Project description:Background: 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. Results: A total of 321 and 368 differentially expressed miRNAs (DEM) were detected during development of pollen and embryo sac in autotetraploid rice, respectively. Gene Ontology enrichment analysis on the targets of miRNAs-enriched during the development of pollen and embryo sac in autotetraploid rice revealed 30 prominent functional gene classes, such as cell differentiation and signal transduction during embryo sac development. However, only 7 prominent functional gene classes, such as flower development and transcription factor activity, were detected during pollen development. The expression levels of 39 DEM, which revealed interaction with meiosis-related genes, showed opposite expression levels in 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. 21nt-phasiRNAs were detected both during pollen and embryo sac development, while 24nt-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. 24nt TEs-siRNAs were found to be up-regulated in embryo sac but down-regulated in pollen development. Conclusion: 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-24nt-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.
Project description: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 autotetraploid 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 were found to be up-regulated and down-regulated, 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 lead to low pollen fertility in autotetraploid rice.
Project description:Analysis of small RNAs revealed differential expressions during the development of pollen and embryo sac and their targets may associate with fertility in autotetraploid rice
Project description:Phosphate starvation/sufficient rice seedling, root or shoot Pi-starvation or Pi-sufficient stresses responsible rice genes, including previously unannotated genes were identified by Illumina mRNA-seq technology. 53 million reads from Pi-starvation or Pi-sufficient root or shoot tissues were uniquely mapped to the rice genome, and these included 40574 RAP3 transcripts in root and 39748 RAP3 transcripts in shoot. We compared our mRNA-seq expression data with that from Rice 44K oligomicroarray, and about 95.5% (root) and 95.4% (shoot) transcripts supported by the array were confirmed expression both by the array and by mRNA-seq, Moreover, 11888 (root) and 11098 (shoot) RAP genes which were not supported by array, were evidenced expression with mRNA-seq. Furthermore, we discovered 8590 (root) and 8193 (shoot) previously unannotated transcripts upon Pi-starvation and/or Pi-sufficient.
Project description:Newly synthetic autotetraploid rice generally shows lower pollen fertility and seed setting rate in comparison to diploid rice, and this can hinder its own domestication and breeding. In this study, cytological analysis was used to demonstrate the imbalanced separation of homologous chromosomes occurred at meiosis I during the pollen mother cell development, and the early degeneration of tapetum cells in autotetraploid rice. We identified a total of 941 differentially expressed proteins (DEPs) in anthers, including 489 up-regulated and 452 down-regulated proteins. KOG analysis showed that the identified DEPs were related to post-translational modifications, such as protein ubiquitination. These modifications are related to chromatin remodeling and homologous recombination abnormalities during meiosis. The key proteins related to the pentose phosphate pathway (BGIOSGA016558, BGIOSGA022166, BGIOSGA028743) are significantly down-regulated, which can affect the failure of autotetraploid rice to provide the energy needed for cell development after polyploidization, and ultimately lead to the early degradation of the tapetum. Proteins (BGIOSGA017346and BGIOSGA027368) related to the degradation of glutenin are up-regulated, indicating that the decomposition of glutenin can lead to the early degradation of tapetum. These cytological and molecular evidences will help to promote our understanding of the differences in anther development between diploid and autotetraploid rice during meiosis.
Project description:Pollen tube growth is essential for successful fertilization and stable crop yields. We constructed loss-of-function/knock-out mutants that simultaneously target two rice genes using the CRISPR/Cas9 mutagenesis system. The selected OsRALF17 and OsRALF19 genes are strongly expressed in rice bicellular/tricellular pollen and have essential functions in the pollen tube growth. For the corresponding transcriptomic analysis, we sampled mature pollen anthers from a control group and an OsRALF17/19 knock-out mutant.