Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (Gossypium barbadense L.)
Abstract
:1. Introduction
2. Results
2.1. Determination of the Pollen Abortion Stage in CMS Line H276A
2.2. Transcriptome Sequencing and Genome Mapping
2.3. Analysis of DEGs between H276A and H276B
2.4. GO Annotation and Pathway Analysis of DEGs
2.5. Confirmation of DEGs by qRT-PCR
3. Discussion
3.1. Cytological Characteristics of Pollen Abortion
3.2. DEGs Related to the Tricarboxylic Acid (TCA) Cycle and Oxidative Phosphorylation
3.3. DEGs Associated with Pentatricopeptide Repeat (PPR) Proteins
3.4. DEGs Involved in MYB Transcription Factors
3.5. Other DEGs
4. Materials and Methods
4.1. Plant Materials
4.2. Paraffin Sections and Microspore Development Observations
4.3. RNA Extraction, cDNA Library Construction and Illumina Deep Sequencing
4.4. Genome Mapping and Novel Transcript Prediction
4.5. Differential Expression Analysis
4.6. Functional Enrichment Analysis and Transcription Factor Prediction of DEGs
4.7. Reverse-Transcription PCR (RT-PCR) and Quantitative Reverse-Transcription PCR (qRT-PCR)
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
CMS | Cytoplasmic male sterility |
DEGs | Differentially expressed genes |
qRT-PCR | Quantitative reverse-transcribed PCR |
PMC | Pollen mother cell |
RNA-Seq | Transcriptome sequencing |
TCA | Tricarboxylic acid cycle |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
GO | Gene ontology |
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Sample | H276A-1 | H276A-2 | H276A-3 | H276B-1 | H276B-2 | H276B-3 |
---|---|---|---|---|---|---|
Total Raw Reads (Mb) | 50.42 | 53.42 | 51.82 | 51.81 | 51.82 | 51.82 |
Total Clean Reads (Mb) | 40.38 | 44.48 | 44.73 | 44.58 | 44.25 | 44.37 |
Clean Reads Q20 (%) | 98.06 | 98.01 | 98.39 | 98.38 | 98.39 | 98.34 |
Total Mapping Ratio (%) | 73.34 | 74.20 | 75.20 | 76.04 | 75.60 | 76.18 |
Total Gene Number | 57,228 | 57,777 | 57,428 | 56,938 | 56,929 | 56,629 |
Total Transcript Number | 86,162 | 87,603 | 85,063 | 85,039 | 84,961 | 84,434 |
Novel Transcript Number | 20,514 | 20,952 | 20,288 | 20,580 | 20,554 | 20,474 |
Pathway | Pathway ID | Genes with Pathway Annotation | p-Value | Q-Value | |
---|---|---|---|---|---|
DEGs (2683) | All Genes (56,363) | ||||
Carbohydrate Metabolism | |||||
Ascorbate and aldarate metabolism | ko00053 | 87 (3.24%) | 747 (1.33%) | 2.1883346 × 10−14 | 2.772849 × 10−12 |
Amino sugar and nucleotide sugar metabolism | Ko00520 | 132 (4.92%) | 1789 (3.17%) | 5.144044 × 10−7 | 1.306587 × 10−5 |
Galactose metabolism | Ko00052 | 93 (3.47%) | 1264 (2.24%) | 2.652517 × 10−5 | 3.142800 × 10−4 |
Pentose and glucuronate interconversions | Ko00040 | 119 (4.44%) | 1711 (3.04%) | 2.72211 × 10−5 | 3.142800 × 10−4 |
Fructose and mannose metabolism | Ko00051 | 48 (1.79%) | 623 (1.11%) | 0.0008306261 | 6.203606 × 10−3 |
Pentose phosphate pathway | Ko00030 | 44 (1.64%) | 562 (1%) | 0.0009706133 | 6.487784 × 10−3 |
Glycolysis/gluconeogenesis | Ko00010 | 62 (2.31%) | 894 (1.59%) | 0.00222321 | 1.344513 × 10−2 |
Global and Overview Map | |||||
Metabolic pathways | ko01100 | 822 (30.64%) | 14,040 (24.91%) | 3.498244 × 10−12 | 2.221385 × 10−10 |
Biosynthesis of secondary metabolites | Ko01110 | 464 (17.29%) | 8032 (14.25) | 3.713275 × 10−6 | 5.239844 × 10−5 |
Biosynthesis of Other Secondary Metabolites | |||||
Flavonoid biosynthesis | Ko00941 | 35 (1.3%) | 266 (0.47%) | 6.13195 × 10−8 | 2.595859 × 10−6 |
Stilbenoid, diarylheptanoid and gingerol biosynthesis | Ko00945 | 31 (1.16%) | 240 (0.43%) | 5.103317 × 10−7 | 1.306587 × 10−5 |
Flavone and flavonol biosynthesis | Ko00944 | 12 (0.45%) | 105 (0.19%) | 0.004277302 | 2.263406 × 10−2 |
