Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of environmentally sensitive genic male sterility genes in neo-tetraploid rice

Abstract Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells（PMCs）abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 440 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( TMS9-1 , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS9-1 and TMS5 as the candidate gene and analysed its pollen fertility in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in pollen fertility value, pollen development process and expression level in H1 and its knock out lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, knockout of environmentally sensitive genic male sterility genes in the present study provides the new useful germplasm for polyploidy rice breeding.

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Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of environmentally sensitive genic male sterility genes in neo-tetraploid rice

10.21203/rs.2.19654/v2 ◽

2020 ◽

Author(s):

Jinwen Wu ◽

Yuanmou Chen ◽

Hong Lin ◽

Yang Chen ◽

Hang Yu ◽

...

Keyword(s):

Male Sterility ◽

Pollen Development ◽

Pollen Fertility ◽

Development Process ◽

Rice Breeding ◽

Rna Seq ◽

High Pollen ◽

Autotetraploid Rice ◽

Environmentally Sensitive ◽

Sterility Genes

Abstract Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells（PMCs）abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 1479 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 433 were annotated as pollen fertility-related genes, and 240 (~55.4%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( TMS5 , CSA etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS5 as a candidate gene and analysed its phenotype in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in phenotypic charts, pollen development process and expression level in H1 and TMS5 knockout lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, knockout of environmentally sensitive genic male sterility genes in the present study provide the new useful germplasm for polyploidy rice breeding.

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Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of related genes in neo-tetraploid rice

10.21203/rs.2.19654/v1 ◽

2019 ◽

Author(s):

Jinwen Wu ◽

Yuanmou Chen ◽

Hong Lin ◽

Yang Chen ◽

Hang Yu ◽

...

Keyword(s):

Pollen Development ◽

Pollen Fertility ◽

Development Process ◽

Yield Potential ◽

Lower Percentage ◽

High Fertility ◽

Rice Breeding ◽

Rna Seq ◽

High Pollen ◽

Autotetraploid Rice

Abstract Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice.Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of PMC cell abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 1483 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 433 were annotated as pollen fertility-related genes, and 240 (~55.4%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( CSA , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS5 as a candidate gene and analysed its phenotype in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in phenotypic charts, pollen development process and expression level in H1 and its TMS5 knockout lines.Conclusions: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, the present study provides a new useful germplasm for polyploidy rice breeding.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21197046 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7046

Author(s):

Jinwen Wu ◽

Yuanmou Chen ◽

Hong Lin ◽

Yang Chen ◽

Hang Yu ◽

...

Keyword(s):

Pollen Development ◽

Pollen Fertility ◽

Development Process ◽

Low Fertility ◽

Abnormal Development ◽

Lower Percentage ◽

High Fertility ◽

Rna Seq ◽

Autotetraploid Rice ◽

Environmentally Sensitive

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22020841 ◽

2021 ◽

Vol 22 (2) ◽

pp. 841

Author(s):

Nabieu Kamara ◽

Yamin Jiao ◽

Zijun Lu ◽

Kelvin Dodzi Aloryi ◽

Jinwen Wu ◽

...

Keyword(s):

Genome Sequencing ◽

Pollen Development ◽

Pollen Fertility ◽

Bulked Segregant Analysis ◽

Low Fertility ◽

High Fertility ◽

Rna Seq ◽

Autotetraploid Rice ◽

Genetic Mechanisms ◽

New Perspective

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.

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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

International Journal of Molecular Sciences ◽

10.3390/ijms21207489 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7489

Author(s):

Zijun Lu ◽

Xiaotong Guo ◽

Zhiyu Huang ◽

Juan Xia ◽

Xiang Li ◽

...

Keyword(s):

Differential Expression ◽

Pollen Development ◽

Pollen Fertility ◽

Expression Patterns ◽

Middle Layer ◽

Low Fertility ◽

Myb Transcription Factor ◽

Pcr Analysis ◽

High Fertility ◽

Autotetraploid 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.

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

PLoS ONE ◽

10.1371/journal.pone.0253244 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0253244

Author(s):

Jinwen Wu ◽

Hao Fan ◽

Yifan Hu ◽

Haibin Guo ◽

Hong Lin ◽

...

Keyword(s):

Reference Genes ◽

Pollen Development ◽

Pollen Fertility ◽

Expression Patterns ◽

Cytochrome B5 ◽

Development Stages ◽

Qrt Pcr ◽

Autotetraploid Rice ◽

Fertility Genes ◽

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.

