MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat
Abstract Background Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-11-01
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| Series: | BMC Plant Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s12870-018-1514-z |
| _version_ | 1818032454321045504 |
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| author | Arun S. K. Shunmugam Venkatesh Bollina Stefanie Dukowic-Schulze Pankaj K. Bhowmik Chris Ambrose James D. Higgins Curtis Pozniak Andrew G. Sharpe Kevin Rozwadowski Sateesh Kagale |
| author_facet | Arun S. K. Shunmugam Venkatesh Bollina Stefanie Dukowic-Schulze Pankaj K. Bhowmik Chris Ambrose James D. Higgins Curtis Pozniak Andrew G. Sharpe Kevin Rozwadowski Sateesh Kagale |
| author_sort | Arun S. K. Shunmugam |
| collection | DOAJ |
| description | Abstract Background Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. Results We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. Conclusion The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species. |
| first_indexed | 2024-12-10T06:07:38Z |
| format | Article |
| id | doaj.art-769933a3de814891a532f277f60e0260 |
| institution | Directory Open Access Journal |
| issn | 1471-2229 |
| language | English |
| last_indexed | 2024-12-10T06:07:38Z |
| publishDate | 2018-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Plant Biology |
| spelling | doaj.art-769933a3de814891a532f277f60e02602022-12-22T01:59:40ZengBMCBMC Plant Biology1471-22292018-11-0118111210.1186/s12870-018-1514-zMeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheatArun S. K. Shunmugam0Venkatesh Bollina1Stefanie Dukowic-Schulze2Pankaj K. Bhowmik3Chris Ambrose4James D. Higgins5Curtis Pozniak6Andrew G. Sharpe7Kevin Rozwadowski8Sateesh Kagale9National Research Council CanadaNational Research Council CanadaDepartment of Horticultural Science, University of MinnesotaNational Research Council CanadaDepartment of Biology, University of SaskatchewanDepartment of Genetics and Genome Biology, University of LeicesterDepartment of Plant Sciences, University of SaskatchewanNational Research Council CanadaAgriculture and Agri-Food CanadaNational Research Council CanadaAbstract Background Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. Results We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. Conclusion The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.http://link.springer.com/article/10.1186/s12870-018-1514-zMeiosisMeiocytesSporogenous archesporial columnWheatCytogeneticsMeiotic prophase |
| spellingShingle | Arun S. K. Shunmugam Venkatesh Bollina Stefanie Dukowic-Schulze Pankaj K. Bhowmik Chris Ambrose James D. Higgins Curtis Pozniak Andrew G. Sharpe Kevin Rozwadowski Sateesh Kagale MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat BMC Plant Biology Meiosis Meiocytes Sporogenous archesporial column Wheat Cytogenetics Meiotic prophase |
| title | MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat |
| title_full | MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat |
| title_fullStr | MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat |
| title_full_unstemmed | MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat |
| title_short | MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat |
| title_sort | meiocapture an efficient method for staging and isolation of meiocytes in the prophase i sub stages of meiosis in wheat |
| topic | Meiosis Meiocytes Sporogenous archesporial column Wheat Cytogenetics Meiotic prophase |
| url | http://link.springer.com/article/10.1186/s12870-018-1514-z |
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