Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line
Abstract
:1. Introduction
2. Results
2.1. Phenotypic Characteristics of the Dwarf and Male Sterile Line P198
2.2. Identification of Candidate Genes with T-DNA Insertion Mutations in P198
2.2.1. Determination of T-DNA Copy Number in P198
2.2.2. Detection of Insertion Sites with Fusion Primer and Nested Integrated PCR (FPNI-PCR)
2.2.3. Detecting Copy Number and Insertion Sites with Oxford Nanopore Technology (ONT) Sequencing
2.2.4. Identification of T-DNA Insertion–Mutated Genes in P198
2.3. Detection of Potential RNAi Target Genes
2.4. Transcriptome Sequencing and DEG Analysis
2.5. GO and KEGG Enrichment Analyses
2.6. Analysis of the DEGs in Shoots Involved in Phytohormone Biosynthesis and Single Transduction
2.7. Analysis of Potential Key DEGs in Buds Associated with Male Sterility
3. Discussion
3.1. LOC111200331 Is an Important Candidate Gene for the P198 Phenotype
3.2. Auxin and BR Are Critical for Curled Leaves and the Dwarf Phenotype in P198
3.3. Alterations in the Expression of Genes Associated with Lipid Metabolism and the Tapetum Secretion Process May Lead to Abnormal Pollen in P198
4. Materials and Methods
4.1. Plant Materials
4.2. Southern Blotting
4.3. qPCR
4.4. ddPCR
4.5. FPNI-PCR
4.6. ONT Sequencing
4.7. Transcriptome Analysis
4.8. RT–PCR
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Meng, X.; Yu, H.; Zhang, Y.; Zhuang, F.; Song, X.; Gao, S.; Gao, C.; Li, J. Construction of a Genome-Wide Mutant Library in Rice Using CRISPR/Cas9. Mol. Plant 2017, 10, 1238–1241. [Google Scholar] [CrossRef] [PubMed]
- Wang, L.; Luo, Y.Z.; Zhang, L.; Jiao, X.M.; Wang, M.B.; Fan, Y.L. Rolling circle amplification-mediated hairpin RNA (RMHR) library construction in plants. Nucleic Acids Res. 2008, 36, e149. [Google Scholar] [CrossRef] [PubMed]
- Wang, L.; Zheng, J.; Luo, Y.; Xu, T.; Zhang, Q.; Zhang, L.; Xu, M.; Wan, J.; Wang, M.B.; Zhang, C.; et al. Construction of a genomewide RNAi mutant library in rice. Plant Biotechnol. J. 2013, 11, 997–1005. [Google Scholar] [CrossRef] [PubMed]
- Zhao, S.; Luo, J.; Zeng, X.; Li, K.; Yuan, R.; Zhu, L.; Li, X.; Wu, G.; Yan, X. Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus. Int. J. Mol. Sci. 2020, 21, 7243. [Google Scholar] [CrossRef] [PubMed]
- Feldmann, K.A.; Marks, M.D.; Christianson, M.L.; Quatrano, R.S. A Dwarf Mutant of Arabidopsis Generated by T-DNA Insertion Mutagenesis. Science 1989, 243, 1351–1354. [Google Scholar] [CrossRef] [PubMed]
- Marks, M.D.; Feldmann, K.A. Trichome Development in Arabidopsis thaliana. I. T-DNA Tagging of the GLABROUS1 Gene. Plant Cell 1989, 1, 1043–1050. [Google Scholar] [CrossRef] [PubMed]
- Koncz, C.; Mayerhofer, R.; Koncz-Kalman, Z.; Nawrath, C.; Reiss, B.; Redei, G.P.; Schell, J. Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. Embo J. 1990, 9, 1337–1346. [Google Scholar] [CrossRef] [PubMed]
- Azpiroz-Leehan, R.; Feldmann, K.A. T-DNA insertion mutagenesis in Arabidopsis: Going back and forth. Trends Genet. 1997, 13, 152–156. [Google Scholar] [CrossRef]
- Jorgensen, R.; Snyder, C.; Jones, J.D. T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives. Mol. General. Genet. MGG 1987, 207, 471–477. [Google Scholar] [CrossRef]
