Integrated microRNA and transcriptome profiling reveal key miRNA-mRNA interaction pairs associated with seed development in Tartary buckwheat (Fagopyrum tataricum)

Abstract

Background: Tartary buckwheat seed development is an extremely complex process involving many gene regulatory pathways. MicroRNAs (miRNAs) have been identified as the important negative regulators of gene expression and performed crucial regulatory roles in various plant biological processes. However, whether miRNAs participate in Tartary buckwheat seed development remains unexplored.

Results: In this study, we first identified 26 miRNA biosynthesis genes in the Tartary buckwheat genome and described their phylogeny and expression profiling. Then we performed small RNA (sRNA) sequencing for Tartary buckwheat seeds at three developmental stages to identify the miRNAs associated with seed development. In total, 230 miRNAs, including 101 conserved and 129 novel miRNAs, were first identified in Tartary buckwheat, and 3268 target genes were successfully predicted. Among these miRNAs, 76 exhibited differential expression during seed development, and 1534 target genes which correspond to 74 differentially expressed miRNAs (DEMs) were identified. Based on integrated analysis of DEMs and their targets expression, 65 miRNA-mRNA interaction pairs (25 DEMs corresponding to 65 target genes) were identified that exhibited significantly opposite expression during Tartary buckwheat seed development, and 6 of the miRNA-mRNA pairs were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and ligase-mediated rapid amplification of 5' cDNA ends (5'-RLM-RACE). Functional annotation of the 65 target mRNAs showed that 56 miRNA-mRNA interaction pairs major involved in cell differentiation and proliferation, cell elongation, hormones response, organogenesis, embryo and endosperm development, seed size, mineral elements transport, and flavonoid biosynthesis, which indicated that they are the key miRNA-mRNA pairs for Tartary buckwheat seed development.

Conclusions: Our findings provided insights for the first time into miRNA-mediated regulatory pathways in Tartary buckwheat seed development and suggested that miRNAs play important role in Tartary buckwheat seed development. These findings will be help to study the roles and regulatory mechanism of miRNAs in Tartary buckwheat seed development.

Keywords: AGO; DCL; Flavonoids; Integrated analysis; RDR; Seed development; Seed size; Target gene; Tartary buckwheat; miRNAs.

Fig. 1

Phylogenetic analysis of Tartary buckwheat RDR, DCL and AGO proteins. The corresponding proteins of Arabidopsis, maize, and rice were also used to construct the phylogenetic tree. The phylogenetic tree was constructed by using MUSCLE and MEGA 7.0 with the Maximum Likelihood method

Fig. 2

Expression profiles of Tartary buckwheat miRNA biosynthesis genes in different organs and during seed development. The different colors represent the expression abundance of the genes

Fig. 3

Seed phenotypes of Tartary buckwheat at the initial filling stage (S1), peak filling stage (S2), and initial maturity stage (S3)

Fig. 4

DEMs during Tartary buckwheat seed development. a Venn diagram represented the overlap of DEMs among the comparisons. b Number of DEMs in the comparisons of S1 vs. S2, S1 vs. S3, and S2 vs. S3. Green and yellow bars represented the number of up- or down-regulated genes, respectively. c Expression heat map of DEMs. The different colors represent the expression abundance of the miRNAs

Fig. 5

Quantitative real-time PCR verification of 9 DEMs at different developmental stages in Tartary buckwheat seeds

Fig. 6

miRNA-mRNA interaction pairs showed significant negative correlation (R ≥ 0.5, P < 0.05) of expression during Tartary buckwheat seed development. Left: Heat map of DEMs. Right: Heat map of the differentially expressed target mRNAs of DEMs. The different colors represent the expression abundance of the miRNAs and their corresponding target mRNAs

Fig. 7

Quantitative real-time PCR validation of six identified key miRNA-mRNA interaction pairs for Tartary buckwheat seed development. Green and blue bars represented the miRNAs and their corresponding target mRNAs, respectively. The error bar represents the error values of three biological replicates

Fig. 8

5′ RLM-RACE verification of six identified key miRNA-mRNA interaction pairs for Tartary buckwheat seed development. The numbers above sequences indicate the detected cleavage site of independent clones