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.
A comprehensive understanding of these processes requires a thorough understanding of their gene regulatory networks and catalytic and structural proteins.
#PROPHASE UNDER MICROSCOPE SERIES#
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.
Prophase I is the longest (taking up to 90% of the total duration) and arguably most important phase of meiosis. The underlying genetic control of strict homologous chromosome pairing in polyploid plants, such as commercial hexaploid bread wheat ( Triticum aestivum, 2n = 6x = 42 AABBDD), tetraploid pasta wheat ( Triticum durum, 2n = 4x = 28 AABB) or canola ( Brassica napus, AACC) is not fully understood yet. Polyploid crops remarkably display diploid-like meiotic behavior and disomic inheritance despite having highly similar homoeologous chromosomes. In plants, polyploidy adds an extra layer of complexity to the meiotic process. Although the cytological events during meiosis are well characterized, the mechanisms controlling meiotic progression, chromosome recognition, pairing between homologous or homoeologous chromosomes and recombination are still poorly understood. The process of meiosis occurs in specialized cells called meiocytes, and involves three principal events that include chromosome pairing, recombination and segregation. Meiosis is a highly conserved process that is essential for fertility in sexually reproducing organisms. The method also provides a foundation for similar studies in other crop species. 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 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. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. 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. 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.
#PROPHASE UNDER MICROSCOPE FREE#
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 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. 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. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis.
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