- Jupe, F.; Rivkin, A.C.; Michael, T.P.; Zander, M.; Motley, S.T.; Sandoval, J.P.; Slotkin, R.K.; Chen, H.; Castanon, R.; Nery, J.R.; et al. The complex architecture and epigenomic impact of plant T-DNA insertions. PLoS Genet. 2019, 15, e1007819. [Google Scholar] [CrossRef]
- Nacry, P.; Camilleri, C.; Courtial, B.; Caboche, M.; Bouchez, D. Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 1998, 149, 641–650. [Google Scholar] [CrossRef] [PubMed]
- Zhu, Q.H.; Ramm, K.; Eamens, A.L.; Dennis, E.S.; Upadhyaya, N.M. Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Sci. 2006, 171, 308–322. [Google Scholar] [CrossRef] [PubMed]
- Galbiati, M.; Moreno, M.A.; Nadzan, G.; Zourelidou, M.; Dellaporta, S.L. Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis. Funct. Integr. Genom. 2000, 1, 25–34. [Google Scholar] [CrossRef] [PubMed]
- Southern, E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 1975, 98, 503–517. [Google Scholar] [CrossRef] [PubMed]
- Yang, L.; Ding, J.; Zhang, C.; Jia, J.; Weng, H.; Liu, W.; Zhang, D. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 2005, 23, 759–763. [Google Scholar] [CrossRef] [PubMed]
- Głowacka, K.; Kromdijk, J.; Leonelli, L.; Niyogi, K.K.; Clemente, T.E.; Long, S.P. An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants. Plant Cell Environ. 2016, 39, 908–917. [Google Scholar] [CrossRef] [PubMed]
- Baker, M. Digital PCR hits its stride. Nat. Methods 2012, 9, 541–544. [Google Scholar] [CrossRef]
- Hindson, C.M.; Chevillet, J.R.; Briggs, H.A.; Gallichotte, E.N.; Ruf, I.K.; Hindson, B.J.; Vessella, R.L.; Tewari, M. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat. Methods 2013, 10, 1003–1005. [Google Scholar] [CrossRef]
- Narancio, R.; John, U.; Mason, J.; Giraldo, P.; Spangenberg, G. Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens). Mol. Biol. Rep. 2021, 48, 3069–3077. [Google Scholar] [CrossRef]
- Liu, Y.G.; Whittier, R.F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 1995, 25, 674–681. [Google Scholar] [CrossRef]
- Ochman, H.; Gerber, A.S.; Hartl, D.L. Genetic applications of an inverse polymerase chain reaction. Genetics 1988, 120, 621–623. [Google Scholar] [CrossRef] [PubMed]
- O’Malley, R.C.; Alonso, J.M.; Kim, C.J.; Leisse, T.J.; Ecker, J.R. An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat. Protoc. 2007, 2, 2910–2917. [Google Scholar] [CrossRef] [PubMed]
- Mellenthin, M.; Ellersiek, U.; Börger, A.; Baier, M. Expression of the Arabidopsis Sigma Factor SIG5 Is Photoreceptor and Photosynthesis Controlled. Plants 2014, 3, 359–391. [Google Scholar] [CrossRef] [PubMed]
- Liu, Y.-G.; Mitsukawa, N.; Oosumi, T.; Whittier, R.F. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 1995, 8, 457–463. [Google Scholar] [CrossRef] [PubMed]
- Settles, A.M.; Latshaw, S.; McCarty, D.R. Molecular analysis of high-copy insertion sites in maize. Nucleic Acids Res. 2004, 32, e54. [Google Scholar] [CrossRef]
- Fu, F.-F.; Ye, R.; Xu, S.-P.; Xue, H.-W. Studies on rice seed quality through analysis of a large-scale T-DNA insertion population. Cell Res. 2009, 19, 380–391. [Google Scholar] [CrossRef] [PubMed]
- Liu, F.; Gong, D.; Zhang, Q.; Wang, D.; Cui, M.; Zhang, Z.; Liu, G.; Wu, J.; Wang, Y. High-throughput generation of an activation-tagged mutant library for functional genomic analyses in tobacco. Planta 2015, 241, 629–640. [Google Scholar] [CrossRef] [PubMed]
