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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   

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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

  1. Pipette tips (10, 100, and 1,000 μl)
  2. 50-ml tube
  3. 1.7-ml tubes with caps
  4. 2.0-ml tubes with caps
  5. 2.3-mm stainless steel beads (Bio Spec Products, catalog number: 11079123ss )
  6. 30-μmnylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2316 )
  7. 20-μmnylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2315 )
  8. 10-μm nylon meshes (Sysmex, CellTrics®, catalog number: 04-0042-2314 )
  9. Glass Petri dish (Corning, PYREX®, catalog number: 423790 )
  10. Leaf tissue of C-value standard (e.g., Nicotiana tabacum L. ‘Xanthi’; 2C = 10.04 pg, Johnston et al., 1999)
  11. Spores of ferns or lycophytes (kept by dry storage and under < 4 °C)
  12. PVP-40 (Sigma-Aldrich, catalog number: PVP40 )
  13. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  14. RNaseA solution (10 mg/ml in ddH2O) (Sigma-Aldrich, catalog number: R5000-100MG )
  15. Triton X-100
  16. Sodium sulfite (Na2SO3)
  17. Tris-HCl (pH 7.5)
  18. Propidium iodide
  19. 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.
  20. PI solution (see Recipes)

Equipment

  1. Pipette (10, 100, and 1,000 μl)
  2. Vortex (Scientific Industries, model: Vortex-Genie 2 )
  3. Razors and razor pen
  4. 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

  1. BD FACSCan system (BD Biosciences, Franklin Lake, NJ, USA)

Procedure

  1. Prepare buffer for use
    1. Allocate appropriate amount of Backmen stock buffer to a 50-ml tube based on an estimation of 1-1.5 ml per sample.
    2. Add 0.04 g PVP-40, 5 μl 2-mercaptoethanol, 1 μl RNase per ml of buffer.
  2. Extract spore nuclei by bead-vortexing
    1. For each sample, weigh ~0.007 g spores into a 1.7-ml tube. Green spores that usually have thin spore walls, 4 times the amount of spores are recommended (Kuo et al., 2017)*.
      *Note: For bead-vortexing, the detailed process can be seen in the video supplied in Kuo et al. (2017): http://onlinelibrary.wiley.com/store/10.1111/nph.14291/asset/supinfo/nph14291-sup-0002-VideoS1.mov?v=1&s=dae6f0590d33413f1444bc0add857d285fb73cd6
    2. Add 16 stainless steel beads into each 1.7-ml tube.
    3. Add 250 μl of buffer into each 1.7-ml tube.
    4. Vortex these tubes at a speed of 1,900 rpm for 1 min. For green spores, a speed of 3,200 rpm and a vortex duration of 0.5 min are recommended (Kuo et al., 2017).
    5. Filter bead-vortexed samples into 2.0-ml tubes through nylon meshes. The size of nylon mesh is selected based on spore sizes to prevent spore being filtered through the mesh.
    6. Add additional buffer to the samples, and ensure each of filtered spore nuclei solutions is greater than 500 μl in volume.
  3. Extract standard nuclei by chopping leaf tissue
    1. Add 500 μl of buffer to a glass Petri dish.
    2. Add a (~400 mm2) piece of young leaf to the Petri dish, and chop it with a razor on ice until most tissue slices are less than 1 mm in size.
    3. Filter the chopped sample into a 2.0-ml tube through a 30-μm nylon mesh.
    4. Add additional buffer to the sample, and ensure that the filtered leaf nuclei solution is greater than 500 μl in volume or more depending on need.
  4. Staining nuclei solutions
    1. Mix spore nuclei and standard leaf nuclei solutions into a 500-μl volume in 2.0-ml tubes.
    2. Add 10 μl PI solution into each of mixed nuclei solutions.
    3. Incubate in the dark at 4 °C for 1 h for staining.

Data analysis

  1. Set up a histogram plot of particle count vs. linear value of relative fluorescence in BD FACSCan system.
  2. After PI staining, measure nuclear DNA content of the samples in BD FACSCan system, and adjust the fluorescence laser voltage to visualize the nuclei peaks on the histogram plot.
  3. Measure > 1,300 particles for each peak, and the coefficient variation for each peak should be lower 5% as the quality criteria suggested by Greilhuber et al. (2007).
  4. Genome size of spore nuclei (pg or Mbp) =  x standard 2C value (Figure 1).


