Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode Meloidogyne incognita

Abstract

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.

Keywords: Meloidogyne incognita; microRNA expression; quantitative polymerase chain reaction; stage-specific.

Figure 1

Flowchart of the miRNA analysis performed in the present study. The encircled numerals (I to V) indicate the order in which the experiments were carried out. The sequencing data from an Ilumina sequencer was used as raw data, which were further cleaned and annotated to segregate as well as identify different types of sRNAs. Known miRNAs were identified using miRBase and identification of novel miRNAs was carried out using MIREAP software, results from both of which were then analyzed to study families, clusters, and the validation of their expression.

Figure 2

Length distribution of small RNAs at different stages (egg, J2, J3, J4, and female). Frequencies of reads were normalized using DESeq2 to illustrate relative abundances of reads with different lengths under the same scale. (A) Clean reads; (B) unique reads.

Figure 3

Annotation of small RNAs of M. incognita. (A) Clean reads; (B) unique reads.

Figure 4

Pre-miRNA structure prediction of novel miRNAs from each stage of the M. incognita life cycle. Figures show structures, free energy values, and stem-loop structures for selected novel miRNAs.

Figure 5

Nucleotide bias at each nucleotide position of known and novel miRNA of all developmental stages combined. (A) Known miRNA from annotation aligned to the miRNA precursor/mature miRNA in miRBase (allowing two mismatches and free gaps); (B) novel miRNAs predicted from the unannotated small RNAs using MIREAP software (Read ≥ 3).

Figure 6

Distribution of miRNA expression across stages of the M. incognita life cycle. (A) Known miRNA (2724); and (B) novel miRNA (383).

Figure 7

Heat map for the DESeq2-normalized expression profiles of known and novel miRNA families in five developmental stages. (A) Known miRNA; (B) novel miRNA. In both cases, sequences with R > 10 were selected for normalization. The heatmap of the normalized data (Tables S11 and S12) was drawn using MeV (v4.8.1) Pearson correlation applied for clustering. The TreeView tool was used to improve the figure clarity.

Figure 8

Validation of sequencing and real-time expression of stage-specific miRNAs. (A) Expression profiles of selected known and novel stage specific miRNA from high-throughput sequencing data. The normalized values of the stage-specific miRNAs are given at the top of the columns. Columns with DESeq2 values not mentioned had a normalized score of 0.1 to 1.6; (B) relative expression levels of known and novel miRNA studied using qPCR. The miRNA MI03018 (miR-58) was used as an internal standard.