- Yuan, H.-M.; Chen, S.; Lin, L.; Wei, R.; Li, H.-Y.; Liu, G.-F.; Jiang, J. Genome-Wide Analysis of a TaLEA-Introduced Transgenic Populus simonii × Populus nigra Dwarf Mutant. Int. J. Mol. Sci. 2012, 13, 2744–2762. [Google Scholar] [CrossRef]
- Zeng, T.; Zhang, D.; Li, Y.; Li, C.; Liu, X.; Shi, Y.; Song, Y.; Li, Y.; Wang, T. Identification of genomic insertion and flanking sequences of the transgenic drought-tolerant maize line “SbSNAC1-382” using the single-molecule real-time (SMRT) sequencing method. PLoS ONE 2020, 15, e0226455. [Google Scholar] [CrossRef]
- Windels, P.; Taverniers, I.; Depicker, A.; Van Bockstaele, E.; De Loose, M. Characterisation of the Roundup Ready soybean insert. Eur. Food Res. Technol. 2001, 213, 107–112. [Google Scholar] [CrossRef]
- Volpicella, M.; Leoni, C.; Costanza, A.; Fanizza, I.; Placido, A.; Ceci, L.R. Genome Walking by Next Generation Sequencing Approaches. Biology 2012, 1, 495–507. [Google Scholar] [CrossRef] [PubMed]
- Guo, B.; Guo, Y.; Hong, H.; Qiu, L.J. Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method. Front. Plant Sci. 2016, 7, 1009. [Google Scholar] [CrossRef] [PubMed]
- Chen, X.; Dong, Y.; Huang, Y.; Fan, J.; Yang, M.; Zhang, J. Whole-genome resequencing using next-generation and Nanopore sequencing for molecular characterization of T-DNA integration in transgenic poplar 741. BMC Genom. 2021, 22, 329. [Google Scholar] [CrossRef] [PubMed]
- Li, S.; Jia, S.; Hou, L.; Nguyen, H.; Sato, S.; Holding, D.; Cahoon, E.; Zhang, C.; Clemente, T.; Yu, B. Mapping of transgenic alleles in soybean using a nanopore-based sequencing strategy. J. Exp. Bot. 2019, 70, 3825–3833. [Google Scholar] [CrossRef] [PubMed]
- Siddique, K.; Wei, J.; Li, R.; Zhang, D.; Shi, J. Identification of T-DNA Insertion Site and Flanking Sequence of a Genetically Modified Maize Event IE09S034 Using Next-Generation Sequencing Technology. Mol. Biotechnol. 2019, 61, 694–702. [Google Scholar] [CrossRef]
- Tang, G.; Zhong, X.; Hong, W.; Li, J.; Shu, Y.; Liu, L. Generation and Identification of the Number of Copies of Exogenous Genes and the T-DNA Insertion Site in SCN-Resistance Transformation Event ZHs1-2. Int. J. Mol. Sci. 2022, 23, 6849. [Google Scholar] [CrossRef]
- Hedden, P. The genes of the Green Revolution. Trends Genet. 2003, 19, 5–9. [Google Scholar] [CrossRef]
- Gautam, R.; Shukla, P.; Kirti, P.B. Male sterility in plants: An overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production. Theor. Appl. Genet. 2023, 136, 195. [Google Scholar] [CrossRef]
- Khush, G.S. Green revolution: The way forward. Nat. Rev. Genet. 2001, 2, 815–822. [Google Scholar] [CrossRef]
- Gaur, V.S.; Channappa, G.; Chakraborti, M.; Sharma, T.R.; Mondal, T.K. ‘Green revolution’ dwarf gene sd1 of rice has gigantic impact. Brief. Funct. Genom. 2020, 19, 390–409. [Google Scholar] [CrossRef]
- Wu, Z.; Tang, D.; Liu, K.; Miao, C.; Zhuo, X.; Li, Y.; Tan, X.; Sun, M.; Luo, Q.; Cheng, Z. Characterization of a new semi-dominant dwarf allele of SLR1 and its potential application in hybrid rice breeding. J. Exp. Bot. 2018, 69, 4703–4713. [Google Scholar] [CrossRef] [PubMed]
- Peng, J.; Richards, D.E.; Hartley, N.M.; Murphy, G.P.; Devos, K.M.; Flintham, J.E.; Beales, J.; Fish, L.J.; Worland, A.J.; Pelica, F.; et al. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 1999, 400, 256–261. [Google Scholar] [CrossRef] [PubMed]