Phenylpropanoid biosynthesis | Ko00940 | 91(3.39%) | 1497 (2.66%) | 0.01086005 | 4.755953 × 10−2 |
Metabolism of Terpenoids and Polyketides | |||||
Zeatin biosynthesis | Ko00908 | 24 (0.89%) | 162 (0.29%) | 8.181436 × 10−7 | 1.731737 × 10−5 |
Limonene and pinene degradation | Ko00903 | 24 (0.89%) | 210 (0.37%) | 7.083635 × 10−5 | 7.496847 × 10−4 |
Carotenoid biosynthesis | Ko00906 | 21 (0.78%) | 244 (0.43%) | 0.006774023 | 3.308850 × 10−2 |
Transcription | |||||
RNA polymerase | Ko03020 | 79 (2.94%) | 962 (1.71%) | 2.302535 × 10−6 | 3.878907 × 10−5 |
Energy Metabolism | |||||
Nitrogen metabolism | Ko00910 | 22 (0.82%) | 149 (0.26%) | 2.443406 × 10−6 | 3.878907 × 10−5 |
Photosynthesis | Ko00195 | 22 (0.82%) | 213 (0.38) | 0.0005760129 | 4.572102 × 10−3 |
Lipid Metabolism | |||||
Sphingolipid metabolism | Ko00600 | 33 (1.23%) | 338 (0.6%) | 8.44879 × 10−5 | 8.543074 × 10−4 |
α-Linolenic acid metabolism | Ko00592 | 27 (1.01%) | 272 (0.48%) | 0.0002803392 | 2.543077 × 10−3 |
Glycerolipid metabolism | Ko00561 | 65 (2.42%) | 895 (1.59%) | 0.0005644657 | 4.572102 × 10−3 |
Amino Acid Metabolism | |||||
Tryptophan metabolism | Ko00380 | 26 (0.97%) | 278 (0.49%) | 0.0008792512 | 6.203606 × 10−3 |
Metabolism of Other Amino Acids | |||||
Taurine and hypotaurine metabolism | Ko00430 | 6 (0.22%) | 30 (0.05%) | 0.002561534 | 1.478704 × 10−2 |
Glycan Biosynthesis and Metabolism | |||||
Glycosaminoglycan degradation | Ko00531 | 23 (0.86%) | 251 (0.45%) | 0.002192842 | 1.344513 × 10−2 |
Other glycan degradation | Ko00551 | 42 (1.57%) | 562 (1%) | 0.002931596 | 1.618751 × 10−2 |
Glycosphingolipid biosynthesis (ganglio series) | Ko00604 | 16 (0.6%) | 164 (0.29%) | 0.005312663 | 2.698833 × 10−2 |
Glycosphingolipid biosynthesis (globo series) | Ko00603 | 8 (0.3%) | 61 (0.11%) | 0.00811891 | 3.818895 × 10−2 |
Transport and Catabolism | |||||
Regulation of autophagy | Ko04140 | 34 (1.27%) | 462 (0.82%) | 0.008579786 | 3.891546 × 10−2 |
Gene ID | Protein Identity | Fold Change | |
---|---|---|---|
RNA-Seq | qRT-PCR | ||
LOC107957220 | Glycine-rich cell wall structural protein-like | 4.92 | 5.02 |
LOC107903993 | PPR protein At2g01860-like | 2.81 | 0.70 |
LOC107893809 | MADS-box CMB1 | 2.81 | 1.65 |
LOC107928908 | Hyoscyamine 6-dioxygenase-like | 2.73 | 2.71 |
LOC107940910 | Ethylene responsive element binding protein 3 | 2.59 | 2.63 |
LOC107916224 | Cell wall/vacuolar inhibitor of fructosidase 1-like | 1.96 | 2.11 |
LOC107919764 | Transcription factor MYB86 | −3.21 | −3.60 |
LOC107908692 | Pentatricopeptide repeat-containing protein At3g11460 | −3.38 | −4.38 |
LOC107940452 | Flavonol synthase/flavanone 3-hydroxylase-like | −3.86 | −1.19 |
LOC107941000 | Tetraketide α-pyrone reductase 1 | −3.94 | −3.50 |
LOC107961143 | Anther-specific protein LAT52 | −4.03 | −2.74 |
LOC107945055 | Chalcone synthase | −5.15 | −5.72 |
LOC107900663 | Bifunctional pinoresinol-lariciresinol reductase 2 | −5.67 | −3.43 |
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Kong, X.; Liu, D.; Liao, X.; Zheng, J.; Diao, Y.; Liu, Y.; Zhou, R. Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (Gossypium barbadense L.). Int. J. Mol. Sci. 2017, 18, 2240. https://doi.org/10.3390/ijms18112240
Kong X, Liu D, Liao X, Zheng J, Diao Y, Liu Y, Zhou R. Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (Gossypium barbadense L.). International Journal of Molecular Sciences. 2017; 18(11):2240. https://doi.org/10.3390/ijms18112240
Chicago/Turabian StyleKong, Xiangjun, Dongmei Liu, Xiaofang Liao, Jie Zheng, Yong Diao, Yiding Liu, and Ruiyang Zhou. 2017. "Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (Gossypium barbadense L.)" International Journal of Molecular Sciences 18, no. 11: 2240. https://doi.org/10.3390/ijms18112240