MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

Shunmugam, Arun S. K.; Bollina, Venkatesh; Dukowic-Schulze, Stefanie; Bhowmik, Pankaj K.; Ambrose, Chris; Higgins, James D.; Pozniak, Curtis; Sharpe, Andrew G.; Rozwadowski, Kevin; Kagale, Sateesh

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat

journal contribution

posted on 2019-09-04, 14:00 authored by 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

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.

Funding

We gratefully acknowledge the funding support from Genome Prairie, Genome Canada, Western Grains Research Foundation, the Saskatchewan Ministry of Agriculture, Saskatchewan Wheat Development Commission, and the Alberta Wheat and Barley Development Commission through the Canadian Triticum Applied Genomics2 (CTAG2) project. SD-S was supported by NSF grants IOS-1025881 and IOS-1546792. JDH is supported by BBSRC grant BB/M014908/1.

History

Citation

BMC Plant Biology, 2018, 18, pp. 293-293 (12)

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Genetics and Genome Biology

Version

VoR (Version of Record)

Published in

BMC Plant Biology

Publisher

BMC (part of Springer Nature)

issn

1471-2229

Acceptance date

2018-10-31

Copyright date

2018

Available date

2019-09-04

Publisher DOI

https://doi.org/10.1186/s12870-018-1514-z

Publisher version

https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-018-1514-z

Notes

All data generated or analysed during the study are included in this published article [and its supplementary information files].;The Correction to this article has been published in BMC Plant Biology 2019 19:178 10.1186/s12870-019-1780-4 Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the ‘Background’. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the ‘Background’ [1]. Thus, the following amendment to the Background section should be noted: Original sentence: “Several methods have been previously described or proposed for the isolation of male meiocytes [6], such as micromanipulation (Plumbago [10, 11], Nicotiana [12], Brassica [13, 14], Arabidopsis [15, 16, 17] and sunflower [18]), capillary collection of meiocytes (CCM) (Arabidopsis [19, 20] and maize [21, 22]), laser capture microdissection (LCM) in rice [23, 24, 25, 26], Percoll gradient separation (Arabidopsis [27, 28], rice [29] and Brassica [30]) and isolation of nuclei tagged in specific cell types (INTACT) in Arabidopsis [31].” Corrected sentence: “Several methods have been previously described or proposed for the isolation of male meiocytes [6], such as micromanipulation (Plumbago [10, 11], Nicotiana [12], Brassica [13], rice [14], Arabidopsis [15, 16, 17] and sunflower [18]), capillary collection of meiocytes (CCM) (Arabidopsis [19, 20] and maize [21, 22]), laser capture microdissection (LCM) in rice [23, 24, 25, 26], Percoll gradient separation (Arabidopsis [27, 28], rice [29] and Brassica [30]) and isolation of nuclei tagged in specific cell types (INTACT) in Arabidopsis [31].”.