- Tang, T.; Botwright Acuña, T.; Spielmeyer, W.; Richards, R.A. Effect of gibberellin-sensitive Rht18 and gibberellin-insensitive Rht-D1b dwarfing genes on vegetative and reproductive growth in bread wheat. J. Exp. Bot. 2021, 72, 445–458. [Google Scholar] [CrossRef] [PubMed]
- Zhang, L.; Ma, C.; Wang, L.; Su, X.; Huang, J.; Cheng, H.; Guo, H. Repression of GhTUBB1 Reduces Plant Height in Gossypium hirsutum. Int. J. Mol. Sci. 2023, 24, 15424. [Google Scholar] [CrossRef] [PubMed]
- Donadio, L.; Lederman, I.; Roberto, S.; Stuchi, E. Dwarfing-canopy and rootstock cultivars for fruit trees. Rev. Bras. Frutic. 2019, 41, e-997. [Google Scholar] [CrossRef]
- Li, X.; Xiang, F.; Zhang, W.; Yan, J.; Li, X.; Zhong, M.; Yang, P.; Chen, C.; Liu, X.; Mao, D.; et al. Characterization and fine mapping of a new dwarf mutant in Brassica napus. BMC Plant Biol. 2021, 21, 117. [Google Scholar] [CrossRef] [PubMed]
- Wang, Y.; He, J.; Yang, L.; Wang, Y.; Chen, W.; Wan, S.; Chu, P.; Guan, R. Fine mapping of a major locus controlling plant height using a high-density single-nucleotide polymorphism map in Brassica napus. Theor. Appl. Genet. 2016, 129, 1479–1491. [Google Scholar] [CrossRef]
- Zhao, B.; Wang, B.; Li, Z.; Guo, T.; Zhao, J.; Guan, Z.; Liu, K. Identification and characterization of a new dwarf locus DS-4 encoding an Aux/IAA7 protein in Brassica napus. Theor. Appl. Genet. 2019, 132, 1435–1449. [Google Scholar] [CrossRef]
- Cheng, H.; Jin, F.; Zaman, Q.U.; Ding, B.; Hao, M.; Wang, Y.; Huang, Y.; Wells, R.; Dong, Y.; Hu, Q. Identification of Bna.IAA7.C05 as allelic gene for dwarf mutant generated from tissue culture in oilseed rape. BMC Plant Biol. 2019, 19, 500. [Google Scholar] [CrossRef]
- Li, H.; Li, J.; Song, J.; Zhao, B.; Guo, C.; Wang, B.; Zhang, Q.; Wang, J.; King, G.J.; Liu, K. An auxin signaling gene BnaA3.IAA7 contributes to improved plant architecture and yield heterosis in rapeseed. New Phytol. 2019, 222, 837–851. [Google Scholar] [CrossRef]
- Tingdong, F.; Yongming, Z. Progress and future development of hybrid rapeseed in China. Eng. Sci. 2013, 11, 13–18. [Google Scholar]
- Dian-rong, L.; Jian-hua, T. Role and function of cultivar Qinyou 2 in rapeseed hybrid breeding and production in China. Chin. J. Oil Crop Sci. 2015, 37, 902. [Google Scholar]
- Yamagishi, H.; Bhat, S.R. Cytoplasmic male sterility in Brassicaceae crops. Breed. Sci. 2014, 64, 38–47. [Google Scholar] [CrossRef] [PubMed]
- Tester, M.; Langridge, P. Breeding technologies to increase crop production in a changing world. Science 2010, 327, 818–822. [Google Scholar] [CrossRef] [PubMed]
- Whitford, R.; Fleury, D.; Reif, J.C.; Garcia, M.; Okada, T.; Korzun, V.; Langridge, P. Hybrid breeding in wheat: Technologies to improve hybrid wheat seed production. J. Exp. Bot. 2013, 64, 5411–5428. [Google Scholar] [CrossRef] [PubMed]
- Chen, L.; Liu, Y.-G. Male Sterility and Fertility Restoration in Crops. Annu. Rev. Plant Biol. 2014, 65, 579–606. [Google Scholar] [CrossRef]
- Singh, S.; Dey, S.S.; Bhatia, R.; Kumar, R.; Behera, T.K. Current understanding of male sterility systems in vegetable Brassicas and their exploitation in hybrid breeding. Plant Reprod. 2019, 32, 231–256. [Google Scholar] [CrossRef]
- Wu, G.; Li, Z.; Wu, Y.; Cao, Y.; Lu, C. Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Brassica napus. J. Agric. Food Chem. 2010, 58, 2812–2817. [Google Scholar] [CrossRef]
