Library construction and sequencing of small RNAs

To identify the miRNAs in ginger, total RNAs were extracted from leaf, stem, root, flower, and rhizome in three different development stages: 1st I_d (first internode), 2nd I_d (second internode), 3rd I_d (third internode) of ginger and then used to construct 7 small RNA libraries. Then, the 7 small RNA libraries were sequenced using the BGI-tech Standard Small RNA sequencing platform and underwent bioinformatics analysis. A total of 28,804,171 raw tag counts from the leaf, 29,640,807 from the root, 29,630,116 from the 1st I_d of the rhizome, 29,084,284 from the 2nd I_d of the rhizome, 28,757,890 from the 3rd I_d of the rhizome, 29,115,222 from the stem, and 28,991,447 from the flower were obtained. After removal of adaptors, low quality reads, and contaminants, 26,648,833 clean tag counts from the leaf, 24,815,451 from the root, 26,324,400 from the 1st I_d of rhizome, 26,825,995 from the 2nd I_d of rhizome, 26,253,384 from the 3rd I_d of rhizome, 26,823,604 from the stem, and 26,988,071 from the flower were kept (Supplementary Table S1).

The clean reads and unique reads of the 7 tissues were subjected to an analysis of the size distribution, as shown in Fig. 1. The lengths of the small RNAs in 7 samples were 15 to 31 nt. The 21 nt class was the most abundant in the stem, followed by 24, 22, and 20 nt classes (Fig. 1a). In the rhizome, 24 nt small RNAs were the most frequent, followed by 21, 22, and 23 nt. The 24-nt peak was found to be dominant at a unique read level in all 7 samples except for the flower and leaf, while the peak of 21 nt small RNAs was found in the flower and leaf (Fig. 1b).

Fig. 1

The length distribution of the clean and unique reads from leaf, stem, root, flower, and rhizome (1st I_d, 2nd I_d, and 3rd I_d) of Z. officinale. a Clean reads; b unique reads

Conserved and novel miRNAs in ginger

To analyze the population of conserved miRNAs in ginger, the miRNA sequences of the 7 libraries were searched against the currently known mature miRNAs from other plants and ginger RNA sequences. As a result, a total of 160 conserved miRNA precursors were identified from 7 sRNA datasets (Supplementary Table S2). All these conserved miRNAs of ginger belong to 28 miRNA families, and at least one precursor was identified in most miRNA families. Among them, MIR396 was the largest family identified with 16 members, followed by MIR319 with 14 members. There were 13 members in the MIR156, MIR169, MIR171, and MIR166 families. However, several miRNA families were found to possess only one precursor, including MIR162, MIR165, MIR477, MIR5179, MIR528, MIR5523, and MIR5532.

The sRNA sequencing results indicated that the clean counts of conserved miRNAs ranged from 1 up to more than 10,000 in 7 samples. Of all conserved miRNAs, the clean counts of MIR159, MIR160, and MIR395 exceeded 10,000 in one tissue. The clean counts of ten miRNA families (MIR156, MIR164, MIR166, MIR167, MIR168, MIR171, MIR319, MIR396, MIR398, and MIR528) ranged from 10,000 to 50,000 at least in one tissue. However, the other miRNA families (MIR157, MIR160, MIR162, MIR165, MIR169, MIR172, MIR390, MIR393, MIR395, MIR399, MIR408, MIR5523, MIR5532, and MIR845) had fewer than 5000 reads in all 7 tissues (Supplementary Table S2).

In addition to the conserved miRNAs, some novel miRNA sequences were also found in the 7 libraries from the remaining sRNA sequences after removal of the tRNAs, rRNAs, snoRNAs, snRNAs, and known miRNAs. According to the criteria for plant miRNAs, a total of 104 novel miRNAs were identified in 7 libraries. The length of novel miRNA sequences ranged from 19 to 30 nt. However, the sequences of most novel miRNAs were 24 nt in length, followed by 25, 21, and 22 nt. The length of pre-miRNAs ranged from 52 to 1439 nt. The average minimum folding free energy value of the hairpin structures was − 160.719 kcal/mol in Z. officinale Roscoe (Supplementary Table S3). The secondary structures of 104 novel miRNA precursors are shown in Supplementary Fig. S1. The actual tag count span of novel miRNAs was large in the 7 libraries, varying from 0 to 74,105. Among these novel miRNAs, novel miR110, miR177, miR192, miR22, miR23, miR3, miR7, and miR90 accounted for more than 2000 counts in some of the 7 libraries.

