Conservation and diversification of the miR166 family in soybean and potential roles of newly identified miR166s

Abstract

Background: microRNA166 (miR166) is a highly conserved family of miRNAs implicated in a wide range of cellular and physiological processes in plants. miR166 family generally comprises multiple miR166 members in plants, which might exhibit functional redundancy and specificity. The soybean miR166 family consists of 21 members according to the miRBase database. However, the evolutionary conservation and functional diversification of miR166 family members in soybean remain poorly understood.

Results: We identified five novel miR166s in soybean by data mining approach, thus enlarging the size of miR166 family from 21 to 26 members. Phylogenetic analyses of the 26 miR166s and their precursors indicated that soybean miR166 family exhibited both evolutionary conservation and diversification, and ten pairs of miR166 precursors with high sequence identity were individually grouped into a discrete clade in the phylogenetic tree. The analysis of genomic organization and evolution of MIR166 gene family revealed that eight segmental duplications and four tandem duplications might occur during evolution of the miR166 family in soybean. The cis-elements in promoters of MIR166 family genes and their putative targets pointed to their possible contributions to the functional conservation and diversification. The targets of soybean miR166s were predicted, and the cleavage of ATHB14-LIKE transcript was experimentally validated by RACE PCR. Further, the expression patterns of the five newly identified MIR166s and 12 target genes were examined during seed development and in response to abiotic stresses, which provided important clues for dissecting their functions and isoform specificity.

Conclusion: This study enlarged the size of soybean miR166 family from 21 to 26 members, and the 26 soybean miR166s exhibited evolutionary conservation and diversification. These findings have laid a foundation for elucidating functional conservation and diversification of miR166 family members, especially during seed development or under abiotic stresses.

Keywords: Evolutionary conservation and diversification; Gene expression pattern; Promoter analysis; Soybean; miR166 family.

Fig. 1

Stem-loop structures of the five newly identified pre-miR166s in soybean. The panel a-e sequentially correspond to pre-miR166v-z. The mature miRNA portion is highlighted in green bar

Fig. 2

Phylogenetic analysis and alignment of miR166 family in soybean. a Phylogenetic analysis of miR166 precursors. b Phylogenetic analysis and alignment of mature miR166s. The five newly identified miR166s in soybean are highlighted with a star, and the identity between paralogous pair is listed

Fig. 3

Chromosomal localization and duplication of MIR166 family genes in soybean. Each colored box represents a chromosome. The approximate distribution of each soybean MIR166 gene is marked on the circle with a short black line. Tandem duplication clusters are indicated with star. Colored lines indicate the linkage group with segmental duplicated MIR166 genes, and segmental duplication regions were determined using the Plant Genome Duplication Database

Fig. 4

Promoter analysis of MIR166 genes in soybean. Phylogenetic relationship of MIR166 promoters is shown in left panel. The 6 classes of cis-elements (I-VI) are responsive to hormones, abiotic stresses, seed development, HD-ZIPIII binding, biosynthesis and metabolism, cell cycle and circadian, respectively. If MIR166 harbors the cis-element in promoter region, the box is labeled with the number 1 and highlighted in blue color, otherwise the box is labeled with the number 0

Fig. 5

Expression analysis of soybean MIR166 genes in various tissues. The transcript profiling data of soybean tissues were extracted from the publicly-available Phytozome database (http://www.phytozome.net) for heatmap generation. The color scale above the heat map indicates gene expression levels, low transcript abundance indicated by green color and high transcript abundance indicated by red color. Maximum FPKM value for each MIR166 is shown

Fig. 6

Predicted targets of miR166 family and validation of miR166 target gene using RLM-RACE PCR. a The alignments of fragments of target mRNAs that have complementarity to miR166s are shown. miR166 sequences are displayed to highlight complementarity to target mRNAs. The targets are classified into seven groups (I, no annotation; II, ATHB-15; III, ATHB14-LIKE; IV, REVOLUTA-LIKE; V, PHYTOENE SYNTHASE; VI, AMMONIUM TRANSPORTER 2-LIKE; VII, two-component response regulator-like APRR2). b RLM-RACE PCR for ATHB14-LIKE. Lane 1 and M show cleaved product of desired size (734 bp) and DNA ladder, respectively. c Mapping of ATHB14-LIKE mRNA cleavage sites by RLM-RACE. The arrows indicate the cleavage sites and the numbers show the frequency of clones sequenced

Fig. 7

Phylogenetic analysis of unique target sequences (UTS) present in plant species and binding sites of miR166 in target genes. Target genes of miR166s in soybean are highlighted with a star. The details of UTSs were listed in Additional file 10: Table S7

Fig. 8

Expression patterns of miR166 target genes in different soybean tissues. The transcript profiling data of 12 annotated target genes in soybean tissues were extracted from the publicly-available Phytozome database (http://www.phytozome.net)

Fig. 9

Expression analysis of the five newly identified MIR166 genes in response to seed development and abiotic stresses. a Expression patterns of the 5 MIR166 genes during seed development (15, 30, 45, 65 days after flowering). b Expression patterns of the 5 MIR166 genes in seedlings exposed to cold stress for 0, 3, 6, 12, 24, 48 and 72 h. c Expression patterns of the 5 MIR166 genes in seedlings exposed to drought stress for 0, 2, 4, 6, 8 and 10 d. d Expression patterns of the 5 MIR166 genes in seedlings exposed to salinity stress for 0, 3, 6, 12, 24 and 72 h. Error bars indicate SE of two biological and three technical replicates. Values were normalized against the SUBI3 gene

Fig. 10

Expression analysis of the 12 target genes in response to seed development and abiotic stresses. Expression patterns of target genes during seed development (a), under cold stress (b), drought stress (c) and salinity stress (d). Stress treatments and seed development stage were used as same as the ones in Fig. 9. PSY, PHYTOENE SYNTHASE; AMT2-LIKE, AMMONIUM TRANSPORTER 2-LIKE. Error bars indicate SE of two biological and three technical replicates. Values were normalized against the SUBI3 gene