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

Abstract

BackgroundMolecular 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.ResultsWe 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1514-z) contains supplementary material, which is available to authorized users.

ASYNAPSIS 1 ensures crossover fidelity in polyploid wheat by promoting homologous recombination and suppressing non-homologous recombination

“…Meiotic stages in wheat are relatively synchronous and correlate with anther length ( Shunmugam et al., 2018 ). Immunolocalization of ASY1 and ZYP1 was performed on pollen mother cells from wild type and asy1 hypomorphic mutants to determine if meiotic prophase I progression was affected ( Figure 2 ).…”

Ubiquitination in Plant Meiosis: Recent Advances and High Throughput Methods

“…Bulk collection of meiotic tissue can introduce variation in even carefully staged samples which may obscure small or rapid changes. Further, development of meiocytes within the same anther may not be fully synchronized but it is possible to overcome this issue by introducing further staging steps such as cytological analysis ( Shunmugam et al, 2018 ; Barakate et al, 2021 ). Recently, Lewandowska et al (2019) developed a micro-proteomic workflow, allowing identification of ∼2,800 and 4,000 proteins from precisely staged single and paired barley anthers.…”

The coordinated regulation of early meiotic stages is dominated by non‐coding RNAs and stage‐specific transcription in wheat

“…Meiocytes were isolated from anthers in developing wheat inflorescences using the MeioCapture method (Shunmugam et al, 2018). Briefly, the spikes were collected along with the leaf sheath, placed in a beaker with distilled water on ice, and transferred to the lab immediately for meiocyte isolation.…”

“…Efforts to profile the male meiotic transcriptome have therefore been limited by the use of whole anthers (Crismani et al, 2006; Martín et al, 2018). Using the MeioCapture method (Shunmugam et al, 2018) for attaining high‐purity male meiocytes presented an opportunity to obtain synchronized populations of cells spanning the entire meiotic progression, including resolved cell populations in each of the five sub‐stages of prophase I. In concert with Illumina and Oxford Nanopore platforms for in‐depth coverage of mRNAs, sncRNAs, and lincRNAs, we aimed to capture a high‐resolution network of the expressed transcripts and regulatory ncRNAs present throughout meiosis.…”