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Comparative small RNA analysis of pollen development in autotetraploid and diploid rice

ABSTRACT: 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.

ORGANISM(S): Oryza sativa Japonica Group

PROVIDER: GSE79344 | GEO | 2016/11/04

SECONDARY ACCESSION(S): PRJNA315549

REPOSITORIES: GEO

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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:Enhancing grain production of rice (Oryza sativa L.) is a top priority in ensuring food security for human being. One approach to increase yield is to delay leaf senescence and to extend the available time for photosynthesis. microRNAs (miRNAs) are key regulators for aging and cellular senescence in eukayotes. However, miRNAs and their roles in rice leaf senescence remain unexplored. Here, we report identification of miRNAs and their putative target genes by deep sequencing of six small RNA libraries, six RNA-seq libraries and two degradome libraries from the leaves of two super hybrid rice, Nei-2-You 6 (N2Y6, age-resistant rice) and Liang-You-Pei 9 (LYP9, age-sensitive rice). Totally 372 known miRNAs and 162 miRNA candidates were identified, and 1145 targets were identified. Compared with the expression of miRNAs in the leaves of LYP9, the numbers of miRNAs up-regulated and down-regulated in the leaves of N2Y6 were 47 and 30 at early stage of grain-filling, 21 and 17 at the middle stage, and 11 and 37 at the late stage, respectively. Six miRNA families, osa-miR159, osa-miR160 osa-miR164, osa-miR167, osa-miR172 and osa-miR1848, targeting the genes encoding APETALA2 (AP2), zinc finger proteins, salicylic acid-induced protein 19 (SIP19), Auxin response factors (ARF) and NAC transcription factors, respectively, were found to be involved in leaf senescence through phytohormone signaling pathways. These results provided valuable information for understanding the miRNA-mediated leaf senescence of rice, and offered an important foundation for rice breeding. [miRNA] sample 1:The flag leaves at early stage of grain-filling of N2Y6 rice; sample 2: The flag leaves at middle stage of grain-filling of N2Y6 rice;sample 3:The flag leaves at late stage of grain-filling of N2Y6 rice; sample 4:The flag leaves at early stage of grain-filling of LYP9 rice; sample 5: The flag leaves at middle stage of grain-filling of LYP9 rice;sample 6:The flag leaves at late stage of grain-filling of LYP9 rice. [DGE]: samples 7-12 [degradome (targets)]: samples 13:The flag leaves at mixed stages of grain-filling of N2Y6 rice; sample 14:The flag leaves at mixed stages of grain-filling of LYP9 rice

Project description:Purpose: Mechanism of gene expression changes in autotetraploid drought resistance variation Methods: Leaves under control and drought treatment of 6h, 12h and 48h were sampled from diploid and the autotetraploid for RNA-seq. Total RNA was extracted from 100mg sample with RNeasy plant mini kit (Omega Biotech, China) and purified with RNase-Free DNase set. Following, cDNA library was constructed and the library concentration and insert size was respectively assessed on the Qubit2.0 (Invitrogen, USA) and Agilent Bioanalyzer 2100 system, and then the effective concentration of the library was accurately quantified through Q-PCR. Prepared high quality libraries were sequenced on Illumina HiSeq X-ten platform to generate paired-end raw reads. qRT–PCR validation was performed using SYBR Green assays. Results: The study findings showed that the autotetraploid sour jujube exhibited a superior drought tolerance and enhanced regrowth potential after dehydration in comparison with the diploid counterpart. The physiological responses gradually triggered important functions in the autotetraploid sour jujube under extreme drought conditions. Furthermore, a comparative transcriptome analysis showed that more differentially expressed genes (DEGs) were detected in autotetraploid after drought stress. Through GO enrichment analysis, many DEGs between the diploid and autotetraploid sour jujube after drought-stress exposure were respectively annotated to the oxidation–reduction process, photosystem, DNA binding transcription factor activity, oxidoreductase activity. Consistently, six reactive oxygen species scavenging-related genes were specifically differentially expressed, positive changes of the genes involved in glutathione metabolism pathways were greater and the lower O2− level and malonaldehyde (MDA) content and higher antioxidant enzymes activity were detected in the autotetraploid under drought-stress conditions. The higher chlorophyll content and differentially enriched genes during photosynthesis suggest that the photosynthetic system in the autotetraploid, having variations in stomatal and cellular characteristics, was enhanced during drought stress. In addition, DEGs in the autotetraploid after stress exposure were significantly enriched in DNA-replication and plant hormone, including auxin, abscisic acid and gibberellin signal-transduction pathways. Under osmotic stress conditions, genes associated with the synthesis and transport of osmotic regulatory substances including anthocyanin biosynthesis were differentially expressed, and the soluble sugar, soluble protein and proline contents were significantly higher in the autotetraploid. Moreover, several genes encoding transcription factors (TFs) including GRAS, Bhlh, MYB, WRKY and NAC were induced specifically or to higher levels in the autotetraploid under drought-stress conditions, and hub genes, LOC107403632, LOC107422279, LOC107434947, LOC107412673 and LOC107432609, related to up-regulated transcription factors in the autotetraploid compared with the diploid were identified. Conclusions: Owing to the whole-genome doubling, many functional genes in the autotetraploid plants were differentially expressed compared with in the diploid during drought stress, resulting in resistance difference.

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Comparative small RNA analysis of pollen development in autotetraploid and diploid rice

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