasmimorpha longicaudata towards useful molecular tools for population management
© The Author(s). 2016
Received: 4 June 2015
Accepted: 21 May 2016
Published: 12 October 2016
Diachasmimorpha longicaudata (Hymenoptera: Braconidae) is a solitary parasitoid of Tephritidae (Diptera) fruit flies of economic importance currently being mass-reared in bio-factories and successfully used worldwide. A peculiar biological aspect of Hymenoptera is its haplo-diploid life cycle, where females (diploid) develop from fertilized eggs and males (haploid) from unfertilized eggs. Diploid males were described in many species and recently evidenced in D. longicaudata by mean of inbreeding studies. Sex determination in this parasitoid is based on the Complementary Sex Determination (CSD) system, with alleles from at least one locus involved in early steps of this pathway. Since limited information is available about genetics of this parasitoid species, a deeper analysis on D. longicaudata’s genomics is required to provide molecular tools for achieving a more cost effective production under artificial rearing conditions.
We report here the first transcriptome analysis of male-larvae, adult females and adult males of D. longicaudata using 454-pyrosequencing. A total of 469766 reads were analyzed and 8483 high-quality isotigs were assembled. After functional annotation, a total of 51686 unigenes were produced, from which, 7021 isotigs and 20227 singletons had at least one BLAST hit against the NCBI non-redundant protein database. A preliminary comparison of adult female and male evidenced that 98 transcripts showed differential expression profiles, with at least a 10-fold difference. Among the functionally annotated transcripts we detected four sequences potentially involved in sex determination and three homologues to two known genes involved in the sex determination cascade. Finally, a total of 4674SimpleSequence Repeats (SSRs) were in silico identified and characterized.
The information obtained here will significantly contribute to the development of D. longicaudata functional genomics, genetics and population-based genome studies. Thousands of new microsatellite markers were identified as toolkits for population genetics analysis. The transcriptome characterized here is the starting point to elucidate the molecular bases of the sex determination mechanism in this species.
Diachasmimorpha longicaudata Ashmead (Hymenoptera: Braconidae) is a solitary endoparasitoid of larval and prepupal stages of fruit flies (Diptera: Tephritidae). It is native to Southeast Asia and has been successfully established in tropical and subtropical regions of the world. This beneficial insect is commonly used worldwide as a biological control agent in integrated pest control programs, mostly against fruit flies of the genera Bactrocera, Anastrepha and Ceratitis [1–5]. Many studies performed on biological parameters , conditions of rearing , and behavior [4, 8, 9] are available for this parasitoid. Inbreeding experiments provided the first description of viable diploid males in this species, and the presence of a multiple-locus CSD mechanism [10, 11] based on cytogenetic and cytological techniques and sex ratio analysis . A deeper knowledge of D. longicaudata genomics is needed for a better understanding of the molecular basis of complex mechanisms, as the sex determination pathway.
Sex determination mechanisms can be very variable between phylogenetically close related species. The same mechanism can apparently be regulated by different genes, suggesting a rapidly evolving system [13–15]. In insects, sex determination pathways are characterized by a gene cascade that is highly variable at the top, but shares a conserved core including a common binary switch, the transformer (tra) gene, and at a least a common executor downstream, the doublesex (dsx) gene [15, 16]. A primary signal initiates one of the two alternative routes in the signaling cascade by leading the differential sex-specific splicing of doublesex (dsx) and sex lethal (slx) genes that control overt sexual differentiation . In Hymenoptera, many models for sex determination mechanisms have been proposed, however functional studies of sex-determining genes have only been performed in few species. The most studied is the social insect species Apis mellifera L. (Hymenoptera: Apidae), the honeybee, which has single-locus complementary sex determination (sl-CSD) . Differential sex development under this mechanism depends on the allelic composition of one sex locus. Heterozygous individuals at the complementary sex determiner (csd) locus develop as female offspring, and homozygous or hemizygous individuals develop into male offspring . The heteroallelic combination of CSD activates the splicing factor transformer (tra), termed feminizer (fem), which mediates the female-specific splicing of the dsx pre-mRNA. In homozygous or hemizygous individuals for the csd locus, no functional TRA/FEM protein is present due to sex-specific splicing of the tra pre-mRNA, hence leading to male development [14, 18]. Other highly studied species is the gregarious parasitoid Nasonia vitripennis Walker (Hymenoptera: Pteromalidae), the jewel wasp. Nasonia has a non-CSD sex-determining system, described as the maternal effect genomic imprinting sex determination system (MEGISD). In this case, female-specific Nvtra (N. vitripennis designation for the tra gene) splicing depends on an autoregulatory loop, where a threshold level of maternally provided messenger is essential for female development .