- Wang, Z.; Ye, S.; Li, J.; Zheng, B.; Bao, M.; Ning, G. Fusion primer and nested integrated PCR (FPNI-PCR): A new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 2011, 11, 109. [Google Scholar] [CrossRef]
- Song, J.-M.; Guan, Z.; Hu, J.; Guo, C.; Yang, Z.; Wang, S.; Liu, D.; Wang, B.; Lu, S.; Zhou, R. Eight high-quality genomes reveal pan-genome architecture and ecotype differentiation of Brassica napus. Nat. Plants 2020, 6, 34–45. [Google Scholar] [CrossRef]
- Koren, S.; Walenz, B.P.; Berlin, K.; Miller, J.R.; Bergman, N.H.; Phillippy, A.M. Canu: Scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017, 27, 722–736. [Google Scholar] [CrossRef] [PubMed]
- Zhao, Z.; Zang, S.; Zou, W.; Pan, Y.-B.; Yao, W.; You, C.; Que, Y. Long Non-Coding RNAs: New Players in Plants. Int. J. Mol. Sci. 2022, 23, 9301. [Google Scholar] [CrossRef] [PubMed]
- Hayashi, K.-i. The Interaction and Integration of Auxin Signaling Components. Plant Cell Physiol. 2012, 53, 965–975. [Google Scholar] [CrossRef] [PubMed]
- Plackett, A.R.; Powers, S.J.; Fernandez-Garcia, N.; Urbanova, T.; Takebayashi, Y.; Seo, M.; Jikumaru, Y.; Benlloch, R.; Nilsson, O.; Ruiz-Rivero, O.; et al. Analysis of the developmental roles of the Arabidopsis gibberellin 20-oxidases demonstrates that GA20ox1, -2, and -3 are the dominant paralogs. Plant Cell 2012, 24, 941–960. [Google Scholar] [CrossRef] [PubMed]
- Jasinski, S.; Piazza, P.; Craft, J.; Hay, A.; Woolley, L.; Rieu, I.; Phillips, A.; Hedden, P.; Tsiantis, M. KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities. Curr. Biol. 2005, 15, 1560–1565. [Google Scholar] [CrossRef] [PubMed]
- Wang, H.; Caruso, L.V.; Downie, A.B.; Perry, S.E. The embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism. Plant Cell 2004, 16, 1206–1219. [Google Scholar] [CrossRef]
- Kim, T.W.; Hwang, J.Y.; Kim, Y.S.; Joo, S.H.; Chang, S.C.; Lee, J.S.; Takatsuto, S.; Kim, S.K. Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis. Plant Cell 2005, 17, 2397–2412. [Google Scholar] [CrossRef] [PubMed]
- Choi, H.; Ohyama, K.; Kim, Y.Y.; Jin, J.Y.; Lee, S.B.; Yamaoka, Y.; Muranaka, T.; Suh, M.C.; Fujioka, S.; Lee, Y. The role of Arabidopsis ABCG9 and ABCG31 ATP binding cassette transporters in pollen fitness and the deposition of steryl glycosides on the pollen coat. Plant Cell 2014, 26, 310–324. [Google Scholar] [CrossRef]
- Yadav, V.; Molina, I.; Ranathunge, K.; Castillo, I.Q.; Rothstein, S.J.; Reed, J.W. ABCG transporters are required for suberin and pollen wall extracellular barriers in Arabidopsis. Plant Cell 2014, 26, 3569–3588. [Google Scholar] [CrossRef]
- Lee, Y.-H.; Tegeder, M. Selective expression of a novel high-affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers. Plant J. 2004, 40, 60–74. [Google Scholar] [CrossRef]
- Rösti, J.; Barton, C.J.; Albrecht, S.; Dupree, P.; Pauly, M.; Findlay, K.; Roberts, K.; Seifert, G.J. UDP-Glucose 4-Epimerase Isoforms UGE2 and UGE4 Cooperate in Providing UDP-Galactose for Cell Wall Biosynthesis and Growth of Arabidopsis thaliana. Plant Cell 2007, 19, 1565–1579. [Google Scholar] [CrossRef] [PubMed]
- Park, J.I.; Ishimizu, T.; Suwabe, K.; Sudo, K.; Masuko, H.; Hakozaki, H.; Nou, I.S.; Suzuki, G.; Watanabe, M. UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana. Plant Cell Physiol. 2010, 51, 981–996. [Google Scholar] [CrossRef] [PubMed]
