Challenges of flow‐cytometric estimation of nuclear genome size in orchids, a plant group with both whole‐genome and progressively partial endoreplication

Abstract

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for $10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. V C 2015 International Society for Advancement of Cytometry Key terms

Karyotype diversity and genome size variation in Neotropical Maxillariinae orchids

“…Previous measurements showed the occurrence of endoreplication in the samples – as commonly cited for Orchidaceae (Trávníček et al . ). It requires a reference standard with GS, which is not so similar to studied species, in order to avoid overlapping peaks (see Fig.…”

Flow cytometric seed screen data are consistent with models of chromosome inheritance in asymmetrically compensating allopolyploids

“…Overall, the potential of estimating flow cytometry DNA content in plant science has not been fully explored. Recent studies based on this method have revealed new phenomena in plant science (70)(71)(72) or new applications of the method (73). The present study highlights the use of FCM methodology, a powerful and precise tool for DNA content estimation, in plant reproduction biology.…”

“…Recent studies based on this method have revealed new phenomena in plant science (70)(71)(72) or new applications of the method (73). Overall, the potential of estimating flow cytometry DNA content in plant science has not been fully explored.…”

Ophrys fuscaandOphrys dyris(Orchidaceae) – constancy of tetraploidy amongst populations in Central Portugal

“…In recent times, flow cytometry has become the preferred method to estimate nuclear DNA contents in plants, not only because it is fast, reproducible and enables analysis of thousands of particles, but also because it only requires small amounts of tissue (Doležel et al, 2007). Nonetheless, this is not always straightforward, and several taxonomic groups pose challenges due to either the presence of cytosolic compounds or unbalanced numbers of nuclei in G1-phase of mitosis, which may lead to misinterpretation of the resulting flow histograms (Trávníček et al, 2015). Orchids are sometimes challenging due to frequent rounds of endoreplication (including partial endoreplication), which might be tissue-specific, hence efforts have focused in searching for alternatives in order to overcome this problem (e.g.…”

“…Orchids are sometimes challenging due to frequent rounds of endoreplication (including partial endoreplication), which might be tissue-specific, hence efforts have focused in searching for alternatives in order to overcome this problem (e.g. Pellicer and Leitch, 2014;Trávníček et al, 2015). In our study, although Ophrys does not seem to represent a major challenge in this respect, we investigated the use of pollinaria for estimating nuclear DNA contents and allocating DNA-ploidies instead of leaves.…”