The next generation sequencing methods (Roche 454, Solexa/Illumina, etc.) provide the opportunity for genomic exploration in non-model arthropods species wherein little or no molecular knowledge is available . In particular, 454-sequencing technology based on the pyrosequencing principle has recently enabled the application of functional genomics to a broad range of arthropods [20–30].
In the current study we applied 454 sequencing technology to characterize the transcriptome of D. longicaudata by de novo assembly of three libraries (third instar male larvae, male, and female adults). Our results show the first global and big picture of D. longicaudata transcriptome as well as the functional annotation of its unigenes (isotigs and singletons). We also present a preliminary scenario of genes differentially expressed between sexes as a first molecular insight into the study of the molecular basis of sex determination in this non-model organism. Additionally, since no microsatellite sequences have been reported for D. longicaudata, we in silico identified and characterized SSRs with potential application as molecular markers in population genetics studies.
Results and Discussion
Transcriptome sequencing and assembly
To cover the transcriptome of D. longicaudata, third instar male larvae and adult individuals of both sexes (three pools) were used for RNA extraction. We chose adult individuals from both sexes in order to assess sex-specific expression patterns. As D. longicaudata is an endoparasitoid, immature stages in this species are very difficult to sample and, impossible to identify and differentiate male or female individuals in early developmental stages. A male larval sample obtained from virgin females was assessed as well in order to improve the transcriptome yield. The 454-pyrosequencing was performed independently on the three pools on a 454 GS FLX Titanium (Roche). A total of 175.7 Mbp of transcriptome data were generated, comprising 469766 raw reads with an average length of 376bp. The raw sequencing dataset was submitted to Sequence Read Archive (SRA) database, accession number SRP072867, under BioProject: PRJNA317427; samples corresponding to raw sequences of each library have been identified as NCBI BioSamples SRS1376223, SRS1376224, and SRS1376239. After filtering for adaptors, primers and low-quality sequences, 99.8 % of the first raw sequences resulted in high quality reads, representing approximately 174.3 Mbp. Low quality sequences (956 reads) were discarded.
D. longicaudata transcriptome summary
Total number of qualified reads
Qualified reads from larval pool
Qualified reads from female adult pool
Qualified reads from male adult pool
Average read length (bp)
Total number of isotigs larger than 200bp
Mean length of contig/isotigs (bp)
Total number of singletons larger than 200bp
Average length of singletons (bp)
Total number of predicted peptides
Average length of peptide (amino acids)
Total of isotigs with BLASTx hits
Total of singletons with BLASTx hits
All 51686 unigenes were subjected to BLASTx similarity search against the NR protein database (National Center for Biotechnology Information, NCBI) followed by the BLAST2Go suite and all 38685 predicted protein sequences were run through the InterproScan full suite (to complement annotation by comparing unigenes to HMM models from PFams for example), to assign a putative function and GO terms [32, 33].
We then merged all results from InterProscan and Blast2GO by their resulting GO annotation.
Examining the BLASTx results, a7021 isotigs (82.7 %) and 20227 singletons sequences (46.8 %) had significant BLASTx matches(Table 1). The frequency of BLASTx hits from isotigs/singletons was 52.72 %, above the values previously reported for de novo transcriptome assemblies of related species that had no sequenced genome range from 20 to 40 %[34, 35].
The majority of matched sequences exhibited high similarity to Apis (11.5 %), Camponotus (11.4 %) and Harpegnathor (11 %). The BLASTx top hit distribution is showed in Additional file 1.
GO terms were assigned to a total of 16930 transcripts, 4483 isotigs (52.8 %) and 12447 singletons (28.8 %). Of the GO annotated isotigs and singletons sequences, 32.65 % of terms were assigned to “Biological Processes”, 42.71 % to “Molecular functions” and 24.63 % to “Cellular components” (Fig. 3).
