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Understanding the regulation of autotetraploid sterility is essential for harnessing the strong advantages in genomic buffer capacity, biodiversity, and heterosis of autotetraploid rice. miRNAs play crucial roles in fertility regulation, yet information about their reproductive roles and target genes in tetraploid rice remains limited. Here, we used three tetraploid lines, H1 (fertile), HF (fertile), and LF (sterile), to investigate cytological features and identify factors associated with autotetraploid sterility. LF showed abnormal meiosis, resulting in low pollen fertility and viability, ultimately leading to scarce fertilization and a low-seed setting compared to H1 and HF. RNA-seq revealed 30 miRNA-candidate target pairs related to autotetraploid pollen sterility. These pairs showed opposite expression patterns, with differential expression between fertile lines (H1 and HF) and the sterile line (LF). qRT-PCR confirmed that miR9564, miR528, and miR27874 were highly expressed in the anthers of H1 and HF but not in LF, while opposite results were obtained in their targets (ARPS, M2T, and OsRPC53). Haplotype and expression pattern analyses revealed that ARPS was specifically expressed in lines with the same haplotype of MIR9564 (the precursor of miR9564) as LF. Furthermore, the Dual-GFP assay verified that miR9564 inhibited the fluorescence signal of ARPS-GFP. The over-expression of ARPS significantly decreased the seed setting rate (59.10%) and pollen fertility (50.44%) of neo-tetraploid rice, suggesting that ARPS plays important roles in autotetraploid pollen sterility. This study provides insights into the cytological characteristic and miRNA expression profiles of tetraploid lines with different fertility, shedding light on the role of miRNAs in polyploid rice. [ABSTRACT FROM AUTHOR]

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2–3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice. [ABSTRACT FROM AUTHOR]

Autotetraploid rice is a useful rice germplasm for polyploid rice breeding. However, low fertility limits its commercial production. A neo-tetraploid rice with high fertility was developed from the progenies of crossing between autotetraploid lines by our research group. Our previous study showed that a myeloblastosis (MYB) transcription factor, MOF1, might be associated with the pollen development in tetraploid rice. However, little information is available about its role in pollen development in tetraploid rice. Here, we identified a new haplotype of MOF1 from neo-tetraploid rice and marked it as MOF1a. Transcriptome and qRT-PCR analysis demonstrated that MOF1a highly expressed in anthers, and displayed differential expression in neo-tetraploid rice compared to tetraploid rice line with low pollen fertility. The mutant (mof1a) of MOF1a, which was generated by CRISPR/Cas9 knockout in neo-tetraploid rice, showed low pollen fertility, and also exhibited abnormal tapetum and middle layer development, and defective chromosome behaviors during meiosis. A total of 13 tapetal related genes were found to be up-regulated in meiotic anthers of MOF1a compared with wild type plants by RNA-seq analysis, including CYP703A3, PTC1, and OsABCG26, which had been demonstrated to affect tapetal development. Moreover, 335 meiosis-related genes displayed differential expression patterns at same stage, including nine important meiosis-related genes, such as metallothionein OsMT1a. These results demonstrated that MOF1a plays an important role in pollen development and provides a foundation for understanding the molecular mechanism underlying MOF1a in reproduction of tetraploid rice. [ABSTRACT FROM AUTHOR]