Genome-wide identification of long noncoding RNA genes and their potential association with fecundity and virulence in rice brown planthopper, Nilaparvata lugens
© Xiao et al. 2015
Received: 14 June 2015
Accepted: 23 September 2015
Published: 5 October 2015
The functional repertoire of long noncoding RNA (lncRNA) has been characterized in several model organisms, demonstrating that lncRNA plays important roles in fundamental biological processes. However, they remain largely unidentified in most species. Understanding the characteristics and functions of lncRNA in insects would be useful for insect resources utilization and sustainable pest control.
A computational pipeline was developed to identify lncRNA genes in the rice brown planthopper, Nilaparvata lugens, a destructive rice pest causing huge yield losses. Strand specific RT-PCR were used to determine the transcription orientation of lncRNAs.
In total, 2,439 lncRNA transcripts corresponding to 1,882 loci were detected from 12 whole transcriptomes (RNA-seq) datasets, including samples from high fecundity (HFP), low fecundity (LFP), I87i and C89i populations, in addition Mudgo and TN1 virulence strains. The identified N. lugens lncRNAs had low sequence similarities with other known lncRNAs. However, their structural features were similar with mammalian counterparts. N. lugens lncRNAs had shorter transcripts than protein-coding genes due to the lower exon number though their exons and introns were longer. Only 19.9% of N. lugens lncRNAs had multiple alternatively spliced isoforms. We observed biases in the genome location of N. lugens lncRNAs. More than 30% of the lncRNAs overlapped with known protein-coding genes. These lncRNAs tend to be co-expressed with their neighboring genes (Pearson correlation, p < 0.01, T-test) and might interact with adjacent protein-coding genes. In total, 19-148 lncRNAs were specifically-expressed in the samples of HFP, LFP, Mudgo, TN1, I87i and C89i populations. Three lncRNAs specifically expressed in HFP and LFP populations overlapped with reproductive-associated genes.
The structural features of N. lugens lncRNAs are similar to mammalian counterparts. Coexpression and function analysis suggeste that N. lugens lncRNAs might have important functions in high fecundity and virulence adaptability.
This study provided the first catalog of lncRNA genes in rice brown planthopper. Gene expression and genome location analysis indicated that lncRNAs might play important roles in high fecundity and virulence adaptation in N. lugens.
The development of high-throughput techniques has accelerated the sequencing of insect genomes and transcriptomes, leading to the rapid accumulation of insect gene data. Currently, 156 insect genomes have been sequenced and were deposited in the NCBI genome database , mainly from Diptera, Lepidoptera, and Hymenoptera. Hundreds of insect transcriptomes have been submitted to the NCBI SRA database . Huge amounts of insect RNA-seq data provide valuable resources to retrieve gene sequences and to estimate gene abundance by counting the read numbers . However, major works on insect genome annotation and RNA-seq analysis have been limited to protein-coding genes.
Increasing evidence has showed that noncoding RNA (ncRNA) genes exist widely in the genomes of almost all organisms [4, 5]. ncRNAs are arbitrary classified into two types based on their sizes. One type is small RNAs, which are shorter than 200 nucleotides (nt), including but not limited to microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and transfer RNAs (tRNAs). The other type is long noncoding RNAs (lncRNAs), with transcripts longer than 200 nt that lack protein-coding potential . The lncRNAs located in the intergenic region are named as long intergenic noncoding RNAs (lincRNAs). LncRNAs with transcripts longer than 50 Kb are defined as very long noncoding RNAs (vlncRNAs) . RNA-sequencing (RNA-seq) data are very useful resources to identify lncRNAs. Several international genome consortia, such as FANTOM, ENCODE, GETx, and modENCODE, have developed several computational approaches and identified thousands of lncRNA genes from a variety of species [8–12]. More than 9000 lincRNA genes were discovered in the human genome [8, 13–17] and >10,000 lincRNAs were found in the mouse genome. By analyzing 93 samples and expressed sequence datasets, 6621 lincRNAs from 4515 gene loci were identified from the pig genome . In a chicken RNA-Seq dataset, Li et al. found 281 novel lincRNA genes associated with muscle development . Jenkins et al. used a computational pipeline to identify lncRNAs from multiple Anopheles gambiae deep RNA-seq data, yielding 2949 lncRNA genes. These lncRNAs showed differential expression across the life stages. The secondary structures of lncRNAs are highly conserved within the Gambiae complex . As an important model organism, Drosophila melanogaster has been extensively investigated for its lncRNA genes. Several efforts have identified 3193 lncRNA genes in D. melanogaster [20–22].