    Figure 1. An example of flow cytometric result of spore and standard nuclei. These peaks are spore nuclei of Dryopteris varia and 2C and 4C leaf nuclei of Nicotiana tabacum L. ‘Xanthi’.

Notes

  1. To dry fertile leaves to release and collect spores, air-drying process under room temperature for 2 to 3 days is recommended. For long-term storage, spore material is better stored in tubes without any solution at a temperature lower than 4 °C.

Recipes

  1. Backmen stock buffer (Ebihara et al., 2005)
    1.0% Triton X-100
    50 mM Na2SO3
    50 mM Tris-HCl (pH 7.5)
    ddH2O (the solvent)
    Note: Store at 4 °C up to 1 year.
  2. PI solution
    2.04 mg/ml propidium iodide
    ddH2O (the solvent)
    Note: Store in the dark at 4 °C for long-term storage.

Acknowledgments

The bead-vortexing condition to extract spore nuclei of seedless vascular plants is accessed and constructed by Kuo et al. (2017). We thank Fay-Wei Li and three anonymous reviewers for providing comments on the draft of this manuscript.

References

  1. Doležel, J., Greilhuber, J. and Suda, J. (2007). Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9): 2233-2244.
  2. Ebihara, A., Ishikawa, H., Matsumoto, S., Lin, S. J., Iwatsuki, K., Takamiya, M., Watano, Y. and Ito, M. (2005). Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. Am J Bot 92(9): 1535-1547.
  3. Greilhuber, J., Temsch, E. M. and Loureiro, J. C. M. (2007). Nuclear DNA Content Measurement. In: Doleel, J., Greilhuber, J. and Suda, J. (Eds.). Flow cytometry with plant cells: analysis of genes, chromosomes and genomes. Wiley, pp: 67-102.
  4. Johnston, J., Bennett, M., Rayburn, A., Galbraith, D. and Price, H. (1999). Reference standards for determination of DNA content of plant nuclei. Am J Bot 86(5): 609-613.
  5. Kuo, L. Y., Huang, Y. J., Chang, J., Chiou, W. L. and Huang, Y. M. (2017). Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach. New Phytol 213(4):1974-1983.
  6. Loureiro, J., Rodriguez, E., Dolezel, J. and Santos, C. (2007). Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100(4): 875-888.

简介

无核维管植物,包括蕨类植物和lycophytes,产生孢子,以启动配子体阶段并完成有性繁殖。其中约10%通过生产基因组未还原的孢子而脱水。因此,能够测量孢子核DNA含量对推断其再生模式是重要的。在这里,我们提出了一个孢子流式细胞术方案,可以有效地确定无核维管植物的繁殖模式。

背景 在无核维管植物中,传统上用于推断核DNA含量以及繁殖模式的孢子发生特征,如减数分裂染色体计数。然而,这些方法是耗时的,或只能提供间接证据。 Kuo等人(2017)尚未建立一种估计这些植物的孢子核DNA含量的有效可靠的方法。在这里,我们描述了使用流式细胞术基于Kuo等人(2017)的工作来评估这些植物的孢子基因组大小的方案。

关键字:无融合生殖, 微珠涡流, 蕨类, 流式细胞术, 石松类, 孢子

材料和试剂

  1. 移液器吸头(10,100和1,000μl)
  2. 50ml管
  3. 带帽的1.7毫升管子
  4. 带帽的2.0毫升管子
  5. 2.3毫米不锈钢珠(Bio Spec Products,目录号:11079123ss)
  6. 30μm尼龙网(Sysmex,CellTrics ®,目录号:04-0042-2316)
  7. 20微米网眼(Sysmex,CellTrics ®,目录号:04-0042-2315)
  8. 10μm尼龙网(Sysmex,CellTrics ®,目录号:04-0042-2314)
  9. 玻璃培养皿(Corning,PYREX ®,目录号:423790)
  10. C值标准的叶组织(例如,,Nicotiana tabacum L。'Xanthi'; 2C = 10.04pg,Johnston等人,1999 )
  11. 蕨类植物或lycophytes孢子(通过干燥储存和<4℃保存)
  12. PVP-40(Sigma-Aldrich,目录号:PVP40)
  13. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  14. RNaseA溶液(10毫克/毫升,ddH 2 O)(Sigma-Aldrich,目录号:R5000-100MG)
  15. Triton X-100
  16. 亚硫酸钠(Na 2 SO 3)
  17. Tris-HCl(pH 7.5)
  18. 碘化丙啶
  19. 后备库存缓冲(见配方)*
    注意:LB01缓冲液(Doležel等人,2007)或GPB缓冲液(Loureiro等人,2007)可以根据植物材料性质而替代使用。
  20. PI解决方案(见配方)