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High Temperatures Cause Male Sterility in Rice Plants with Transcriptional Alterations During Pollen Development

Plant and Cell Physiology ◽

10.1093/pcp/pcp135 ◽

2009 ◽

Vol 50 (11) ◽

pp. 1911-1922 ◽

Cited By ~ 129

Author(s):

Makoto Endo ◽

Tohru Tsuchiya ◽

Kazuki Hamada ◽

Shingo Kawamura ◽

Kentaro Yano ◽

...

Keyword(s):

Male Sterility ◽

High Temperatures ◽

Pollen Development ◽

Rice Plants ◽

Transcriptional Alterations

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OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways

Plant and Cell Physiology ◽

10.1093/pcp/pcaa025 ◽

2020 ◽

Vol 61 (5) ◽

pp. 988-1004 ◽

Cited By ~ 3

Author(s):

Xiaoying Pan ◽

Wei Yan ◽

Zhenyi Chang ◽

Yingchao Xu ◽

Ming Luo ◽

...

Keyword(s):

Pollen Development ◽

Male Fertility ◽

Gene Networks ◽

Affinity Purification ◽

Anther Development ◽

Rna Seq ◽

Male Sterile ◽

New Information ◽

Gel Shift Assay

Abstract Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

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A cross-species approach for the identification of Drosophila male sterility genes

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab183 ◽

2021 ◽

Author(s):

Kimihide Ibaraki ◽

Mihoko Nakatsuka ◽

Takashi Ohsako ◽

Masahide Watanabe ◽

Yu Miyazaki ◽

...

Keyword(s):

Male Sterility ◽

Focal Point ◽

Fruit Fly ◽

Male Reproduction ◽

Model Organisms ◽

Germline Stem Cells ◽

New Genes ◽

Starting Point ◽

Sperm Development ◽

Sterility Genes

Abstract Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

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Identification and Verification of Genes Related to Pollen Development and Male Sterility Induced by High Temperature in Thermo-sensitive Genic Male Sterile Wheat Line YanZhan 4110S

10.21203/rs.3.rs-119009/v1 ◽

2020 ◽

Author(s):

Xuetong Yang ◽

Jiali Ye ◽

Fuqiang Niu ◽

Yi Feng ◽

Xiyue Song

Keyword(s):

Gene Silencing ◽

Male Sterility ◽

Mosaic Virus ◽

Pollen Development ◽

Bioinformatics Analysis ◽

Virus Induced Gene Silencing ◽

Wheat Line ◽

Pollen Abortion ◽

Hub Genes ◽

Genic Male Sterility

Abstract Background: Environment-sensitive genic male sterility is of vital importance to hybrid vigor in crop production and breeding, therefore, it is meaningful to identify and study the function of the genes related to pollen development and male sterility, which still not fully understanding currently. In this study, Yanzhan 4110S, a new thermo-sensitive genic male sterility (TGMS) wheat line, and its near isogenic line Yanzhan 4110 were carried out cytological features observation, bioinformatics analysis to investgate the abortion state and identified the genes involved in pollen development which have fertility regulation function. Barely stripe mosaic virus-induced gene silencing was used to verify the genes function.Results: Cytological analysis showed pollen abortion event of Yanzhan 4110S occur at the later uninucleate stage (Lun) under higher temperature induction (day/night temperatures of 22 °C/20 °C), when the anthers were collected and assessed for transcriptomic profiling through high-throughput sequencing. We then in-depth analyzed the differentially expressed genes (DEGs) by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, the results showed that the occurrence of Yanzhan 4110S male-sterility most likely related to metabolic pathway, including phenylpropanoid biosynthesis in the biosynthesis of other secondary metabolites, starch and sucrose metabolism in carbohydrate metabolism, carbon fixation in photosynthetic organisms as well as carbon metabolism in energy metabolism. The weighted gene co-expression network analysis in the transcriptome profiles further identified some hub genes, where the key genes involved in those pathways were intersection between the unique DEGs of Yanzhan 4110S in anther and hub genes, totally 228 genes, which were highly related to pollen development including TaMut11 and TaSF3. Moreover, further verification through barely stripe mosaic virus-induced gene silencing elucidated that the silencing of TaMut11 and TaSF3 caused pollen abortion, finally resulting in the declination of fertility. So, the genes TaMut11 and TaSF3 are related to fertility conversion of Yanzhan 4110S.Conclusion: Through comparative transcriptome bioinformatics analysis, the genes TaMut11 and TaSF3 associated with pollen development and male sterility induced by high temperature were identified in Yanzhan 4110S, and verificated by barely stripe mosaic virus-induced gene silencing. These findings provided researching the abortive mechanism in environment-sensitive genic male sterility wheat.

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