Genome-wide identification of long noncoding RNA genes and their potential association with fecundity and virulence in rice brown planthopper, Nilaparvata lugens

Abstract

Background: 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.

Methods: 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.

Results: 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.

Discussion: 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.

Conclusions: 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.

Fig. 1

The computational pipeline for identifying lncRNA genes in N. lugens from RNA-seq data

Fig. 2

Strand-specific PCR of 17 randomly selected lncRNAs to determine the transcription orientation. BPHOGS10035448-AS-RA was not amplified with a correct band. So, 16 lncRNAs were successfully amplified and confirmed by sequencing (see Figure S2 for RT-PCR validation). The results indicated that 12 lncRNA transcribed from the antisense strand, and four from the sense strand. F: forward primer (sense); R: reverse primer (antisense); RT: reverse transcriptase

Fig. 3

Structural gene features of N. lugens lncRNA genes. a The number of exons per transcript of lncRNAs and protein-coding genes. The majority of lncRNAs have only two exons. b The transcript lengths of lncRNA and protein-coding gene. On average, lncRNAs have short transcripts. c The distribution of exon sizes of lncRNA and protein-coding genes. d The distribution of intron sizes of lncRNA and protein-coding genes. The density in Y-axis means the area under the curve of a density function represents the probability of getting an x value between a range of x values. Red: lncRNA, green: protein-coding genes

Fig. 4

Gene structures of three lncRNA genes that had the most alternatively spliced isoforms. BPHOGS10000007-OT and BPHOGS10002343-OT have 11 spliced isoforms and BPHLNC-unc241 has 10 spliced isoforms

Fig. 5

RT-PCR validation of alternatively spliced transcripts of BPHLNC-unc241. Isoform-specific primers were designed for ten isoforms. Five of them were successfully amplified followed by sequencing. The PCR product size and the sequencing result of variant J was not as expect (a). The expressions of five isoforms in the third instar to fifth instar nymph and adult were measured, suggesting transcript variants vary in their expression profiles (b,c)

Fig. 6

The heatmap of the lncRNA expression patterns in virulent and fecund populations. a Expression profile changes of lncRNA transcripts across the fat body and salivary gland in the Mudgo and TN1 populations. b Hierarchical clustering of expressional abundance of lncRNA transcripts in the nymph and adult of the HFP and LFP population. The lncRNAs showing tissue specific expression in the Mudgo and TN1 populations are listed in Additional file 6, 7, 8 and 9: Tables S2, S3, S4 and S5. The lncRNAs specifically expressed in the fifth instar nymph and adult were listed in Additional file 10, 11, 12 and 13: Tables S6, S7, S8 and S9

Fig. 7

The genome location of lncRNA in N. lugens, demonstrating that lncRNAs tend to located adjacently to protein-coding genes (<5 Kb). A high number of lncRNAs overlapped with protein-coding genes

Fig. 8

The distribution of Pearson product–moment correlation coefficient between lncRNA and protein-coding genes. lncRNAs had higher coefficients with their neighboring protein-coding genes than with non-neighboring genes, suggesting that lncRNAs tends to be co-expressed with neighboring genes. The density in Y-axis means the area under the curve of a density function represents the probability of getting an x value between a range of x values

Fig. 9

Gene structures of five lncRNA genes. These lncRNA genes had multiple isoforms and overlapped with two adjacent protein-coding genes

Fig. 10

Exon and intron structures of three lncRNA genes that were specifically expressed in the HFP or LFP population. These lncRNAs overlapped with reproduction-associated protein genes encoding glucose dehydrogenase, gastrulation defective, and GALNT7. F: Forward primer, R: Reverse primer

Fig. 11

Amplification of three lncRNAs overlapped with reproduction-associated protein genes. Different combinations of primers pairs indicated that BPHOGS10005591-OT2 (a) and BPHOGS10007976-OT (b) were independently transcribed whereas BPHOGS10035598-OT (c) might share a same transcript with its adjacent protein-coding gene BPHOGS10035598