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

Abstract

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.

Cytological Observation and RNA-Seq Analyses Reveal miR9564 and Its Target Associated with Pollen Sterility in Autotetraploid Rice

“…In recent years, various factors affecting autotetraploidization sterility have been identified, including meiotic genes, miRNAs, long non-coding RNA, and DNA methylation sites [ 6 , 10 , 11 , 12 , 13 , 14 , 19 , 33 , 34 ]. Among these regulators, miRNAs with variable expression patterns have been proposed as an important factor for low fertility of autotetraploid rice.…”

“…Relative to 02428-2x, 321 and 368 miRNAs were differentially expressed in anthers and ovaries of autotetraploid rice 02428-4x, respectively [ 13 ]. Additionally, some differentially expressed miRNAs in the meiotic anther relative to the fertile neo-tetraploid lines were identified, such as osa-miR408-3p and osa-miR528-5p [ 16 , 34 ].…”

“…One of these coDEMs, miR528 , has been previously reported to regulate pollen intine formation by targeting OsUCL23 to influence flavonoid metabolism [ 21 ]. The STTM (Short Tandem Target Mimic) lines of miR528 in neo-tetraploid rice exhibited a significant reduction in seed setting and pollen fertility [ 34 ]. Here, we identified the OsINH2 gene as a novel candidate target of miR528 , which functions in regulating pollen viability and grain numbers [ 35 ].…”

Seed development-related genes contribute to high yield heterosis in integrated utilization of elite autotetraploid and neo-tetraploid rice

“…To characterize the endosperm structure of mature seeds, WE-CLSM observations were performed as follows: The brown rice was cut by a sharp blade and stained by 4% eosin B solution for 5 min, hyalinized via pure methyl salicylate before observation under WE-CLSM. WE-CLSM observations were also performed to characterize the endosperm and embryo development in 5P ovaries, as described in our previous study ( Li et al., 2023 ). The collected samples were fixed in FAA solution (70% ethanol: acetic acid: methanal = 89:5:5, v/v), went through gradient rehydration, stained by 4% eosin B solution, dehydrated by gradient ethanol, and hyalinized via 50% and pure methyl salicylate before observation under WE-CLSM.…”

Lectin Receptor-Like Protein Kinase OsNRFG6 is Required for Embryo Sac Development and Fertilization in Neo-Tetraploid Rice

“…Tetraploid hybrid rice holds substantial promise for yield increase via superposing polyploidy advantage and heterosis (Chen et al 2019 ; Ghaleb et al 2020 ). But its breeding process was limited by reproductive dysfunction associated with polyploidy, including pollen sterility, embryo sac sterility, delayed fertilization, irregular embryogenesis, as well as abnormal endosperm development (Wu et al 2015 ; Li et al 2017 , 2023 ). Relative to extensive studies in polyploidy pollen sterility, it is more difficult in cytological observation and genetic analysis about embryo sac or embryo, resulting in limited understanding about the regulation of embryo sac development and embryogenesis in autotetraploid rice (ATR).…”