Partial pollen and embryo sac sterilities are the two main reasons for low fertility in autotetraploid rice. Our previous study revealed that small RNAs changes may associate with pollen fertility in autotetraploid rice. However, knowledge on comparative analysis between the development of pollen and embryo sac by small RNAs in autotetraploid rice is still unknown. In the present study, WE-CLSM (whole-mount eosin B-staining confocal laser scanning microscopy) and high-throughput sequencing technology was employed to examine the cytological variations and to analyze small RNAs changes during pollen and embryo sac development in autotetraploid rice compared with its diploid counterpart. A total of 321 and 368 differentially expressed miRNAs (DEM) were detected during pollen and embryo sac development in autotetraploid rice, respectively. Gene Ontology enrichment analysis on the targets of DEM associated with embryo sac and pollen development revealed 30 prominent functional gene classes, such as cell differentiation and signal transduction during embryo sac development, while only 7 prominent functional gene classes, such as flower development and transcription factor activity, were detected during pollen development in autotetraploid rice. The expression levels of 39 DEM, which revealed interaction with meiosis-related genes, showed opposite expression patterns during pollen and embryo sac development. Of these DEM, osa-miR1436_L + 3_1ss5CT and osa-miR167h-3p were associated with the female meiosis, while osa-miR159a.1 and osa-MIR159a-p5 were related with the male meiosis. 21 nt-phasiRNAs were detected during both pollen and embryo sac development, while 24 nt-phasiRNAs were found only in pollen development, which displayed down-regulation in autotetraploid compared to diploid rice and their spatial-temporal expression patterns were similar to osa-miR2275d. 24 nt TEs-siRNAs were found to be up-regulated in embryo sac but down-regulated in pollen development. The above results not only provide the small RNAs changes during four landmark stages of pollen and embryo sac development in autotetraploid rice but also have identified specifically expressed miRNAs, especially meiosis-related miRNAs, pollen-specific-24 nt-phasiRNAs and TEs-siRNAs in autotetraploid rice. Together, these findings provide a foundation for understanding the effect of polyploidy on small RNAs expression patterns during pollen and embryo sac development that may lead to different abnormalities in autotetraploid rice.

Autotetraploid rice hybrids have great potential to increase the production, but hybrid sterility is a major hindrance in the utilization of hybrid vigor in polyploid rice, which is mainly caused by pollen abortion. Our previous study showed that double pollen fertility neutral genes, Sa-n and Sb-n, can overcome hybrid sterility in autotetraploid rice. Here, we used an autotetraploid rice line harboring double neutral genes to develop hybrids by crossing with auto- and neo-tetraploid rice, and evaluated heterosis and its underlying molecular mechanism. All autotetraploid rice hybrids, which harbored double pollen fertility neutral genes, Sa-n and Sb-n, displayed high seed setting and significant positive heterosis for yield and yield-related traits. Cytological observations revealed normal chromosome behaviors and higher frequency of bivalents in the hybrid than parents during meiosis. Transcriptome analysis revealed significantly higher expressions of important saccharides metabolism and starch synthase related genes, such as OsBEIIb and OsSSIIIa, in the grains of hybrid than parents. Furthermore, many meiosis-related and specific genes, including DPW and CYP703A3, displayed up-regulation in the hybrid compared to a parent with low seed setting. Many non-additive genes were detected in the hybrid, and GO term of carbohydrate metabolic process was significantly enriched in all the transcriptome tissues except flag leaf (three days after flowering). Moreover, many differentially expressed genes (DEGs) were identified in the yield-related quantitative trait loci (QTLs) regions as possible candidate genes. Our results revealed that increase in the number of bivalents improved the seed setting of hybrid harboring double pollen fertility neutral genes. Many important genes, including meiosis-related and meiosis-specific genes and saccharides metabolism and starch synthase related genes, exhibited heterosis specific expression patterns in polyploid rice during different development stages. The functional analysis of important genes will provide valuable information for molecular mechanisms of heterosis in polyploid rice.