The first nucleotide at the 5′-terminus of mature miRNA had a strong base preference. The conserved miRNAs had a predominance of U at the 5′-terminus except for the 18-nt clade (Fig. 2a). These results are in agreement with previous reports. We also found that miRNAs with a length of 22 nt possessed the lowest number of G bases, and the largest base species at the 5′-terminus existed in 21-nt miRNAs, which is in accordance with the characteristics of miRNA base preferences (Fig. 2a). In the conserved miRNA, uracil was the first preferred base of the 5′-terminus of the 21 potential novel miRNA sequences (Fig. 2b). As with the novel miRNAs, additional base species at the 5′-terminus were found in miRNAs with lengths of 21 and 27 nt.

Fig. 2

Statistics of the first base of conserved and novel miRNAs

miRNA expression profile in different tissues of ginger

miRNAs are known to play an important role in the growth and development of plants. Several miRNA genes are differentially expressed in specific tissues/organs. The expression profiles of miRNAs were normalized by transcripts per million (UMI) for further comparative analysis. Based on a criteria of |log2 (fold change)| ≥ 1 and qvalue< 0.005, differential expression patterns of 90 of the most abundant conserved miRNAs and 62 novel miRNAs in 7 libraries were analyzed.

We found that 17, 9, 8, 4, 13, 8, and 3 conserved miRNAs exhibited significant tissue-specific expression patterns in leaf, stem, root, flower, 1st I_d, 2nd I_d, and 3rd I_d of the rhizome, respectively (Fig. 3). In leaves, one or more isoforms of miR156, miR157, miR160, miR167, miR169, miR171, miR319, miR396, and miR528 were abundant, indicating that these miRNAs were involved in leaf development. miR167h, miR529-3p, miR529-5p, miR166b-5p-2, miR156c-3p-2, miR396b-3p-3, miR168-5p, miR390e, and miR396g-3p were more abundant in the flower than in other tissues. The expression of miR166k, miR396g-5p_1, miR171i-1, miR164a_1, miR408d, miR393-5p, and miR156a_2 was upregulated in roots. We also detected 3 miRNA families with more abundance in the stem (miR166, miR166m_2, miR168a-5p, miR168, miR168b_1, miR169_1, and miR169e_3).

Fig. 3

Expression profiles of conserved miRNAs in 7 different tissues. Fw: flower; Lf: leaf; St: stem; Rt: root; Rz_1: Rhizome 1st I_d; Rz_2: Rhizome 2nd I_d; Rz_3: Rhizome 3rd I_d

We found that 5, 2, 4, 7, 2, 5, and 2 novel miRNAs exhibited significant tissue-specific expression patterns in the leaf, stem, root, flower, 1st I_d, 2nd I_d, and 3rd I_d of the rhizome, respectively (Fig. 4 and Supplementary Table S2). The expression levels of novel_miR234, novel_miR108, novel_miR62, novel_miR149, and novel_miR158 in the flower were higher than those in other tissues. Novel_miR22, novel_miR129, novel_miR31, and novel_miR224 were detected with higher expression in leaves. Novel_miR97, novel_miR179, novel_miR5, and novel_miR9 were more abundant in roots, whereas novel_miR181, novel_miR14, novel_miR71, novel_miR17, and novel_miR58 were more abundant in stems.

Fig. 4

Expression profiles of novel miRNAs in 7 different tissues. Fw: flower; Lf: leaf; St: stem; Rt: root; Rz_1: Rhizome 1st I_d; Rz_2: Rhizome 2nd I_d; Rz_3: Rhizome 3rd I_d

Cluster analysis of differentially expressed miRNAs in ginger rhizome development

Among three different rhizome development stages (1st I_d, 2nd I_d, and 3rd I_d), approximately 22 conserved miRNAs were downregulated, and 8 miRNAs were conserved in the 2nd I_d in comparison with the 1st I_d. Additionally, 8 conserved miRNAs were downregulated, and 15 conserved miRNAs were upregulated in the 3rd I_d in comparison with the 2nd I_d (Supplementary Fig. S2). For example, the expression ratios (2nd/1st I_d) of miR164k and miR398 were 91.39 and 37.15, respectively. However, the 3rd/2nd I_d ratios of the two miRNAs were 0.011 and 0.0.026, respectively. Interestingly, miR169v_1 was gradually decreased during rhizome development.

The novel miRNAs were similar to the conserved miRNAs in that approximately 12 novel miRNAs were upregulated and 7 were downregulated in the 2nd I_d as compared to the 1st I_d stage. There were no changes in the other 14 novel miRNAs (Supplementary Table S3). Of the novel miRNAs, 12 were downregulated, while 11 novel miRNAs were upregulated, and most of them were upregulated in the 3rd I_d compared to the 2nd I_d. Novel miR90 was gradually decreased in rhizome development. There was higher expression of novel miR13 in the 1st I_d and decreased expression in the 2nd I_d stage, which then increased once more in the 3rd I_d. These results suggest that these miRNAs may play a major role in the rhizome growth and development of ginger (Supplementary Fig. S3).

