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.
Highlights
Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis
Transmission electron microscopy (TEM) images of anthers ranging from 0.5 to 1.4 mm in length revealed that the Sporogenous archesporial column (SAC) retains its integrity throughout the prophase I process but eventually disintegrates as the meiocytes develop into microspores (Fig. 3, Additional file 1: Figure S1)
An intact SAC is present at the pre-meiotic stage (0.5 mm anther; Fig. 3a and b), but a disintegrated SAC with free floating meiocytes was observed after the completion of the meiosis-I process (1.4 mm anther; Fig. 3c)
Summary
Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. The process of meiosis occurs in specialized cells called meiocytes, and involves three principal events that include chromosome pairing, recombination and segregation [1]. Prophase I is the longest (taking up to 90% of the total duration) and arguably most important phase of meiosis It is divided into the five sub-stages leptotene, zygotene, pachytene, diplotene and diakinesis, during which a series of closely integrated and spatiotemporally controlled events occur, including condensation and reorganization of the chromosomes, pairing and synapsis of homologs, recombination and crossing over [4]. A prerequisite for the application of global genomic and proteomic profiling approaches to elucidate genetic interactions and pathways controlling meiosis is the availability of methods that allow isolation of high purity meiocytes from plant reproductive tissues. The male meiocytes are present in large numbers within the anther tissues, the complex morphological structure of the anther makes the isolation of male meiocytes challenging
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