- Niewiadomski, P.; Knappe, S.; Geimer, S.; Fischer, K.; Schulz, B.; Unte, U.S.; Rosso, M.G.; Ache, P.; Flügge, U.-I.; Schneider, A. The Arabidopsis Plastidic Glucose 6-Phosphate/Phosphate Translocator GPT1 Is Essential for Pollen Maturation and Embryo Sac Development. Plant Cell 2005, 17, 760–775. [Google Scholar] [CrossRef] [PubMed]
- Li, X.-C.; Zhu, J.; Yang, J.; Zhang, G.-R.; Xing, W.-F.; Zhang, S.; Yang, Z.-N. Glycerol-3-Phosphate Acyltransferase 6 (GPAT6) Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility. Mol. Plant 2012, 5, 131–142. [Google Scholar] [CrossRef] [PubMed]
- Zheng, Z.; Xia, Q.; Dauk, M.; Shen, W.; Selvaraj, G.; Zou, J. Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility. Plant Cell 2003, 15, 1872–1887. [Google Scholar] [CrossRef] [PubMed]
- Casatejada-Anchel, R.; Muñoz-Bertomeu, J.; Rosa-Téllez, S.; Anoman, A.D.; Nebauer, S.G.; Torres-Moncho, A.; Fernie, A.R.; Ros, R. Phosphoglycerate dehydrogenase genes differentially affect Arabidopsis metabolism and development. Plant Sci. 2021, 306, 110863. [Google Scholar] [CrossRef] [PubMed]
- Chen, W.; Yu, X.H.; Zhang, K.; Shi, J.; De Oliveira, S.; Schreiber, L.; Shanklin, J.; Zhang, D. Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis. Plant Physiol. 2011, 157, 842–853. [Google Scholar] [CrossRef]
- De Buck, S.; Windels, P.; De Loose, M.; Depicker, A. Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable β-glucuronidase accumulation levels. Cell. Mol. Life Sci. CMLS 2004, 61, 2632–2645. [Google Scholar] [CrossRef]
- De Buck, S.; Podevin, N.; Nolf, J.; Jacobs, A.; Depicker, A. The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Plant J. 2009, 60, 134–145. [Google Scholar] [CrossRef]
- Sallaud, C.; Meynard, D.; van Boxtel, J.; Gay, C.; Bès, M.; Brizard, J.P.; Larmande, P.; Ortega, D.; Raynal, M.; Portefaix, M.; et al. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor. Appl. Genet. 2003, 106, 1396–1408. [Google Scholar] [CrossRef]
- Bubner, B.; Baldwin, I.T. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Rep. 2004, 23, 263–271. [Google Scholar] [CrossRef] [PubMed]
- Ingham, D.J.; Beer, S.; Money, S.; Hansen, G. Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 2001, 31, 132–134, 136–140. [Google Scholar] [CrossRef] [PubMed]
- Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef] [PubMed]
- Mieog, J.C.; Howitt, C.A.; Ral, J.P. Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay. BMC Plant Biol. 2013, 13, 71. [Google Scholar] [CrossRef] [PubMed]
- Lei, L.; Li, S.; Du, J.; Bashline, L.; Gu, Y. Cellulose synthase INTERACTIVE3 regulates cellulose biosynthesis in both a microtubule-dependent and microtubule-independent manner in Arabidopsis. Plant Cell 2013, 25, 4912–4923. [Google Scholar] [CrossRef] [PubMed]
- Wolters, H.; Jürgens, G. Survival of the flexible: Hormonal growth control and adaptation in plant development. Nat. Rev. Genet. 2009, 10, 305–317. [Google Scholar] [CrossRef] [PubMed]
- Reinhardt, D. Vascular Patterning: More Than Just Auxin? Curr. Biol. 2003, 13, R485–R487. [Google Scholar] [CrossRef]
- Vanneste, S.; Friml, J. Auxin: A trigger for change in plant development. Cell 2009, 136, 1005–1016. [Google Scholar] [CrossRef]