Sequences involved in sex determination functions and SSRs distribution
Markers and position
HWRCMJ02HFI99; HWRCMJ02HFCXO; HWRCMJ02FPEMS; HWRCMJ02IL9LL
Primary sex determination
Primary sex determination soma
Primary sex determination germ-line
Germarium-derived egg chamber formation
Dorsal appendage formation
Ovarian follicle cell differentiation
Primary spermatocyte growth
Comparative transcriptome analysis
Validation of RNA sequencing results
The next generation sequencing approach gives us a massive amount of information that must be validated in order to determine a reliable criterion for data-set comprehension. To confirm differential expression tendencies between sexesand to compare results with those obtained by RNA sequencing, we assessed a group of transcripts using RT-qPCR.
Specific primers used for RT-qPCR. Gene ID and main BLAST matches
Accession no. of top BLAST hits
Product size (bp)
Amplification efficiency (%)
α 1-elongation factor
Odorant binding receptor
Sex determination associated transcripts
As one of the aims of this work was to detect candidate transcripts potentially involved in the sex determination mechanism of D. longicaudata, we looked forhomologues of genes that had already been characterized in several species as involved in sex determination [13, 17, 37, 38]. Through the sequence similarity approach (BLASTx) we searched for homologue sequences to the upstream known regulator fem[NP_001153369; EFN87550; NM001159897.1; XM008553448.1; XM003704454.1; XP008559611.1; EFN89777.1; XP003703392.1; XP006608847.1; EFN60633],and the known and highly conserved downstream regulator dsx[gi|383849166; gi|383849165; XP003395766; ABW99103.1; NP001128407.1; XP_003700217]. We found 3 sequences in the D. longicaudata transcript dataset that showed homology to the mentioned genes at the protein level (BLASTx): isotig07202 and isotig06880were homologues to fem and were identified as different transcript variants from the same locus, and isotig08283 was homologue to dsx.
The expression plots of RNA sequencing for both fem homologues show a tendency for higher expression in females (Figs. 5 g and i). However, as the normalized read count for isotig06880 was under the established cut-off for RNA sequencing (10-fold difference), the tendencies had to be confirmed by RT-qPCR.Isotig07202 higher expression in females was corroborated (p = 0.02) (Fig. 5 g-h).Isotig06880 showed higher expression in females by RNA sequencing and a 1.8-fold higher expression in males (p = 0.0003) by RT-qPCR(Fig. 5i-j). These opposite expression patterns detected for isotig06880 can be explained due to the low read counts provided by RNA sequencing. RT-qPCR has a robust statistical analysis and is, therefore, a more reliable technique. The patterns found between both fem variants could respond to their particular function in the different sexes. This requires further and deeper analysis and transcripts characterization.
For isotig08283, dsx homologue, no significant expression difference between sexes was detected (Fig. 5 k-l). The difference was lower than a 10-fold by RNA sequencing analysis and showed a p-value of 0.09 for RT-qPCR data analysis. This may be due to alternative splicing regulation of this transcripts rather than differential expression.
Finding genes involved in the sex determination mechanism and assessing their expression patterns in both sexes may help us have a better understanding in how and when sexual faith is determined in this non model organism. In biological control strategies, the female parasitoidis the responsible of producing the control effect, as the female lays eggs in immature stage of fruit flies species and prevent its adult emergence, thus, controlling the pest population density . The knowledge about the presence of genes engaged in the sex determination cascade in this parasitoid species take a step forward towards potential sex-ratio manipulation in favor of female production which may constitute one important genetic contribution to improve the productivity and efficiency of artificial rearing of this parasitoid species. To shed light in this matter there are ongoing works on the characterization of the full transcript sequences of the sex determination genes discovered and on the analysis of their expression profiles throughout development for this species .
Genetic diversity: molecular markers prediction
In silico mining of simple sequence repeats (SSR)
Using the MISA software we identified and characterized SSR (microsatellite) motifs in the D. longicaudata transcriptome as potential molecular markers for this species. 1233 putative SSRs within 8483isotigs and 3441 putative SSRs within the 43203 singletons were identified. The frequencies of SSR occurrence considering multiple occurrences were 15 % in Isotigs and of 8 % for singletons. For isotigs, the percentage was higher than that reported in Spalangia endiusWalker (Hymenoptera: Pteromalidae)(8.51 %) and in Laodelphax striatellus(Fallén) (Homoptera: Delphacidae)(7 %) . A total of 982 (12 %) isotigs contained at least one SSR, and 693 SSRs (70.6 %) had sufficient flanking sequences to allow the design of appropriate unique primers. For singletons a total of 3441 contained at least one SSR, and 2082 SSRs (60.5 %) with appropriated flanking sequences characteristics to the design of unique primers.
