Genome-wide profiling of miRNAs during seed development reveals their functional relevance in seed size/weight determination in chickpea

Abstract

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at transcriptional and post-transcriptional levels. The role of miRNAs in seed development and seed size/weight determination is poorly understood in legumes. In this study, we profiled miRNAs at seven successive stages of seed development in a small-seeded and a large-seeded chickpea cultivar via small RNA sequencing. In total, 113 known and 243 novel miRNAs were identified. Gene ontology analysis revealed the enrichment of seed/reproductive/post-embryonic development and signaling pathways processes among the miRNA target genes. A large fraction of the target genes exhibited antagonistic correlation with miRNA expression. The sets of co-expressed miRNAs showing differential expression between the two cultivars were recognized. Known transcription factor (TF) encoding genes involved in seed size/weight determination, including SPL, GRF, MYB, ARF, HAIKU1, SHB1, KLUH/CYP78A5, and E2Fb along with novel genes were found to be targeted by the predicted miRNAs. Differential expression analysis revealed higher transcript levels of members of SPL and REVOLUTA TF families and lower expression of their corresponding miRNAs in the large-seeded cultivar. At least 19 miRNAs known to be involved in seed development or differentially expressed between small-seeded and large-seeded cultivars at late-embryogenesis and/or mid-maturation stages were located within known quantitative trait loci (QTLs) associated with seed size/weight determination. Moreover, 41 target genes of these miRNAs were also located within these QTLs. Altogether, we revealed important roles of miRNAs in seed development and identified candidate miRNAs and their target genes that have functional relevance in determining seed size/weight in chickpea.

Keywords: Cicer arietinum; co‐expression network; miRNA; modules; seed development; seed size/weight; transcription factors.

FIGURE 1

Number of identified miRNAs and their size distribution during various stages of seed development in the chickpea cultivars. (a) Number of total, known, and novel miRNAs identified in JGK 3 and Himchana 1. (b) Length and 5’‐end base distribution in the identified miRNAs. (c, d) Size (20–22 nt) distribution of known and novel miRNAs identified in leaf and stages of seed development (S1–S7) in Himchana 1 (c) and JGK 3 (d)

FIGURE 2

Functional annotation of miRNA target genes. (a) Top 10 enriched biological process and molecular function gene ontology (GO) terms associated with the targets of miRNAs. Left panel represents P‐value of enrichment and right panel represents the fraction of targets for each biological process and molecular function GO term. (b) Distribution of various transcription factor families represented among the predicted target genes is given

FIGURE 3

Correlation of expression of miRNAs and their target genes. (a) Density plot showing correlation between expression levels of miRNAs and their target genes in Himchana 1 and JGK 3. The solid straight line represents a negative correlation value of ≤−0.25. (b) Enriched GO (biological process) terms among the target genes showing negative correlation (≤−0.25) with miRNA expression in the two chickpea cultivars. The enriched GO terms for up‐ and down‐regulated genes in Himchana 1 and JGK 3 are shown separately. The color scale and values in the colored boxes represent P‐value of enrichment

FIGURE 4

Differentially expressed miRNAs between JGK 3 and Himchana 1 and their targets. (a) Number of differentially expressed miRNAs between Himchana 1 and JGK 3 at each stage of seed development. (b) Boxplot showing expression of genes that are targeted by differentially expressed (upregulated and downregulated) miRNAs. (c) Enriched GO (biological process) terms in the target genes of differentially expressed miRNAs at each stage of seed development. Differential expression (up or down‐regulation) in JGK 3 at different stages of seed development are given on the top of each column. The color scale and values in the colored boxes represent P‐value of enrichment. (d) Number of differentially expressed miRNAs and their differentially expressed target genes in stages of seed development showing positive and antagonistic correlation. (e) Heatmap showing the differential expression of miRNAs and their target genes at the given stage (indicated on the right). The values in colored boxes (green, up‐regulated, and red, down‐regulated) represent a fold‐change expression of miRNAs and their target genes between the cultivars

FIGURE 5

Co‐expression network and stage‐specificity of miRNAs modules, and GO enrichment analysis. (a) Dendrogram showing the hierarchical clustering of miRNAs based on co‐expression network analysis. The color rows below the dendrogram indicate module membership. (b) Heatmap showing stage‐specificity of co‐expressed miRNA modules based on their eigengene expression values. The colors on the left represent different modules. (c) Bar‐plot of the modules exhibiting opposite eigengene expression pattern at S3 or S5 stages of seed development between Himchana 1 (HS3/HS5) and JGK 3 (JS3/JS5) cultivars. (d) Heatmap showing expression profiles of miRNAs (included in the modules shown in c) at S3 and S5 stages in the two cultivars. (e) Heatmaps showing expression profiles of the selected miRNAs at S3 and S5 stages of seed development in JGK 3 and Himchana 1 chickpea cultivars based on the smRNA‐seq (left) and RT‐qPCR (right) methods. Correlation between the expression values obtained in smRNA‐seq and RT‐qPCR data is given in the middle. (f,g) Enriched GO (biological process) terms among the targets of miRNAs included in modules showing opposite eigengene expression pattern at S3 (f) and S5 (g) stages. The color scale and values in the colored boxes represent P‐value of enrichment

FIGURE 6

miRNAs and their target genes located in the known seed size/weight associated QTLs. (a) Heatmap showing expression of miRNAs located within the known seed size/weight associated QTLs and exhibit differential expression between S3 and/or S5 stages of chickpea cultivars. (b) Enriched biological process GO terms among the targets of QTLs located miRNAs shown in (a). (c) The network of miRNAs (red triangles) given in (a) and their targets (green ovals) are shown. (d) Heatmap showing expression profile of the target genes of the QTL located miRNAs along with their annotation. The color scales shown in (a) and (d) represent Z‐score determined based on the normalized expression values of miRNAs and target genes, respectively