MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice.

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.

Background: Neo-tetraploid rice, which is a new germplasm developed from autotetraploid rice, has a powerful biological and yield potential and could be used for commercial utilization. The length of panicle, as a part of rice panicle architecture, contributes greatly to high yield. However, little information about long panicle associated with heterosis or hybrid vigor is available in neo-tetraploid rice. Results: In the present study, we developed a neo-tetraploid rice line, Huaduo 8 (H8), with long panicles and harboring wide-compatibility genes for pollen and embryo sac fertility. All the hybrids generated by H8 produced significant high-parent yield heterosis and displayed long panicles similar to H8. RNA-seq analysis detected a total of 4013, 7050, 6787 and 6195 differentially expressed genes uniquely belonging to F₁ and specifically (DEGFu-sp) associated with leaf, sheath, main panicle axis and spikelet in the two hybrids, respectively. Of these DEGFu-sp, 279 and 89 genes were involved in kinase and synthase, and 714 cloned genes, such as GW8, OsGA20ox1, Ghd8, GW6a, and LP1, were identified and validated by qRT-PCR. A total of 2925 known QTLs intervals, with an average of 1~100 genes per interval, were detected in both hybrids. Of these, 109 yield-related QTLs were associated with seven important traits in rice. Moreover, 1393 non-additive DEGs, including 766 up-regulated and 627 down-regulated, were detected in both hybrids. Importantly, eight up-regulated genes associated with panicle were detected in young panicles of the two hybrids compared to their parents by qRT-PCR. Re-sequencing analysis depicted that LP (a gene controlling long panicle) sequence of H8 was different from many other neo-tetraploid rice and most of the diploid and autotetraploid lines. The qRT-PCR results showed that LP was up-regulated in the hybrid compared to its parents at very young stage of panicle development. Conclusions: These results suggested that H8 could overcome the intersubspecific autotetraploid hybrid rice sterility caused by embryo sac and pollen sterility loci. Notably, long panicles of H8 showed dominance phenomenon and played an important role in yield heterosis, which is a complex molecular mechanism. The neo-tetraploid rice is a useful germplasm to attain high yield of polyploid rice.

Polyploidization has played an important role in plant evolution and is a pathway for plants to increase genetic diversification and to get higher heterosis comparing with that of diploid does. This study was undertaken to assess the genetic variation and relationships among 40 autotetraploid rice genotypes and their counterpart diploid cultivars with 99 SSR markers screened from published rice genome. The 99 SSR markers detected polymorphism among autotetraploid genotypes and revealed a total of 291 alleles with an average of 2.949 alleles per locus. Autotetraploid lines showed higher genetic diversity and significant variation in agronomic traits than diploid cultivars. Phylogenetic analysis revealed that most of autotetraploid lines were genetically different from their diploid parents, and inter-subspecific hybrids were prepared on the basis of genetic distance between parents. Inter-subspecific autotetraploid hybrids showed a higher and positive heterobeltiosis and competitive heterosis than diploid hybrids, especially for grain yield. Genetic distance appeared not to predict heterosis in diploid rice for all traits; however, it showed a significant correlation with grain yield, grain length and grain length to width ratio in autotetraploid rice. This extensive research on autotetraploid heterosis and genetic diversity will be useful for the development of autotetraploid rice hybrids.

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.