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Modified protocol for obtaining isolated and high-resolution pachytene chromosomes

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Abstract

The expansion of molecular cytogenetics has called for the evaluation of pachytene chromosomes as targets for in situ hybridization (ISH) and micro-dissection. These techniques represent powerful tools for chromosome identification, karyotype comparison, physical mapping and phylogenetic analyses. Because cytogenetic preparations showing well-individualized and cytoplasm-free chromosomes are pre-requisites for ISH and micro-dissection, this work was performed with the aim of developing an improved methodology to obtain individualized pachytene chromosomes of maize (Zea mays L.). The used protocol, based on two steps of enzymatic treatment for establishment of cell suspension, released the pollen mother cells (PMCs) from maize anthers. All slides, prepared from maize PMC suspensions, showed pachytene cells with at least one individualized bivalent. This result was considered a major methodological advance, since it facilitated the scattering of all bivalents. These chromosomes were digitally straightened and four morphological parameters were established to characterize all ten maize bivalents. Considering the conditions of an excellent pachytene cytogenetic preparation for ISH and micro-dissection, the results showed that the applied methodology provided adequate pachytene chromosomes for distinct purposes and can be adapted for other plant species.

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Acknowledgements

We thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for providing the financial support for this work.

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Authors and Affiliations

  1. Embrapa Café, Parque Estação Biológica, Av. W3 Norte, CEP: 70770-901, Brasília, DF, Brazil

    Eveline Teixeira Caixeta

  2. Instituto de Biotecnologia Aplicada à Agropecuária, Universidade Federal de Viçosa, CEP: 36570-000, Viçosa, MG, Brazil

    Eveline Teixeira Caixeta

  3. Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Federal de Viçosa, CEP: 36570-000, Viçosa, MG, Brazil

    Carlos Roberto Carvalho

  4. Laboratório de Microscopia, Departamento de Produção Vegetal, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, CEP: 29500-000, Alegre, ES, Brazil

    Wellington Ronildo Clarindo

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  1. Eveline Teixeira Caixeta
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  2. Carlos Roberto Carvalho
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  3. Wellington Ronildo Clarindo
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Correspondence to Carlos Roberto Carvalho.

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Caixeta, E.T., Carvalho, C.R. & Clarindo, W.R. Modified protocol for obtaining isolated and high-resolution pachytene chromosomes. Nucleus 54, 3–7 (2011). https://doi.org/10.1007/s13237-011-0023-3

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  • DOI: https://doi.org/10.1007/s13237-011-0023-3

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