MiRNA targets prediction in ginger rhizome

Plant miRNAs function with a mechanism of suppressing the translation of target genes or cleaving target mRNAs in diverse vital biological processes. The putative target gene prediction analyses showed that 365 target genes for 44 conserved miRNA families and 27 target genes for 8 novel miRNAs were predicted (Fig. 5 and Supplementary Table S4). It was found that the majority of the target genes for conserved miRNAs were transcription factors. A number of these target genes were conserved between ginger and other plants, such as MYB, TCP, auxin response factor (ARF), LOB domain-containing protein, meristem (NAM) protein, DCL1, NAC domain transcription factor, AGO1, APETALA2, ATP-dependent RNA helicase, squamosa promoter-binding proteins (SPL), and growth-regulating factors (GRF), which are involved in various aspects of secondary metabolic substance biosynthesis and the tissue growth and development of plants. However, some predicted target genes of several conserved miRNAs in ginger were different from those in other plants, including pirin-like protein, (MIR156) homeobox-leucine zipper protein (MIR166), and TIFY (MIR167) (Supplementary Table S4). It was mainly predicted that the target genes of novel miRNAs were MYB transcriptional factor, serine/threonine-protein kinase, multidrug resistance protein, F-box/LRR-repeat protein, polyol transporter, and LRR receptor-like serine/threonine-protein kinase (Supplementary Table S4).

Fig. 5

The regulatory network of miRNA and targeted genes in 3 rhizome developing stages. A red circle denotes miRNA, and a cyan hexagon denotes the predicted targets

Gene ontology (GO) of miRNA target genes in ginger rhizome

In order to further analyze the biological function of miRNA targets in rhizome development, GO analysis was implemented for all the miRNA target genes and differentially expressed genes between the 2nd and 1st I_d, and 3rd and 2nd I_d, respectively (Fig. 6). As shown in Fig. 6a, there are some specific functions with large inferences in the number of target genes in the three GO terms, such as metabolic regulation of biological process, cell process, cell part, organelle in cellular components, and binding in molecular function.

Fig. 6

GO analysis of miRNA targets involving rhizome development. a The GO analysis between the 2nd I_d and 1st I_d; b the GO analysis between the 3rd I_d and 2nd I_d. The ordinate is classified as GO, with the number of genes on the right side of the terms

Comparing the 3rd and 2nd I_d, some specific functions with large differences were found in the number of target genes in the three GO terms, such as nucleoid in cellular components and cell junction (Fig. 6b). Most of the miRNAs were downregulated in the 2nd I_d and then upregulated in the 3rd I_d, and thus, the significant difference in the number of genes for these secondary functions was probably caused by cell proliferation and expansion.

The expression of miRNAs and their target genes during ginger rhizome development

To better understand the biological function of miRNAs in the development of ginger rhizome, miRNA target genes were predicted and are shown in Supplementary Table S4. Many studies have shown that miRNAs negatively regulate target gene expression in the regulation of plant growth and development. To validate the regulation of target expression by miRNA, 8 miRNAs (miR156_2, miR156a, miR171b_2, miR172_2, miR164a_1, miR319e_1, miR319g-5p_1, and miR529) were selected, and their target gene expression in the 1st I_d, 2nd I_d, and 3rd I_d of ginger was detected using qRT-PCR analysis (Fig. 7).

Fig. 7

Validation of target genes by miRNAs using qRT-PCR

Maker00022469, Maker00027061, and Maker000044264 are members of the SPL family and were predicted targets of miR156_2. These three genes were found more abundantly in the rhizome 1st I_d, and then decreased in the 2nd and 3rd I_d stage, while tmiR156_2 exhibited the opposite expression pattern in comparison with these targets. Maker00000910, Maker00021643, and Maker00069047 were predicted targets of miR171a_2. miR319e-1 and miR319g-5p_1 were significantly upregulated during the enlargement of ginger rhizome, whereas their target genes, which encode MYB33-like (Maker00030506) and GRF4-like (Maker00009062), were significantly downregulated, respectively. In contrast, miR529-5p and miR172_2 were significantly down-regulated during the development of ginger rhizome, and their targets were significantly up-regulated. Like miR156a, miR529-5p also targeted SPL13 transcription factor genes (Maker00008769, Maker00052423, and Maker00060741) with different target sites.

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