Identification of MicroRNAs in Meloidogyne incognita Using Deep Sequencing

Abstract

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.

Fig 1. General description of the small RNA sequences of M. incognita.

(A) Size distribution of the raw reads of M. incognita small RNAs. (B) Classification of the small RNA reads.

Fig 2. Size distribution of reads from microRNA, protein encoding genes and others.

Fig 3. Distribution of bases at each position of mature microRNAs.

The first base of mature microRNA tend to be A and U.

Fig 4. min-miR-1 hairpin structure and sequencing profile.

(A) Hairpin structure predicted with RNAfold. (B) Proportion of small RNA tags mapped to the pre-miRNA of min-miR-1.

Fig 5. The expression abundance of microRNAs detected by real time RT-PCR (bars) and by high-throughput sequencing (lines).

Fig 6. Conservation of miR-100, in sequence and genomic organization.

(A) Sequence alignment of miR-100 microRNAs among different species (asu-: A. suum, bma-: B. malayi, bxy-: B. xylophilus, min-: M. incognita, dme-: D. melanogaster, has-: H. sapiens). The seed sequences indicated with red square area. (B) miR-100 and let-7 were clustered together in diverse animals but the miR-100 had lost in the common ancestor of C. elegans and P. pacificus.

Fig 7. The top 10 abundance miRNAs in M. incognita J2 library

Fig 8. M. incognita lacks Piwi-clade argonaute proteins, which is essential for Pi-RNA biosynthesis.

All of argonaute proteins, containing Piwi and PAZ domain, were identified from the genome of C. elegans, B. xylophilus, B. malayi, M. hapla and M. incognita and the multiple sequences alignment were carried out using mafft with default parameters. The phylogenetic tree was constructed using PhyML and the number on the branch indicated the bootstrap values.