Distinct roles have been characterized for only a small subset of lncRNAs and the function of the vast majority of lncRNAs remains unknown. Several studies have shown that lncRNAs play essential roles in a wide variety of fundamental biological processes, such as cell differentiation , pluripotency maintenance , transcription regulation , epigenetic regulation [25, 26], dosage compensation , and tumorigenesis . In D. melanogaster, a yellow-achaete intergenic RNA (yar) affects sleep behavior. Yar is conserved in Drosophila species . A neural-specific lncRNA, CRG, regulates the locomotor activity and climbing ability in Drosophila . These studies suggested that lncRNAs have much more important roles than expected.
The rice brown planthopper, N. lugens, is one of the most destructive insect pests in rice production. It directly sucks the phloem sap and transmits viruses, causing huge yield losses. The rice brown planthopper has two types of wings, long wing and short wing. The wing dimorphism is regulated by insulin receptors . The long-winged brown planthopper migrates from tropical to temperate regions in summer and then back to the tropics in the autumn. In the immigrant areas, the brown planthopper population increases very quickly in one or two generations. This notorious pest has repeatedly adapted to resistant rice varieties used for pest control . The high fecundity and virulence adaptation of N. lugens are major factors causing the high damage to rice. Insecticides are one of the most widely used methods to control rice brown planthopper. However, overuse of insecticides has resulted in resistance, resurgence, and residues. Understanding the mechanism of high fecundity and virulence adaptation is important to develop alternative pest control strategies. Here, we constructed a computational pipeline to identify lncRNAs from RNA-seq datasets of 12 samples of rice brown planthopper. We identified several lncRNAs specifically expressed in a high fecundity N. lugens population and found that expression patterns of lncRNAs varied between N. lugens strains/populations, suggesting that lncRNAs might have key roles in the fecundity and virulence of the rice brown planthopper.
Identification and validation of lncRNAs in N. lugens
The numbers of lncRNAs in individual RNA-seq datasets of different N. lugens strains/populations and in the comprehensive RNA-seq dataset
lncRNA in 12 samples
Number of lncRNA
LFPb adult 2 d
HFPb adult 2 d
LFP 5th instar larva
HFP 5th instar larva
Mudgoc fat body
TN1c fat body
Mudgo salivary gland
TN1 salivary gland
Intronic overlap3 (−)
Exonic overlap4 (+)
Exonic overlap5 (−)
Splice junction overlap6
Structural features of lncRNAs in N. lugens
Specifically-expressed and differentially-expressed lncRNAs
LncRNAs associated with fecundity
Specifically expressed N. lugens lncRNA in varied RNA-seq datasets
Specific expressed lncRNA distribution according to the distance with the closest gene (lncRNA transcript number/closest gene number)
HFP adult 2 d
LFP adult 2 d
HFP 5th instar larva
LFP 5th instar larva
Mudgo fat body
TN1 fat body
Mudgo salivary gland
TN1 salivary gland
In the HFP population, there were 6992 protein-coding genes that were co-expressed with lncRNAs, among which 46 protein-coding genes involve in energy metabolism. In the LFP population, there were 7089 protein-coding genes that were co-expressed with lncRNAs, among which 48 protein-coding genes involve in energy metabolism. The protein coding genes associated with energy metabolisms were not located adjacently or overlapped with any lncRNAs.
We identified 2439 lncRNA transcripts corresponding to 1882 loci from 12 N. lugens RNA-seq datasets including four transcriptome datasets of LFP or HFP fecundity strains, five transcriptomes from the fat body, salivary gland, and antennae of the virulence strain and three other different populations. BLASTN searching of N. lugens lncRNAs against the NCBI nr and NONCODE databases found no highly similar sequences, demonstrating that lncRNAs lack sequence conservation. However, it has been reported that an lncRNA, yar, is conserved in Drosophila species . The lncRNA secondary structures of A. gambiae were conserved within the Gambiae complex . Here, we found that structural features of N. lugens lncRNAs are similar to mammalian counterparts. We also performed RT-PCR and strand-specific PCR to confirm the expression of 20 randomly selected lncRNAs. The results indicated that >80 % identified lncRNAs were reliable. They were unlikely to be the artifacts of full-length coding sequences.