Characterization of microsatellite motifs and distribution
The most frequent type of microsatellite corresponded to tetrameric (38.8 %), dimeric (24.3 %) and trimeric (14.9 %) motifs, being penta,hexanucleotideand composed repeats present at much lower frequencies (6.6 %,4.2 % and 11.1 % respectively, Additional file 3). These results are in contrast to those found in S. endius and L. striatellus, where di and trinucleotide repeats are more frequent [26, 40].
SSR motif combinations can be grouped into unique classes based on DNA base complementarities. Tetranucleotides were grouped into 27 unique classes and the numbers of unique classes possible for di and trinucleotide repeats are 4 and 10, respectively (Additional file 3). The most frequent combination for tetranucleotideswas 3 repeats of AAAT/ATTT; five repeats of AG/CT for dinucleotides and five repeats of AAT/ATT for trinucleotides (Additional file 3). These motifs are mainly AT repeats in concordance with the characterized AT-rich genomes of Hymenopteranspecies .
Information on the isotig and singleton identification (ID), marker ID, repeat motifs, repeat length, primer sequences, positions of forward and reverse primers, and expected fragment length are included in Additional file 3.
The microsatellite markers discovered have the potential to be used inthe characterization of the genetic variability of natural and laboratory populations of D. longicaudata, since little genetic information is currently available of this species [42, 43]. This parasitoid species was successfully established under artificial rearing conditions in several American countries to be used in biological control programs against Tephritid fruit flies of economic importance [44, 45]. Presently, the parasitoid is also being mass-rearedin Argentina with the same purpose . The presence of established populations of this parasitoid in the wild has been reported by Schliserman et al.  and Oroño &Ovruski ,originated from previous releases in the frame of biological control strategies in Argentina. The markers discovered here could be potentially useful to describe these populations at genetic level and also could be of help to develop diagnostic molecular tools to analyze the genetic quality of laboratory populations and mass rearing production of this parasitoid species (eg. inbreeding or deleterious genotypes analyses).
We analyzed the distribution of SSRs among sex determination related transcripts as a subgroup of the biological process transcripts, finding 10 potential markers associated to this subgroup. These markers are located throughout the different transcript sequences previously mentioned above (Table 2). Structure and length of terms involved and percentage of SSR found in each group are showed in Additional file 3 and SSRs distribution in sex determination associated transcripts are shown in Additional file 4. We obtained flanking sequences long enough to design primers for 4 of the 10 described SSRs. Primers, motifs and localization of these SSRs are shown in Additional file 3. Further analysis of the remaining transcript sequences by RACE PCR will increase the number of markers available for future studies on markers associated tothe sex determination pathway.
In addition, the use of microsatellite markers to construct genetic maps and to perform linkage analysis to phenotypic and behavioral traits has been explored in other insect species [48, 49] and represent a challenge to D. longicaudata genetics and genomics,revealing information about the regulation of main physiological pathways and understanding the genetic bases of complex traits (eg. sex determination, parasitization behavior, host allocation, host preference, etc.) all data amenable to be used for the improvement of mass-rearing and release protocols of this biological control agent [44, 45, 50].
We present here the first transcriptome analysis inD. longicaudata. The generated transcripts dataset represents a major contribution to this non-model organism genomics and genetics, andconstitutes the first genome effort to compare the expression patterns between sexes in this parasitoid wasp. During the characterization of this transcriptome we identified two orthologues to components of sex determination pathways: femand dsx, and at least two splicing variants of femthat showed differential expression between sexes. As the trigger of the sex determination cascade remains to be identified, the information provided herecould be a stepping stonefor the identification of these early signals. In addition, SSRs markers distributed throughout the transcriptome were identified, several ones associated to the sex determination group of transcripts. This work highlights the utility of transcriptome high performance sequencing as a fast and cost effective way for obtaining information on expression of species-specific genes, and for generating resources to be used in population genetic studies. The identification of genes involved in theD. longicaudata sex determination system together with SSR and sex determination linked SSRs discovery, are key factors for the early identification and characterization of females (responsible for fly larvae control) and its populations. This knowledge may allow performing improvements in the applied fruit fly control strategies for an effective parasitoid mass rearing toward the sex-ratio manipulation in order to increase the female production in biofactories. Finally, the possibility of identifying putative genes involved in biological processesas immunity, parasitoid-host interaction, and host preference- among others—will also provide genomic tools to improve the artificial rearing protocolsof this parasitoid, used worldwide as a biological control agent for fruit flies of economic importance.