- Zhao, Y.; Christensen, S.K.; Fankhauser, C.; Cashman, J.R.; Cohen, J.D.; Weigel, D.; Chory, J. A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 2001, 291, 306–309. [Google Scholar] [CrossRef]
- Geisler, M.; Bailly, A.; Ivanchenko, M. Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins. Plant Sci. 2016, 245, 1–10. [Google Scholar] [CrossRef]
- Lavy, M.; Estelle, M. Mechanisms of auxin signaling. Development 2016, 143, 3226–3229. [Google Scholar] [CrossRef] [PubMed]
- Liscum, E.; Reed, J.W. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol. Biol. 2002, 49, 387–400. [Google Scholar] [CrossRef] [PubMed]
- Overvoorde, P.J.; Okushima, Y.; Alonso, J.M.; Chan, A.; Chang, C.; Ecker, J.R.; Hughes, B.; Liu, A.; Onodera, C.; Quach, H.; et al. Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana. Plant Cell 2005, 17, 3282–3300. [Google Scholar] [CrossRef] [PubMed]
- Wang, J.; Yan, D.W.; Yuan, T.T.; Gao, X.; Lu, Y.T. A gain-of-function mutation in IAA8 alters Arabidopsis floral organ development by change of jasmonic acid level. Plant Mol. Biol. 2013, 82, 71–83. [Google Scholar] [CrossRef] [PubMed]
- Tan, Y.; Ren, L.; Wang, J.; Ran, S.; Wu, L.; Cheng, Z.; Qu, C.; Li, J.; Liu, L. Identification and characterization of a curly-leaf locus CL1 encoding an IAA2 protein in Brassica napus. Crop J. 2023, 11, 756–765. [Google Scholar] [CrossRef]
- Wei, T.; Zhang, L.; Zhu, R.; Jiang, X.; Yue, C.; Su, Y.; Ren, H.; Wang, M. A Gain-of-Function Mutant of IAA7 Inhibits Stem Elongation by Transcriptional Repression of EXPA5 Genes in Brassica napus. Int. J. Mol. Sci. 2021, 22, 9018. [Google Scholar] [CrossRef] [PubMed]
- Zheng, M.; Hu, M.; Yang, H.; Tang, M.; Zhang, L.; Liu, H.; Li, X.; Liu, J.; Sun, X.; Fan, S.; et al. Three BnaIAA7 homologs are involved in auxin/brassinosteroid-mediated plant morphogenesis in rapeseed (Brassica napus L.). Plant Cell Rep. 2019, 38, 883–897. [Google Scholar] [CrossRef]
- Gutierrez, L.; Mongelard, G.; Floková, K.; Pacurar, D.I.; Novák, O.; Staswick, P.; Kowalczyk, M.; Pacurar, M.; Demailly, H.; Geiss, G.; et al. Auxin controls Arabidopsis adventitious root initiation by regulating jasmonic acid homeostasis. Plant Cell 2012, 24, 2515–2527. [Google Scholar] [CrossRef]
- Sun, T.-p. The molecular mechanism and evolution of the GA–GID1–DELLA signaling module in plants. Curr. Biol. 2011, 21, R338–R345. [Google Scholar] [CrossRef]
- Khripach, V.; Zhabinskii, V.; de Groot, A. Twenty Years of Brassinosteroids: Steroidal Plant Hormones Warrant Better Crops for the XXI Century. Ann. Bot. 2000, 86, 441–447. [Google Scholar] [CrossRef]
- Mitchell, J.W.; Mandava, N.; Worley, J.F.; Plimmer, J.R.; Smith, M.V. Brassins—A new family of plant hormones from rape pollen. Nature 1970, 225, 1065–1066. [Google Scholar] [CrossRef] [PubMed]
- Quilichini, T.D.; Douglas, C.J.; Samuels, A.L. New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana. Ann. Bot. 2014, 114, 1189–1201. [Google Scholar] [CrossRef] [PubMed]
- Polowick, P.L.; Sawhney, V.K. Differentiation of the Tapetum During Microsporogenesis in Tomato (Lycopersicon esculentum Mill.), with Special Reference to the Tapetal Cell Wall. Ann. Bot. 1993, 72, 595–605. [Google Scholar] [CrossRef]
- Narayanan, S.; Prasad, P.V.V.; Welti, R. Alterations in wheat pollen lipidome during high day and night temperature stress. Plant Cell Environ. 2018, 41, 1749–1761. [Google Scholar] [CrossRef] [PubMed]
