Cytological observation and RNA-seq analysis reveal novel miRNAs high expression associated with the pollen fertility of neo-tetraploid rice

BACKGROUND: Neo-tetraploid rice lines exhibit high fertility and strong heterosis and harbor novel specific alleles, which are useful germplasm for polyploid rice breeding. However, the mechanism of the fertility associated with miRNAs remains unknown. In this study, a neo-tetraploid rice line, termed Huaduo21 (H21), was used. Cytological observation and RNA-sequencing were employed to identify the fertility-related miRNAs in neo-tetraploid rice. RESULTS: H21 showed high pollen fertility (88.08%), a lower percentage of the pollen mother cell (PMC) abnormalities, and lower abnormalities during double fertilization and embryogenesis compared with autotetraploid rice. A total of 166 non-additive miRNAs and 3108 non-additive genes were detected between H21 and its parents. GO and KEGG analysis of non-additive genes revealed significant enrichments in the DNA replication, Chromosome and associated proteins, and Replication and repair pathways. Comprehensive multi-omics analysis identified 32 pairs of miRNA/target that were associated with the fertility in H21. Of these, osa-miR408-3p and osa-miR528-5p displayed high expression patterns, targeted the phytocyanin genes, and were associated with high pollen fertility. Suppression of osa-miR528-5p in Huaduo1 resulted in a low seed set and a decrease in the number of grains. Moreover, transgenic analysis implied that osa-MIR397b-p3, osa-miR5492, and osa-MIR5495-p5 might participate in the fertility of H21. CONCLUSION: Taken together, the regulation network of fertility-related miRNAs-targets pairs might contribute to the high seed setting in neo-tetraploid rice. These findings enhance our understanding of the regulatory mechanisms of pollen fertility associated with miRNAs in neo-tetraploid rice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-023-04453-y.