Interfered Chromosome Pairing Promotes Meiosis Instability of Autotetraploid Arabidopsis by High Temperatures

Alterations of environmental temperature affect multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication (WGD) have enhanced genetic plasticity and tolerance to environmental stress, but meanwhile face a challenge for organization and segregation of doubled chromosome sets. In this study, we investigated the impact of increased environmental temperature on male meiosis in autotetraploid Arabidopsis thaliana. Under low to mildly-increased temperatures (5-28°C), irregular chromosome segregation universally takes place in synthesized autotetraploid Columbia-0 (Col-0). Similar meiosis lesions occur in autotetraploid rice (Oryza sativa L.) and allotetraploid canola (Brassica napus cv. Westar), but not in evolutionary-derived hexaploid wheat (Triticum aestivum). As temperature increases to extremely high, chromosome separation and tetrad formation are severely disordered due to univalent formation caused by suppressed crossing-over. We found a strong correlation between tetravalent formation and successful chromosome pairing, both of which are negatively correlated with temperature elevation, suggesting that increased temperature interferes with crossing-over prominently by impacting homolog pairing. Besides, we showed that loading irregularities of axis proteins ASY1 and ASY4 co-localize on the chromosomes of syn1 mutant, and the heat-stressed diploid and autotetraploid Col-0, revealing that heat stress affects lateral region of synaptonemal complex (SC) by impacting stability of axis. Moreover, we showed that chromosome axis and SC in autotetraploid Col-0 are more sensitive to increased temperature than that of diploid Arabidopsis. Taken together, our study provide evidence suggesting that WGD without evolutionary and/or natural adaption negatively affects stability and thermal tolerance of meiotic recombination in Arabidopsis thaliana.

A Genome Doubling Event Reshapes Rice Morphology and Products by Modulating Chromatin Signatures and Gene Expression Profiling

Evolutionarily, polyploidy represents a smart method for adjusting agronomically important in crops through impacts on genomic abundance and chromatin condensation. Autopolyploids have a relatively concise genetic background with great diversity and provide an ideal system to understand genetic and epigenetic mechanisms attributed to the genome-dosage effect. However, whether and how genome duplication events during autopolyploidization impact chromatin signatures are less understood in crops. To address it, we generated an autotetraploid rice line from a diploid progenitor, Oryza sativa ssp. indica 93-11. Using transposase-accessible chromatin sequencing, we found that autopolyploids lead to a higher number of accessible chromatin regions (ACRs) in euchromatin, most of which encode protein-coding genes. As expected, the profiling of ACR densities supported that the effect of ACRs on transcriptional gene activities relies on their positions in the rice genome, regardless of genome doubling. However, we noticed that genome duplication favors genic ACRs as the main drivers of transcriptional changes. In addition, we probed intricate crosstalk among various kinds of epigenetic marks and expression patterns of ACR-associated gene expression in both diploid and autotetraploid rice plants by integrating multiple-omics analyses, including chromatin immunoprecipitation sequencing and RNA-seq. Our data suggested that the combination of H3K36me2 and H3K36me3 may be associated with dynamic perturbation of ACRs introduced by autopolyploidization. As a consequence, we found that numerous metabolites were stimulated by genome doubling. Collectively, our findings suggest that autotetraploids reshape rice morphology and products by modulating chromatin signatures and transcriptional profiling, resulting in a pragmatic means of crop genetic improvement.

Identification of stable pollen development related reference genes for accurate qRT-PCR analysis and morphological variations in autotetraploid and diploid rice

Autotetraploid rice exhibited hybrid vigor and greater genetic variation compared to diploid rice, but low pollen fertility is a major hindrance for its utilization. Our previous analysis revealed that large number of pollen fertility genes were exhibited down-regulation in autotetraploid rice. Hence, it is of utmost importance to reveal the expression patterns of pollen fertility genes with high accuracy. To find stable reference genes for autotetraploid rice, we compared the pollen development stages between diploid and autotetraploid rice, and 14 candidate genes were selected based on transcriptome analysis to evaluate their expression levels. Autotetraploid rice (i.e. Taichung65-4x) displayed lower seed set (40.40%) and higher percentage of abnormalities during the pollen development process than its diploid counterpart. To detect the candidate reference genes for pollen development of autotetraploid and diploid rice, we used five different algorithms, including NormFinder, BestKeeper, ΔCt method, geNorm and Re-Finder to evaluate their expression patterns stability. Consequently, we identified two genes, Cytochrome b5 and CPI, as the best candidate reference genes for qRT-PCR normalization in autotetraploid and diploid rice during pre-meiosis, meiosis, single microspore and bicellular pollen development stages. However, Cytochrome b5 was found to be the most stably expressed gene during different pollen development stages in autotetraploid rice. The results of our study provide a platform for subsequent gene expression analyses in autotetraploid rice, which could also be used in other polyploid plants.