Autotetraploid rice is a useful germplasm that has four chromosome sets and strong biological advantages; however, low fertility limits its commercial utilization. Little information is available about the DNA variation and differential gene expressions associated with low fertility in autotetraploid rice. In the present study, 81 SNPs and 182 InDels were identified in T449 (an autotetraploid rice line with low fertility) compared to E249 (diploid counterpart) by whole-genome re-sequencing. We detected only three non-synonymous SNPs and six large-effect InDels, which were associated with three and six genes, respectively. A total of 75 meiosis-related differentially expressed genes were detected during the meiosis stage by transcriptome analysis, including OsMTOPVIB, which is essential for meiotic DSB formation, and OsMOF, which takes part in homologous chromosome pairing and synapsis. Approximately 20.69% lagging chromosome at metaphase I and 4.65% abnormal tetrad were observed in T449. Moreover, transcriptome analysis revealed down-regulation of a sucrose transporter (OsSUT5) and two monosaccharide transporters (OsMST1 and OsMST8) in T449 at the single microspore stage, and their expression levels were verified by qRT-PCR. Cytological observation of saccharide distribution showed abnormal accumulation of saccharides in T449 and the contents of fructose and glucose were markedly higher in T449 than E249 at the single microspore stage. Our results suggested that polyploidy not only induces abrupt expression changes in the meiosis-related genes that lead to abnormal chromosome behavior, but also causes changes in the saccharide distribution and expression patterns of saccharide-related genes, which jointly causes sterility in the autotetraploid rice.

The improvement of the saccharification of rice straw is one of the strategies to reduce the sophisticated pretreatment that results in high cost and is unfriendly to the environment. We explored the cell wall features in tetraploid rice and highlighted the enhanced saccharification of tetraploid with large biomass. Results showed that lignin content and S/G ratio reduced to 17.09% and 0.37, respectively, in tetraploid straw by the determination of the pyGC-MS method. After the pretreatment, the cellulose crystallinity index decreased from 63.22% to 57.65% in tetraploid straw, which is lower than that of pretreated diploid straw. Surface topological analysis of SEM images indicated that tetraploid straw was more susceptible to the pretreatment. Tetraploid straw showed a strong advantage in the process of enzymatic hydrolysis. The enzyme efficiency reached the highest value of 77.60%, and the rate of enzyme reaction was improved to make the reaction saturated earlier than conventional rice. We concluded that the high saccharification has resulted from the alteration of lignin and cellulose in tetraploid rice. Our research provides an improved green feedstock for bioenergy, and the tetraploid rice straw shows the potential utilization value in bioethanol production.

Neo-tetraploid rice, which developed from the progenies of autotetraploid hybrid by our research group, is a useful germplasm with high fertility and strong heterosis when they crossed with other autotetraploid rice lines. The CRISPR/Cas9-mediated TMS5 gene editing system has been widely used in diploid rice, but there are few reports in tetraploid rice. Here, we used CRISPR/Cas9 technology to edit the TMS5 gene, which is a temperature sensitive gene controlling the fertility in diploid rice, in neo-tetraploid rice to develop male sterile lines. Two mutant lines, H2s and H3s, were developed from the gene editing and displayed characteristics of thermo-sensitive genic male sterility. The daily mean temperatures of 23 °C to 26 °C were found to be critical for sterility (restrictive temperature) in H2s and H3s under both controlled (growth chambers) and natural growing conditions (field). Cytological observation showed the anther dysplasia appeared later in H2s and H3s than that of the TMS5 mutant of diploid rice (E285s) under the same conditions. Then these mutant lines, H2s and H3s, were crossed with tetraploid rice to generate F₁ hybrids, which exhibited obvious advantages for effective number of panicles, total grains and seed setting. The high levels of hybrids heterosis were maintained for several generations that can save seed cost. Our research provides an effective way of developing thermo-sensitive genic male sterility (TGMS) lines of tetraploid rice using gene editing, which will accelerate the utilization of polyploid heterosis.

The developmental process of inflorescence and gametophytes is vital for sexual reproduction in rice. Multiple genes and conserved miRNAs have been characterized to regulate the process. The changes of miRNAs expression during the early development of rice inflorescence remain unknown. In this study, the analysis of miRNAs profiles in the early stage of rice inflorescence development identified 671 miRNAs, including 67 known and 44 novel differentially expressed miRNAs (DEMs). Six distinct clusters of miRNAs expression patterns were detected, and Cluster 5 comprised 110 DEMs, including unconserved, rice-specific osa-miR5506. Overexpression of osa-miR5506 caused pleiotropic abnormalities, including over- or under-developed palea, various numbers of floral organs and spikelet indeterminacy. In addition, the defects of ovaries development were frequently characterized by multiple megasporocytes, ovule-free ovary, megasporocyte degenerated and embryo sac degenerated in the transgenic lines. osa-miR5506 targeted REM transcription factor LOC_Os03g11370. Summarily, these results demonstrated that rice-specific osa-miR5506 plays an essential role in the regulation of floral organ number, spikelet determinacy and female gametophyte development in rice.