It has been reported that the functions of lncRNAs can be inferred by analyzing their co-expression networks and genome locations [32, 33]. In D. melanogaster, the lncRNA CRG positively regulates its neighboring Ca(2+)/calmodulin-dependent protein kinase, which is essential for locomotor activity and climbing ability . 19–148 N. lugens lncRNAs were specifically-expressed in the HFP, LFP, TN1 and Mudgo populations, respectively. However, less than ten lncRNAs were differentially-expressed in various samples. At least three specifically-expressed lncRNAs, BPHOGS10005591-OT2, BPHOGS100007976-OT, and BPHOGS10035598-OT, overlap with reproduction-associated genes that have important functions in sperm storage and embryo dorsoventral polarity. These lncRNAs are also co-expressed with the reproduction-associated genes. According to the evidence of co-expression and genome-location, lncRNAs might have important roles in regulating fecundity in N. lugens. We did not find any lncRNA to be located adjacently to protein-coding genes associated with the virulence adaptation of N. lugens, possibly because the mechanism of virulence remains largely unknown. High fecundity and virulence adaptability are two main factors that underlie the great damage caused by N. lugens [31, 34]. We found indication that lncRNA might participate in the regulation of at least one of these two important biological processes, which should provide new insights into developing alternative eco-friendly pest-control policies for the rice brown planthopper. However, it should be noticed that the evidence presented here are not direct.
A computational pipeline was constructed to identify lncRNA genes from the rice brown planthopper, yielding 2439 lncRNA transcripts corresponding to 1882 loci. Insect lncRNAs shared similar structural gene features with mammalian lncRNAs. 19–148 lncRNAs were specifically-expressed in high fecundity or low fecundity populations. At least three of them were overlapped with reproductive-associated genes. In terms of genome-location and gene-expression, we presented some indications that lncRNAs might play important roles in fecundity and virulence adaptation in N. lugens. Function analysis of lncRNAs is required to elucidate their roles in regulating fecundity and virulence adaptation.
The rice brown planthoppers were collected from rice fields in Nanjing area, Jiangsu Province, China and maintained on rice seedlings at 27 ± 1 °C, under a 16-h light/8-h dark photoperiod and 70–80 % relative humidity. The insects were transferred to fresh seedlings every 5–7 days to ensure sufficient nutrition.
The draft genome sequences of N. lugens were kindly provided by Professor Chuanxi Zhang in Zhejiang University . We annotated the genome sequences using the OMIGA pipeline  and deposited the annotation information in InsectBase (http://www.insect-genome.com/). We obtained 12 transcriptomes of N. lugens, including transcriptome of the 5th instar nymph of a low fecundity population (LFP) and a high fecundity population (HFP), two-days old adults of LFP and HFP population and a wild population. These populations had similar genetic background because they were selected from a starting population. All insects were maintained at same conditions and the transcriptomes were sequenced with a same protocol. The detailed method procedures of sequencing the transcriptome of LFP, HFP and the control population have been reported in . The other seven transcriptomes included the salivary glands of the Mudgo and TN1 population, the fat body of the Mudgo and TN1 population, the antennal of the TN1 population, I87i and C89i population. The transcriptome data were downloaded from the NCBI SRA database [38, 39]. The accession numbers were SRX276866 (the salivary glands of the Mudgo population), SRX276865 (the salivary glands of the TN1 population), SRX360414 (the fat body of the Mudgo population), SRX360412 (the fat body of the TN1 population), SRX290503 (the antennal of the TN1 population), DRX014540 (I87i strain), and DRX014541 (C89i strain).
All these transcriptomes were sequenced using the Illumina sequencing platform (GAII). Random hexamers were used in the cDNA synthesis. Total RNA were used for sequencing the transcriptomes of TN1, Mudgo, I87i and C89i populations whereas poly(A) + RNA were used for constructing the cDNA libraries in sequencing the transcriptomes of LFP, HFP and the control population. In this case, only those lncRNAs with poly (A) tails can be found from the transcriptomes of LFP, HFP and the control populations. It should be noted that many lncRNAs do not have poly (A) tail. These lncRNAs cannot be found from these transcriptomes.
Developing a computational pipeline to identify lncRNAs
A computational pipeline was constructed to identify lncRNA genes from the RNA-seq data. First, the RNA-seq reads of 12 N. lugens RNA-seq datasets were mapped to the genome using TopHat . For the first run, the reads from each RNA-seq dataset were mapped to the genome independently. The junction outputs from each RNA-seq dataset were pooled together as a Pooled Junction Set. This allowed TopHat to use junction information from all RNA-seq datasets. For the second run, TopHat was run on each RNA-seq dataset separately using the Pooled Junction Set. The output of this second run produced the final junction set for transcript assembly using Cufflinks . Second, the assembled transcripts of the 12 RNA-seq datasets were combined together by Cuffcompare, using N. lugens genome-annotation information. The transcripts that satisfied two criteria were retained: length ≥ 200 nt and exon numbers ≥ 2. This step produced 94,388 transcripts corresponding to 43,474 loci. Third, their protein coding potentials were examined by the software getorf (http://emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html). Transcripts with an open reading frame ≥ 300 nt were removed. Fourth, the remaining transcripts were searched against the SWISS-PROT database using BLASTX. Those transcripts that had BLAST hits with known proteins (e-value < 0.001) were regarded as mRNA transcripts and removed. We also removed the putative untranslated region fragments of known mRNA transcripts by sequence alignments, producing 9392 transcripts corresponding to 6734 loci. Fifth, all 9392 transcripts were estimated by the software Coding Potential Calculator (CPC, http://cpc.cbi.pku.edu.cn/). Only those transcripts with a CPC score ≤ −1 were kept, yielding 6175 transcripts corresponding to 4490 loci. Sixth, the remaining transcripts were used to search against the Pfam database using the software Hmmer . Those transcripts that had the potential to encode conserved domains or motifs were removed. In the last step, we removed known tRNAs), ribosomal RNAs (rRNAs), snoRNA, and small nuclear RNAs (snRNAs) by searching the Rfam database using Infernal  and BLASTN against the NONCODE database , producing the final lncRNA gene sets.
