Determining Genome Size from Spores of Seedless Vascular Plants
无种子维管束植物孢子基因组大小的测定
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2322 浏览次数: 8753
评审: Scott A M McAdamEmily CopeAnonymous reviewer(s)
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The authors used this protocol in:
Mar 2017
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Abstract
Seedless vascular plants, including ferns and lycophytes, produce spores to initiate the gametophyte stage and to complete sexual reproduction. Approximately 10% of them are apomictic through the production of genomic unreduced spores. Being able to measure the spore nuclear DNA content is therefore important to infer their reproduction mode. Here we present a protocol of spore flow cytometry that allows an efficient determination of the reproductive modes of seedless vascular plants.
Keywords: Apomixis (无融合生殖)
Bead-vortex (微珠涡流)
Fern (蕨类)
Flow cytometry (流式细胞术)
Lycophyte (石松类)
Spore (孢子)
Background
In seedless vascular plants, sporogenesis features, such as meiotic chromosome counts, were traditionally used to infer nuclear DNA content as well as reproductive modes. However, these approaches are time-consuming, or can only provide indirect evidence. An efficient and reliable method to estimate spore nuclear DNA content of these plants had not been established until Kuo et al. (2017). Herein, we describe a protocol using flow cytometry to evaluate spore genome sizes of these plants based on the work of Kuo et al. (2017).
Materials and Reagents
- Pipette tips (10, 100, and 1,000 μl)
- 50-ml tube
- 1.7-ml tubes with caps
- 2.0-ml tubes with caps
- 2.3-mm stainless steel beads (Bio Spec Products, catalog number: 11079123ss )
- 30-μmnylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2316 )
- 20-μmnylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2315 )
- 10-μm nylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2314 )
- Glass Petri dish (Corning, PYREX®, catalog number: 423790 )
- Leaf tissue of C-value standard (e.g., Nicotiana tabacum L. ‘Xanthi’; 2C = 10.04 pg, Johnston et al., 1999)
- Spores of ferns or lycophytes (kept by dry storage and under < 4 °C)
- PVP-40 (Sigma-Aldrich, catalog number: PVP40 )
- 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
- RNaseA solution (10 mg/ml in ddH2O) (Sigma-Aldrich, catalog number: R5000-100MG )
- Triton X-100
- Sodium sulfite (Na2SO3)
- Tris-HCl (pH 7.5)
- Propidium iodide
- Backmen stock buffer (see Recipes)*
*Note: LB01 buffer (Doležel et al., 2007) or GPB buffer (Loureiro et al., 2007) can be alternatively used depending on plant material properties. - PI solution (see Recipes)
Equipment
- Pipette (10, 100, and 1,000 μl)
- Vortex (Scientific Industries, model: Vortex-Genie 2 )
- Razors and razor pen
- Flow cytometer (BD, BD Biosciences, model: FACScan )*
*Note: FACScan with a 15 microW blue argon ion laser of an emission wave length of 488 nm.
Software
- BD FACSCan system (BD Biosciences, Franklin Lake, NJ, USA)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kuo, L. and Huang, Y. (2017). Determining Genome Size from Spores of Seedless Vascular Plants. Bio-protocol 7(11): e2322. DOI: 10.21769/BioProtoc.2322.
Download Citation in RIS Format
分类
植物科学 > 植物细胞生物学 > 组织分析
植物科学 > 植物分子生物学 > DNA > DNA 提取
细胞生物学 > 单细胞分析 > 大量细胞计数法
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