Genomics Analyses Reveal Unique Classification, Population Structure and Novel Allele of Neo-Tetraploid Rice

Background Neo-tetraploid rice (NTR) is a useful new germplasm that developed from the descendants of the autotetraploid rice (ATR) hybrids. NTR showed improved fertility and yield potential, and produced high yield heterosis when crossed with indica ATR for commercial utilization. However, their classification, population structure and genomic feature remain elusive. Results Here, high-depth genome resequencing data of 15 NTRs and 18 ATRs, together with 38 publicly available data of diploid rice accessions, were analyzed to conduct classification, population structure and haplotype analyses. Five subpopulations were detected and NTRs were clustered into one independent group that was adjacent to japonica subspecies, which maybe the reason for high heterosis when NTRs crossed with indica ATRs. Haplotype patterns of 717 key genes that associated with yield and other agronomic traits were revealed in these NTRs. Moreover, a novel specific SNP variation was detected in the first exon of HSP101, a known heat-inducible gene, which was conserved in all NTRs but absent in ATRs, 3KRG and RiceVarMap2 databases. The novel allele was named as HSP101–1, which was confirmed to be a heat response factor by qRT-PCR, and knockout of HSP101–1 significantly decreased the thermotolerance capacity of NTR. Interestingly, HSP101–1 was also specifically expressed in the anthers of NTR at pre-meiotic and meiosis stages under optimal environment without heat stress, and its loss-of-function mutant showed significant decrease in fertility of NTR. Conclusion The construction of first genomic variation repository and the revelation of population structure provide invaluable information for optimizing the designs of tetraploid rice breeding. The detection of specific genomic variations offered useful genomic markers and new directions to resolve high fertility mechanism of NTR.

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.

Transcriptome and Gene Editing Analyses Reveal MOF1a Defect Alters the Expression of Genes Associated with Tapetum Development and Chromosome Behavior at Meiosis Stage Resulting in Low Pollen Fertility of Tetraploid Rice

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.

iTRAQ-Based Quantitative Glutelin Proteomic Analysis Reveals Differentially Expressed Proteins in the Physiological Metabolism Process during Endosperm Development and their Impacts on Yield and Quality in Autotetraploid Rice

Background: Autotetraploid rice, which is developed through chromosome set doubling using diploid rice, produces high-quality kernels that are rich in storage proteins. However, little information is available about the content of different proteins in autotetraploid rice and their proteomic analysis. Results: The dynamic changes in four storage proteins, albumin, globulin, prolamin, and glutelin, were analyzed in the endosperm of autotetraploid rice (AJNT-4x) and in that of its diploid counterpart (AJNT-2x) for comparison. The contents of the four proteins were all higher during endosperm development in AJNT-4x than in AJNT-2x, but their change and composition were almost the same in the two materials. Then, iTRAQ was employed to analyze the glutelin profiles of AJNT-4x and AJNT-2x at 10 DAF, 15 DAF, and 20 DAF. A total of 1,326 proteins were identified in AJNT-4x and AJNT-2 x using high-throughput LC-MS/MS. Among the 1,326 identified proteins, there were 362 DEPs in AJNT-4x compared with those in AJNT-2x and 372 DEPs between different development stages in AJNT-4x. Eight important upregulated proteins were identified by qRT-PCR, including B8AM24, B8ARJ0, B8AQM6, A2ZCE6, and Q40689. Among them, B8AM24 and B8ARJ0 were related to the lysine biosynthesis process. GO enrichment analysis revealed that the critical functions of DEPs exhibited little overlap between the 10, 15, and 20 DAF groups. Endosperm glutelin accumulation was regulated mainly by different DEPs during the late stage, and 15 DAF was a critical regulating point for glutelin accumulation. KEGG pathway analysis showed that ribosomal proteins were significantly higher in AJNT-4x than in AJNT-2x at 10 DAF, and the protein processing, biosynthesis, and metabolism of amino acids were higher and more active in AJNT-4x at 15 DAF, while the peroxisome was richer in AJNT-4x at 20 DAF. The PPI network showed that ribosomal proteins gradually decreased with increasing endosperm development. Conclusions: These results provide new insights into dynamic glutelin expression differences during endosperm development in autotetraploid rice, which will help in developing rice cultivars with increased yield and improved grain nutritional quality.

Comparative Cytological and Transcriptome Analysis Revealed the Normal Pollen Development Process and Up-Regulation of Fertility-Related Genes in Newly Developed Tetraploid Rice

Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for its utilization. Here, we reported the development of a new tetraploid rice, Huoduo1 (H1), which has the characteristic of high fertility, from crossing generations of autotetraploid rice. Cytological observations displayed the high fertility of the pollen (95.62%) in H1, a lower percentage of pollen mother cell (PMC) abnormalities, and stable chromosome configurations during the pollen development process compared with its parents. Using RNA-seq analysis, we detected 440 differentially expressed genes (DEGs) in H1 compared with its parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant up-regulation in H1 compared with the parents, including three environmentally sensitive genic male sterility genes (TMS9-1, TMS5, and CSA), one meiosis gene (RAD51D), and three tapetal-related genes (MIL2, OsAP25, and OsAP37), which were validated by qRT-PCR in this study. Two genes, TMS9-1 and TMS5, were knocked out using CRISPR/Cas9 technology, and their mutants displayed low fertility and the abnormal development of pollen. Our findings provide evidence for the regulatory mechanisms of fertility in tetraploid rice and indicated that the up-regulation of pollen fertility-related genes may contribute to the high fertility in new tetraploid rice.