Kuo Li-Yaung, Huang Yi-Jia, Chang JenYu, Chiou Wen-Liang, Huang Yao-Moan
Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, 10617, Taiwan.
Department of Forensic Science, Central Police University, Taoyuan City, 33304, Taiwan.
New Phytol. 2017 Mar;213(4):1974-1983. doi: 10.1111/nph.14291. Epub 2016 Nov 7.
Ferns and lycophytes produce spores to initiate the gametophyte stage for sexual reproduction. Approximately 10% of these seedless vascular plants are apomictic, and produce genomic unreduced spores. Genome size comparisons between spores and leaves are a reliable, and potentially easier way to determine their reproductive mode compared to traditional approaches. However, estimation of the spore genome sizes of these plants has not been attempted. We attempted to evaluate the spore genome sizes of ferns and lycophytes using flow cytometry, collected spores from selected species representing different spore physical properties and taxonomic groups, and sought to optimize bead-vortexing conditions. By evaluating the spore and sporophyte genome sizes, we examined whether reproductive modes could be ascertained from these flow cytometry results. We proposed two separate sets of optimized bead-vortexing conditions for the nuclear extraction of green and nongreen spores. We further successfully extracted spore nuclei of 19 families covering most orders, and the qualities and quantities of these extractions satisfied the C-value criteria. These evaluated genome sizes further supported the reproductive modes reported previously. In the current study, flow cytometry was used for the first time to evaluate the spore genome sizes of ferns and lycophytes. This use of spore flow cytometry provides a new, efficient approach to ascertaining the reproductive modes of these plants.
蕨类植物和石松类植物产生孢子以启动有性生殖的配子体阶段。这些无籽维管植物中约有10%是无融合生殖的，并产生基因组未减数的孢子。与传统方法相比，孢子和叶片之间的基因组大小比较是一种可靠且可能更简便的确定其繁殖方式的方法。然而，尚未尝试估计这些植物的孢子基因组大小。我们试图使用流式细胞术评估蕨类植物和石松类植物的孢子基因组大小，从代表不同孢子物理特性和分类群的选定物种中收集孢子，并寻求优化珠子涡旋条件。通过评估孢子和孢子体的基因组大小，我们研究了是否可以从这些流式细胞术结果中确定繁殖方式。我们针对绿色和非绿色孢子的核提取提出了两组不同的优化珠子涡旋条件。我们进一步成功提取了涵盖大多数目19个科的孢子核，这些提取物的质量和数量满足C值标准。这些评估的基因组大小进一步支持了先前报道的繁殖方式。在本研究中，首次使用流式细胞术评估蕨类植物和石松类植物的孢子基因组大小。这种孢子流式细胞术的应用为确定这些植物的繁殖方式提供了一种新的有效方法。
