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

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.

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

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.

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

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.

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

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 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.

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

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.

scholarly journals 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

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.

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

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hang Yu ◽  
Qihang Li ◽  
Yudi Li ◽  
Huijing Yang ◽  
Zijun Lu ◽  
...  
Keyword(s):  
Population Structure ◽  
Agronomic Traits ◽  
Genomic Variation ◽  
Yield Potential ◽  
High Yield ◽  
Genomic Feature ◽  
High Fertility ◽  
Loss Of Function ◽  
Autotetraploid Rice ◽  
Novel Allele

Abstract 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.

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

PLoS ONE ◽  
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.

scholarly journals Characterization of nucleic acids from extracellular vesicle-enriched human sweat

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Geneviève Bart ◽  
Daniel Fischer ◽  
Anatoliy Samoylenko ◽  
Artem Zhyvolozhnyi ◽  
Pavlo Stehantsev ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Human Genome ◽  
Body Fluids ◽  
Lower Percentage ◽  
Rna Seq ◽  
Protein Coding ◽  
Human Sweat ◽  
Dna And Rna ◽  
Ribonucleoprotein Complexes ◽  
Eccrine Glands

Abstract Background The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. Results We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7–45%), with 50–60% of those reads mapping to unannotated region of the genome and 30–55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. Conclusions Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

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

2020 ◽  
Vol 61 (5) ◽  
pp. 988-1004 ◽  
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.

scholarly journals Utilization of Molecular Markers for Rice Breeding

2021 ◽  
Vol 17 (1) ◽  
pp. 45
Author(s):  
Tasliah Tasliah
Keyword(s):  
Molecular Markers ◽  
Abiotic Stresses ◽  
Rice Production ◽  
Phosphate Deficiency ◽  
Yield Potential ◽  
Rflp Markers ◽  
Eating Quality ◽  
Rice Breeding ◽  
Rice Varieties ◽  
Early Maturity

Rice is the staple food for more than half of the world's population. Rice production in 2050 must increase by at least 50% to keep up with the population growth. Efforts to increase rice production continue using various strategies. Breeders apply multiple approaches including application of molecular markers in developing varieties better than the previous ones. Since the discovery of the restriction fragment length polymorphism (RFLP) markers in 1980s and the development of polymerase chain reaction (PCR) method, many types of molecular markers have been developed and applied to various crops including rice. Various molecular approaches to map genetic loci associated with rice superior traits were conducted. The mapped loci are very useful for rice breeding purposes. This paper reports the results of mapping and breeding economically important traits in rice, mainly those related to abiotic stresses, agronomic traits, yield, and yield quality. These included characters of semidwarf stature, aromatic grain, high yield potential, eating quality, higher Zn and Fe grain, more tolerant to abiotic stresses, such as salinity, drought, phosphate deficiency, Al toxicity and Fe toxicity, submergence, as well as early maturity character. The mapped characters can be transferred using marker-assisted backcrossing (MABC) method into cultivated rice genotypes well-adopted by farmers. Several countries including Indonesia have benefited from this breeding method, and Indonesia have released several rice varieties developed through MABC. These include rice varieties such as Code, Angke, Inpari 30, Inpari Blas, Inpari HDB, Bio Patenggang Agritan, and Bioni 63 Ciherang Agritan

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