Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species

Abstract

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome.

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Acknowledgments

Research funds were provided by Spanish National projects (BIO2007-29814 and BIO2010-12302-E) and Generalitat Valenciana (Prometeo/2009/075). The research leading to these results has also received funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement No. 289841 (ProCoGen). We thank M. A. Morcillo for technical assistance, Dr. José Juarez (Instituto Valenciano de Investigaciones Agrarias, IVIA, Spain) and Dr. Vesselina Nikolova (Vegetable Crops Research Institute, Plovdiv, Bulgaria) for their support in the flow cytometry and karyological analyses, respectively.

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Correspondence to I. Arrillaga.

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: Profiles of SSR marker genetic analysis of lines generated from megagametophyte cultures. Oria 6, mother tree; L1-L12 and L14-L24 putative haploid lines; L13, L25 and L26 diploid controls. (JPEG 1055 kb)

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Arrillaga, I., Guevara, M.A., Muñoz-Bertomeu, J. et al. Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species. Plant Cell Tiss Organ Cult 118, 147–155 (2014). https://doi.org/10.1007/s11240-014-0470-z

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Keywords

  • Chromosome counting
  • Flow cytometry
  • Genome sequencing
  • Haploid callus
  • Microsatellites
  • Pinus pinaster