Diachasmimorpha longicaudata individuals used in this study came from the Instituto de Genética experimental strain (INTA Hurlingham, Buenos Aires, Argentina). This strainwas founded with individuals imported from Mexico to Tucumán (Argentina) in 1998 (SENASA, exp n° 14054/98)and introduced to our laboratory in 2001. The colony was maintained in glass flasks with water and honey and reared on third instar larvae of Ceratitiscapitata(Wiedemann) (Diptera: Tephritidae) as host,according to previously established protocols [6, 10].
RNA preparation, cDNA libraries construction and 454-pyrosequencing
Total RNA was extracted using TRIzol® reagent (Invitrogen) from three pools of D. longicaudata individuals. Pools consisted of approximately 90mg of tissue. In each case this corresponded to 30 adult females, 30 adult males (both samples15 days after emergence), and 5 third instar male larvae (8 days after oviposition of virgin females). The larval pool was included for a more representative data set. The resultant RNA was resuspended in 50 μl of DEPC treated water. Quantity and quality of RNA were assessed using a Nanodrop (Thermo Scientific Nanodrop 2000) spectrophotometer and agarose gel electrophoresis (1 % P/V). Approximately 90μg of total RNA were processed in INDEAR (Rosario Biotechnology Institute, Rosario, Argentina) for cDNA synthesis, mRNA enrichment, libraries construction, and subsequent 454-pyrosequencing. Diachasmimorpha longicaudata cDNA libraries were subjected to a half plate production run on the 454-GS-FLX (Roche) sequencing instrument.
Transcript assembly and annotation
After removing low quality sequences, filtering for adaptors and primers, all curated raw 454 read sequences from all libraries were assembled into isotigs (aligned reads from a single transcript), isotigs (alignment variants of transcripts) and isogroups (isotigs representing a genomic locus) using Newbler v2.6 Assembler software (Roche, IN, USA). Reads identified like singletons (i.e., reads not assembled into isotigs), were subjected to CD-HIT-454 clustering algorithm using a sequence identity cut-off of 90 %, which eliminates redundant sequences or artificial duplicates. BLASTx (cut-off e-value ≤ 10−10) searches were performed against the NCBI nr protein database in order to make an assessment of the putative identities of the sequences. Annotation and mapping were done using the software BLAST2GO, which assigns Gene Ontology terms  (http://www.geneontology.org), KEGG maps (Kyoto Encyclopedia of Genes and Genomes, KASS) and an enzyme classification number (EC number) using a combination of similarity searches and statistical analysis . GO annotation was further completed by running the full suite of InterProScan under default parameters .In order to identify the most probable open reading frame (ORF) in every isotig and singleton, the program TransDecoder  was used with default settings. The best and most probable peptides were used as input for InterProScan. InterProScan combines different protein signature recognition methods native to the InterPro member databases into one resource that searches for the corresponding InterPro and GO annotations.
For transcriptome comparison with other model organisms the MUMMER software package, specifically Nucmer,  and the Circos visualization tool  were used. The Honey Bee Genome Sequencing Consortium assembly Amel_4.5 and Official Gene Set OGSv3.2 (www.beebase.org) and the NasoniaBase genome Assembly Nvit_1.0 (http://hymenopteragenome.org/nasonia/) databases were used.
Differentially expressed transcripts identification (gene mining and RT-qPCR)
Total RNA was extracted from adult female and male D. longicaudata pools of individuals (each pool consisting of 15 adult insects of the same sex and age) using TRIzol® (Invitrogen) following the manufacturer’s protocol. The total RNA obtained was resuspended in 30 μl of DEPC treated water and quantity/quality was assessed as previously described. About 1μg of total RNA was used as template to synthesize first-strand cDNA using ImProm-II Reverse Transcriptase (Promega) and Oligo (dT) primers (Promega) following the manufacturer’s protocol. The resultant cDNA was diluted 1/10 for further use in RT-qPCR.