LncRNA gene expression analysis in 12 N. lugens RNA-seq datasets
The transcript abundance of the identified lncRNA genes were estimated by counting reads and normalizing with the software Cuffdiff , which used T-test to measure the significance of the expressional difference. A heatmap was produced by analyzing the expression abundance of lncRNA genes with the software Clustering . The average linkage method was used and the results were viewed using Java TreeView . If the expression of a lncRNA meets following criteria, we defined it as the specifically-expressed lncRNA: 1) the expression is > 3 FPKM in one sample whereas it is < 1 FPKM in other samples; 2) the FPKM of this lncRNA in one sample is at least 10-fold higher than those in other samples. For finding differentially-expressed lncRNAs, the cutoff was set as p-value <0.01 and q-value < 0.05. q-value means the FDR-adjusted p-value of the test statistic.
Co-expression analysis of protein-coding genes and lncRNAs
Co-expression analysis was performed between lncRNAs and protein-coding genes using all 12 transcriptome RNA-seq datasets. Pearson product–moment correlation coefficient was used to estimate the co-expression relationship by using a R script. The lncRNA: mRNA relationship with |r| > 0.8 were treated as the strong correlation.
Structural gene features of N. lugens lncRNAs
Gene structures of lncRNA genes were constructed by aligning lncRNAs with the N. lugens genome. The protein-coding gene information was obtained by the OMIGA annotation. The lengths of exons and introns were calculated. We wrote a Perl scalable vector graphics module to draw the exon-intron structures of the lncRNA genes. The software Geneious was used to show the transcript structure of lncRNA and protein-coding genes .
Total RNA isolation and cDNA synthesis
The third to the fifth instar of N. lugens nymph and adult were chose for gene expression analysis. Total RNA was extracted from 50 individuals of a wild population using the TRIzol® reagent, following the manufacturer’s instructions (Life Technologies, CA, USA). RNA integrity was checked by electrophoresis using 1.2 % agarose gels. The RNA purity was examined using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA synthesis was performed following the manufacturer’s instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan). Random primers were used in the cDNA synthesis for RT-PCR amplification of lncRNAs. Gene-specific primers (GSP) were used in the cDNA synthesis for the strand-specific RT-PCR.
We randomly selected 20 lncRNA genes for validation. The rice brown planthoppers from a wild population were used for extracting total RNA. The strand-specific RT-PCR was used to determine the transcript orientation. In the cDNA synthesis, three reactions were used: Forward (F) primer with reverse transcriptase (RT), reverse (R) primer with RT, both F and R primers without RT. To validate the alternative splicing of lncRNAs, we selected BPHLNC-unc241 for isoform-specific PCR. This lncRNA gene has ten alternatively spliced transcripts. The transcription of three reproduction-associated protein-coding genes (BPHOGS10005591, BPHOGS10007976 and BPHOGS10035598) and their overlapping lncRNA genes (BPHOGS10005591-OT2, BPHOGS10007976-OT and BPHOGS10035598-OT) were also confirmed by RT-PCR.
The primers were designed using an Integrated DNA Technologies online tool (IDT, Coralville, IA, USA; http://www.idtdna.com/Scitools/) and the primer sequences are shown in Additional files 14, 15 and 16: Tables S10, S11 and S12. Premix Taq® Version 2.0 kit (Takara) was used for the PCR reactions, which were performed in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). PCR conditions were 94 °C for 5 min; followed by five cycles of 94 °C for 30 s, 60 °C (reduced by 1 °C/cycle) for 30 s and 72 °C for 1 min; and then 28 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. The last step was followed by final extension at 72 °C for 10 min. The PCR products were checked by electrophoresis using 1.5 % agarose gels. The PCR products were purified by using Wizard HSV Gel (Promega, Madison, WI, USA), following the manufacturer’s instructions. The PCR products were sequenced by the GeneScript Company (Nanjing, China).
This work was supported by National Basic Research Program of China (2012CB114102).
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