Identification of miRNAs Mediating Seed Storability of Maize during Germination Stage by High-Throughput Sequencing, Transcriptome and Degradome Sequencing

Abstract

Seed storability is an important trait for improving grain quality and germplasm conservation, but little is known about the regulatory mechanisms and gene networks involved. MicroRNAs (miRNAs) are small non-coding RNAs regulating the translation and accumulation of their target mRNAs by means of sequence complementarity and have recently emerged as critical regulators of seed germination. Here, we used the germinating embryos of two maize inbred lines with significant differences in seed storability to identify the miRNAs and target genes involved. We identified a total of 218 previously known and 448 novel miRNAs by miRNA sequencing and degradome analysis, of which 27 known and 11 newly predicted miRNAs are differentially expressed in two maize inbred lines, as measured by Gene Ontology (GO) enrichment analysis. We then combined transcriptome sequencing and real-time quantitative polymerase chain reaction (RT-PCR) to screen and confirm six pairs of differentially expressed miRNAs associated with seed storability, along with their negative regulatory target genes. The enrichment analysis suggested that the miRNAs/target gene mediation of seed storability occurs via the ethylene activation signaling pathway, hormone synthesis and signal transduction, as well as plant organ morphogenesis. Our results should help elucidate the mechanisms through which miRNAs are involved in seed storability in maize.

Keywords: degradome sequencing; maize; miRNA; seed storability; transcriptome.

Figure 1

Changes of germination percentage and water content of two inbred lines Dong 156 and Dong 237. (A) The change in Dong156 and Dong237 seed germination during accelerated aging. The seeds were aged under 45 °C and 95% RH. The error bars indicate ± SE (n = 3), (B) the moisture content change curves of Dong156 and Dong237 of the control group, (C) the moisture content change curves of in the fourth day artificial aging treatment group Dong156 and Dong237.

Figure 2

Summary of miRNA sizes and differentially expressed miRNAs and the number of identified known miRNAs. (A) The size distribution of unique sRNAs. (B) Differential expression heat maps of known miRNAs and putative miRNA s in maize. R+n means that there are n more bases on the right end of the miRNA included in the miRBase, R−n means that there are n fewer bases on the right end of the miRNA included in the miRBase, L+n means that there are n more bases on the left end of the miRNA included in the miRBase, L+n means that there are n more bases on the left end of the miRNA included in the miRBase, 2ss12AT20TA means that the 12th base T is replaced by A (ss, substitution), and the 20th base T is replaced by A, a total of 2. New miRNAs are labeled with PC (Predicted Candidate).

Figure 3

The miRNA, target, and degradation product abundances by RNA seq and validation by alternative methods. (A) high throughput sequencing to verify miRNA, the orange bars indicate the up-regulated miRNA and the blue bars indicate the down-regulated miRNA. (B) qRT-PCR to verify miRNA, the orange bars indicate the up-regulated miRNA and the blue bars indicate the down-regulated miRNA. (C) high throughput sequencing to verify target genes, the orange bars indicate the up-regulated target genes and the blue bars indicate the down-regulated target genes. (D) qRT-PCR to verify target genes, the orange bars indicate the up-regulated target genes and the blue bars indicate the down-regulated target genes. (E–L) the relative abundance of zma-miR169o-5p, zma-miR390a-5p, zma-miR396c_L-1, zma-miR397b-p5, zma-miR444, and novel-miR4 and their target genes. The red dot is the same position of the predicted target gene and the degradation site obtained from the degradome sequencing, and the black line is the actual degradation site measured in the degradome sequencing. (M) zma-miR169o-5p_R-1 acts on the cleavage site of the target gene GRMZM2G165488 were confirmed by 5′RLM-RACE. (N) zma-miR444a_1ss12TC acts on the cleavage site of the target gene GRMZM2G399072 were confirmed by 5′RLM-RACE. The yellow bars represent the exon part of the gene, the green bars represent the cutting site part of the target gene, the black broken line represents the intron part of the gene, and the blue bars represent other structures of the gene.

Figure 4

Differential expression of the known and putative miRNAs and target genes in maize. FC, fold change. Orange bars represent expression of miRNAs; blue bars represent expression of target genes.

Figure 5

Analysis of the targets of identified miRNAs in maize seed storability. (A) Hierarchical clustering of DEGs expression. (B) Profile of GO analysis of the targets of identified responsive miRNAs in maize seed storability. (C) Profile of KEGG analysis of the targets of identified responsive miRNAs in maize seed storability.

Figure 6

Analysis of differentially expressed genes in transcriptome. (A) Gene ontology LoopCircos of DEGs. (B) The volcano map of differentially expressed genes. (C) Hierarchical clustering of DEGs expression. (D) KEGG enrichment analysis of DEGs.

Figure 7

Possible microRNAs-dependent regulatory pathways that participate in seed storability during maize germination.