Small RNA sequencing provides candidate miRNA-target pairs for revealing the mechanism of apomixis in Zanthoxylum bungeanum

Xitong Fei,Anzhi Wei,Haichao Hu,Shujie Wang,Yu Lei,Yichen Qi

doi:10.1186/s12870-021-02935-5

Abstract

BackgroundApomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Therefore, apomixis technology has great application potential in plant breeding. To identify the apomixis types and critical period, embryonic development at different flower development stages of Zanthoxylum bungeanum was observed by cytology.ResultsThe results show that the S3 stage is the critical period of apomixis, during which the nucellar cells develop into an adventitious primordial embryo. Cytological observations showed that the type of apomixis in Z. bungeanum is sporophytic apomixis. Furthermore, miRNA sequencing, miRNA-target gene interaction, dual luciferase reporter assay, and RT-qPCR verification were used to reveal the dynamic regulation of miRNA-target pairs in Z. bungeanum apomixis. The miRNA sequencing identified 96 mature miRNAs, of which 40 were known and 56 were novel. Additionally, 29 differentially expressed miRNAs were screened according to the miRNAs expression levels at the different developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that the target genes of the differentially expressed miRNAs were mainly enriched in plant hormone signal transduction, RNA biosynthetic process, and response to hormone pathways.ConclusionsDuring the critical period of apomictic embryonic development, miR172c significantly reduces the expression levels of TOE3 and APETALA 2 (AP2) genes, thereby upregulating the expression of the AGAMOUS gene. A molecular regulation model of miRNA-target pairs was constructed based on their interactions and expression patterns to further understand the role of miRNA-target pairs in apomixis. Our data suggest that miR172c may regulates AGAMOUS expression by inhibiting TOE3 in the critical period of apomixis.

Highlights

Apomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother
The 35S::AP2-GUS, 35S::TOE3-GUS, and 35S:: miR172c-GUS produced a large amount of GUS protein after injection into tobacco leaves, while 35S::AP2-GUS + 35S::miR172c-GUS and 35S::TOE3-GUS + 35S:: miR172c-GUS significantly reduced GUS protein activity (Fig. 6a and b). These results suggest that miR172c could cleave TOE3 and APETALA 2 (AP2) genes and block the synthesis of the GUS protein, confirming that TOE3 and AP2 were its target genes
Using miRNA sequencing, miRNA-target gene interaction experiments, and RT-qPCR verification, we showed that miR172 inhibits the activity of TOE3 and AP2 during S3, thereby increasing the expression level of AGAMOUS

Summary

Introduction

Apomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Apomixis technology has great application potential in plant breeding. The male and female gametophytes combine to form a new plant [1]. Another mode of plant reproduction can produce offspring without sexual reproduction, known as apomixis. This is a reproductive mode that directly produces offspring, without combining the male and female gametes. Apomixis has considerable application potential in crop genetic breeding

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