An uncharacterized protein NY1 targets EAT1 to regulate anther tapetum development in polyploid rice.

BACKGROUND: Autotetraploid rice is a useful germplasm for the breeding of polyploid rice; however, low fertility is a major hindrance for its utilization. Neo-tetraploid rice with high fertility was developed from the crossing of different autotetraploid rice lines. Our previous research showed that the mutant (ny1) of LOC_Os07g32406 (NY1), which was generated by CRISPR/Cas9 knock-out in neo-tetraploid rice, showed low pollen fertility, low seed set, and defective chromosome behavior during meiosis. However, the molecular genetic mechanism underlying the fertility remains largely unknown. RESULTS: Here, cytological observations of the NY1 mutant (ny1) indicated that ny1 exhibited abnormal tapetum and middle layer development. RNA-seq analysis displayed a total of 5606 differentially expressed genes (DEGs) in ny1 compared to wild type (H1) during meiosis, of which 2977 were up-regulated and 2629 were down-regulated. Among the down-regulated genes, 16 important genes associated with tapetal development were detected, including EAT1, CYP703A3, CYP704B2, DPW, PTC1, OsABCG26, OsAGO2, SAW1, OsPKS1, OsPKS2, and OsTKPR1. The mutant of EAT1 was generated by CRISPR/Cas9 that showed abnormal tapetum and pollen wall formation, which was similar to ny1. Moreover, 478 meiosis-related genes displayed down-regulation at same stage, including 9 important meiosis-related genes, such as OsREC8, OsSHOC1, SMC1, SMC6a and DCM1, and their expression levels were validated by qRT-PCR. CONCLUSIONS: Taken together, these results will aid in identifying the key genes associated with pollen fertility, which offered insights into the molecular mechanism underlying pollen development in tetraploid rice.

Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice.

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.

Global identification and analysis revealed differentially expressed lncRNAs associated with meiosis and low fertility in autotetraploid rice.

BACKGROUND: Autotetraploid rice is a useful germplasm for polyploid rice breeding. Our previous research showed that non-coding RNAs might be associated with low fertility in autotetraploid rice. However, little information is available on long non-coding RNAs (lncRNAs) involved in the low fertility of autotetraploid rice. In the present study, RNA-seq was employed to detect the differentially expressed meiosis-related lncRNAs in autotetraploid rice, and gene overexpression and knock out experiments were used to validate the potential function of candidate lncRNA. RESULTS: A total of 444 differentially expressed lncRNAs (DEL) were detected during anther and ovary meiosis in autotetraploid rice. Of these, 328 DEL were associated with the transposable elements, which displayed low expression levels during meiosis in autotetraploid rice. We used rapid amplification of cDNA ends (RACE) assay to validate 10 DEL and found that the lncRNAs were not assembly artifacts, and six of them were conserved in tetraploid rice. Moreover, 237 and 20 lncRNAs were associated with pollen mother cell (PMC) and embryo sac mother cell (EMC) meiosis in autotetraploid rice, respectively. The differential expressions of some meiosis-related targets and its DEL regulator, including MEL1 regulated by TCONS_00068868, LOC_Os12g41350 (meiotic asynaptic mutant 1) by TCONS_00057811 in PMC, and LOC_Os12g39420 by TCONS_00144592 in EMC, were confirmed by qRT-PCR. TCONS_00057811, TCONS_00055980 and TCONS_00130461 showed anther specific expression patterns and were found to be highly expressed during meiosis. CRISPR/Cas9 editing of lncRNA57811 displayed similar morphology compared to wild type. The overexpression of lncRNA57811 resulted in low pollen fertility (29.70%) and seed setting (33%) in rice. CONCLUSION: The differential expression levels of lncRNAs, associated with transposable elements and meiosis-regulated targets, might be endogenous noncoding regulators of pollen/embryo sac development that cause low fertility in autotetraploid rice. The results enhance our understanding about rice lncRNAs, and facilitate functional research in autotetraploid rice.

Comparative cytological and transcriptome analyses of ny2 mutant delayed degeneration of tapetal cells and promotes abnormal microspore development in neo-tetraploid rice.

We aimed to investigate the genetic defects related to pollen development and infertility in NY2, a novel tetraploid rice germplasm known as Neo-tetraploid rice. This rice variety was created through the crossbreeding and selective breeding of various autotetraploid rice lines and has previously shown high fertility. Our previous research has revealed that the NY2 gene, encoding a eukaryotic translation initiation factor 3 subunit E, regulates pollen fertility. However, the underlying mechanism behind this fertility is yet to be understood. To shed light on this matter, we performed a combined cytological and transcriptome analysis of the NY2 gene. Cytological analysis indicated that ny2 underwent abnormal tapetal cells, microspore, and middle layer development, which led to pollen abortion and ultimately to male sterility. Genetic analysis revealed that the F1 plants showed normal fertility and an obvious advantage for seed setting compared to ny2. Global gene expression analysis in ny2 revealed a total of 7545 genes were detected at the meiosis stage, and 3925 and 3620 displayed upregulation and downregulation, respectively. The genes were significantly enriched for the gene ontology (GO) term "carbohydrate metabolic process. Moreover, 9 genes related to tapetum or pollen fertility showed down-regulation, such as OsABCG26 (ATP Binding Cassette G26), TMS9-1 (Thermosensitive Male Sterility), EAT1 (Programmed cell death regulatory), KIN14M (Kinesin Motor), OsMT1a (Metallothionein), and OsSTRL2 (Atypical strictosidine synthase), which were validated by qRT-PCR. Further analyses of DEGs identified nine down-regulated transcription factor genes related to pollen development. NY2 is an important regulator of the development of tapetum and microspore. The regulatory gene network described in this study may offer important understandings into the molecular processes that underlie fertility control in tetraploid rice.

Genome-wide analysis of DNA polymorphisms, the methylome and transcriptome revealed that multiple factors are associated with low pollen fertility in autotetraploid rice.

Autotetraploid rice is a useful germplasm with high biomass production; however, low fertility is the main barrier in commercial utilization. In our previous study, differential expression of meiosis-related miRNAs was found to be involved in the pollen sterility of autotetraploid rice. However, genome-wide DNA variations and methylomes associated with low fertility of autotetraploid rice are still poorly understood. Here, we measured both global DNA variations and the methylome and compared them with the transcriptome during pollen development in autotetraploid rice by high-throughput sequencing. A total of 34416 SNPs, 6993 InDels, 1003 SVs and 25 CNVs were detected, and 11367 and 41117 differentially methylated regions showed hypermethylation and hypomethylation in 02428-4x. In total, 1110 genes displayed differentially expression in 02428-4x during meiosis, of these six harbored CNVs, including four upregulated genes with gain CNVs, such as LOC_Os11g38620. We identified 122 genes by comparing with the previous data that might be associated with low fertility during pollen development in 02428-4x. Of the 122 gens, 98 were displayed methylation and differential expression, including OsMADS98, CYP703A3 and OsABCG26. The downregulation of these three genes were confirmed by qPCR during meiosis of 02428-4x, which played pivotal roles in pollen fertility. These results indicate that the low fertility of autotetraploid rice is not only caused by the differential expression of genes involved in pollen development, but also by sequence variation and differential methylation, suggesting that the reason for pollen sterility in autotetraploid rice is complex and might be affected by multiple factors.