To identify differentially expressed contigs, reads from each library were mapped back to all the contigs which were previously assembled using all libraries together. To assess differential expression between sexes from RNA sequencing results, the relative expression level for a given transcript was calculated normalizing the read counts by the length of that transcript and divided by the number of sequence reads in the library. This value was then multiplied by the largest library sequenced. This result is then regarded as Expression Units (EU). For the identification of differentially expressed transcripts, we restricted our analysis to the transcripts expressed in all three libraries. Any isotig with less than 3 counts was discarded due to the large variability in estimating expression from low covered genes. To assess the tendencies for differential expression between male and female libraries a cut-off value of the previously mentioned quotient was set at a 10-fold difference between sexes. Female and male number of reads was normalized to larvae libraryandis represented in logarithmic scale.
We performed a Genes of Interest (GI) selection based on two criteria:1) A set of sequences which included isotigs 01415, 02512 and 03151, was selected according to significant differential expression detected trough female/male RNA sequencing resultscomparison;2) the set was selected according to sequence homology (BLASTx) that comprised three sequences isotigs 06880, 07270 and 08283. All GI were subjected to RT-qPCR analysis using IQTMSYBR®GreenSupermix (Bio-Rad). Primers were designed using Primer-BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The cycling parameters were 95 °C for 5 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 45 s ending with a melting curve product amplification. Relative gene expression was analyzed by the multiple reference gene method . Elongation factor 1-alpha (ef1-α) and β actin of D. longicaudata were used as the internal reference genes, as has been used in other insect [56, 57]. Statistical analysis was performed using the Student t-test.
In order to identify SSRs markers, we ran the software MISA [ref, http://pgrc.ipk-gatersleben.de/misa/misa.html].For SSR selection the criteria used werebased on the minimum number of repeats as follows: five for dinucleotide, four for trinucleotide, and three for tetra, penta and hexanucleotide motives.
To develop all pairs of primers which amplify the corresponding SSR marker in every unigene, we ran an in-house script which uses EPRIMER32 from the EMBOSS package (http://emboss.sourceforge.net/apps/cvs/emboss/apps/eprimer32.html).
CSD, Complementary Sex Determination; SSR, Simple Sequence Repeats; SNP, Single Nucleotide Polymorphism; MEGISD, Maternal Effect Genomic Imprinting Sex Determination; RT-qPCR, reverse transcription quantitative polymerase chain reaction; GI, Gene of Interest; ID, Identification; INTA, Instituto Nacional de Tecnología Agropecuaria, Argentina; SENASA, Servicio Nacional de Sanidad y Calidad Agroalimentaria, Argentina; EU, Expression Units
We are indebted to the two anonymous reviewers for their valuable comments and suggestion that helped us to improve the manuscript. We wish to thank Dr. V. Romanowski and his staff at Institute of Biotechnology and Molecular Biology (UNLP, La Plata, Argentina). We are also very grateful to Dr. Carabajal Paladino (Laboratory of Molecular Cytogenetics, Biology Centre ASCR, Ceske Budejovice, Czech Republic) for her invaluable contribution to improve this manuscript. We thank German Crippa for his technical assistance in D. longicaudata rearing. We would also like to thank INDEAR (Rosario Biotechnology Institute, Rosario, Argentina) for technical assistance.
This work was partially supported by the National Institute of Agricultural Technology (INTA) through the project AEBIO-242411 (module pests) to SBL and by the
Agencia Nacional de Promoción Científica y Tecnológica de Argentina (ANPCyT) through Fondo Nacional deCiencia y Tecnología (FONCyT) (grants PICT/2008 0502) to JLC.
Availability of supporting data
Raw sequence reads of D. longicaudata have been submitted to NCBI Sequence Read Archive under the accession number SRP072867; Bioproject PRJNA317427; Biosamples SRS1376223, SRS1376224 and SRS1376239 (corresponding to raw sequences generated from larvae, female and male libraries respectively). This Transcriptome Shotgun Assembly project has been deposited at GenBank under the accession GELG00000000. The version described in this paper is the first version, GELG01000000.
MCM designed the study, carried the laboratory work with ACS help, worked on sequence analysis, and drafted the manuscript. ACS helped in laboratory work and drafted the manuscript. JLC helped in the design of the study and to draft the manuscript, MR, SG, MF participated in all bioinformatic work, and helped to draft the manuscript. SBL designed the study and the analysis, wrote the manuscript. All the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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