- Wan, X.; Wu, S.; Li, Z.; An, X.; Tian, Y. Lipid Metabolism: Critical Roles in Male Fertility and Other Aspects of Reproductive Development in Plants. Mol. Plant 2020, 13, 955–983. [Google Scholar] [CrossRef]
- Wei, S.; Ma, L. Comprehensive Insight into Tapetum-Mediated Pollen Development in Arabidopsis thaliana. Cells 2023, 12, 247. [Google Scholar] [CrossRef]
- Liu, S.; Li, Z.; Wu, S.; Wan, X. The essential roles of sugar metabolism for pollen development and male fertility in plants. Crop J. 2021, 9, 1223–1236. [Google Scholar] [CrossRef]
- Dai, C.; Li, Y.; Li, L.; Du, Z.; Lin, S.; Tian, X.; Li, S.; Yang, B.; Yao, W.; Wang, J.; et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. Mol. Breed. 2020, 40, 96. [Google Scholar] [CrossRef]
- Aboul-Maaty, N.A.-F.; Oraby, H.A.-S. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull. Natl. Res. Cent. 2019, 43, 25. [Google Scholar] [CrossRef]
- Li, H. New strategies to improve minimap2 alignment accuracy. Bioinformatics 2021, 37, 4572–4574. [Google Scholar] [CrossRef]
- Danecek, P.; Bonfield, J.K.; Liddle, J.; Marshall, J.; Ohan, V.; Pollard, M.O.; Whitwham, A.; Keane, T.; McCarthy, S.A.; Davies, R.M.; et al. Twelve years of SAMtools and BCFtools. GigaScience 2021, 10, giab008. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.; Langmead, B.; Salzberg, S.L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef] [PubMed]
- Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef]
- Wu, T.; Hu, E.; Xu, S.; Chen, M.; Guo, P.; Dai, Z.; Feng, T.; Zhou, L.; Tang, W.; Zhan, L.; et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation 2021, 2, 100141. [Google Scholar] [CrossRef]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
Method | Primer Set Name | Concentration (Copy/μL) | Ratio to CruA | T-DNA Copy Number |
---|---|---|---|---|
qPCR | CruA | 9,057,666.67 ± 549,797.54 | 1.00 ± 0.05 | - |
HTP II | 17,803,333.33 ± 1,201,013.46 | 1.97 ± 0.08 | 7.87 ± 0.3 | |
NOS | 20,300,000.00 ± 2,714,479.69 | 2.23 ± 0.14 | 8.94 ± 0.56 | |
P35S | 17,373,333.33 ± 489,114.85 | 1.92 ± 0.05 | 7.68 ± 0.21 | |
ddPCR | CruA | 47.98 ± 1.69 | 1.00 ± 0.04 | - |
HTP II | 172.00 ± 1.22 | 3.59 ± 0.13 | 7.18 ± 0.27 | |
NOS | 82.95 ± 1.69 | 1.73 ± 0.09 | 6.93 ± 0.36 | |
P35S | 84.73 ± 2.51 | 1.77 ± 0.11 | 7.08 ± 0.43 |
Chromosome | Insertion Sites | Adjacent T-DNA Board | Strand |
---|---|---|---|
scaffoldA04 | 23,593,294 | RB | + |
scaffoldA04 | 23,593,903 | LB | − |
scaffoldC07 | 58,081,718 | LB | − |
scaffoldA07 | 30,827,977 | RB | − |
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Zhao, S.; Luo, J.; Tang, M.; Zhang, C.; Song, M.; Wu, G.; Yan, X. Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line. Int. J. Mol. Sci. 2024, 25, 174. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25010174
Zhao S, Luo J, Tang M, Zhang C, Song M, Wu G, Yan X. Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line. International Journal of Molecular Sciences. 2024; 25(1):174. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25010174
Chicago/Turabian StyleZhao, Shengbo, Junling Luo, Min Tang, Chi Zhang, Miaoying Song, Gang Wu, and Xiaohong Yan. 2024. "Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line" International Journal of Molecular Sciences 25, no. 1: 174. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25010174