设备

  1. 移液器(10,100和1,000μl)
  2. Vortex(科学工业,型号:Vortex-Genie 2)
  3. 剃须刀和剃须刀笔
  4. 流式细胞仪(BD,BD Biosciences,型号:FACScan)*
    注意:FACScan具有发射波长为488 nm的15微米蓝色氩离子激光器。

软件

  1. BD FACSCan系统(BD Biosciences,Franklin Lake,NJ,USA)

程序

  1. 准备使用缓冲区
    1. 根据每个样本1-1.5毫升的估计,将适量的Backmen储备缓冲液分配到50ml管中。
    2. 加入0.04g PVP-40,5μl2-巯基乙醇,每ml缓冲液1μlRNA酶
  2. 通过珠 - 漩涡提取孢子核
    1. 对于每个样品,将〜0.007g孢子称重至1.7ml管中。推荐通常具有细孢子壁的绿色孢子,是孢子量的4倍(Kuo等人,2017)*。
      注意:对于珠流漩涡,详细的过程可以在Kuo等人提供的视频中看到。 (2017): http://onlinelibrary.wiley.com/store/10.1111/nph.14291/asset/supinfo/nph14291-sup-0002-VideoS1.mov?v = 1&amp; s = dae6f0590d33413f1444bc0add857d285fb73cd6
    2. 在每个1.7 ml管中加入16个不锈钢珠
    3. 在每个1.7 ml管中加入250μl缓冲液
    4. 以1900rpm的速度涡旋这些管1分钟。对于绿色孢子,推荐速度为3200rpm,涡流持续时间为0.5分钟(Kuo等人,2017)。
    5. 通过尼龙网将滤珠涡旋样品过滤成2.0-ml管。根据孢子大小选择尼龙网的尺寸,以防止孢子通过网筛过滤
    6. 向样品添加额外的缓冲液,并确保每个过滤孢子核溶液的体积大于500μl
  3. 通过切碎叶组织提取标准细胞核
    1. 向玻璃培养皿中加入500μl缓冲液。
    2. 在培养皿中加入(〜400毫米 2 )青叶,用冰刀切碎,直至大部分组织切片的大小小于1毫米。
    3. 将切碎的样品通过30μm尼龙网筛过滤成2.0 ml管
    4. 向样品添加额外的缓冲液,并确保过滤的叶核溶液体积大于500μl或更多,具体取决于需要。
  4. 染色核溶液
    1. 将孢子核和标准叶核溶液在2.0 ml管中混合成500μl体积
    2. 在每个混合细胞核溶液中加入10μlPI溶液
    3. 在黑暗中在4℃下孵育1小时进行染色。

数据分析

  1. 在BD FACSCan系统中设置粒子计数与相对荧光线性值的直方图
  2. PI染色后,测定BD FACSCan系统中样品的核DNA含量,并调整荧光激光电压,直观绘制出可视化核峰。
  3. 测量&gt;每个峰值1,300个粒子,每个峰值的系数变化应该按照Greilhuber等人(2007)提出的质量标准降低5%。
  4. 孢子核的基因组大小(pg或Mbp)= &nbsp; x标准2C值图1)

    图1.孢子和标准细胞核的流式细胞计数结果的一个实例。这些峰是Dry is are are are are are are are are are i i i i i i i i i i i i的烟草(Nicotiana tabacum) L。 'Xanthi'。

笔记

  1. 干燥可育叶片以释放和收集孢子,建议在室温下进行2至3天的空气干燥过程。对于长期储存,孢子材料在低于4°C的温度下更好地储存在管中,无任何溶液。

食谱

  1. 后备库存缓冲区(Ebihara等人,2005)
    1.0%Triton X-100
    50mM Na 2 SO 3
    50mM Tris-HCl(pH7.5)
    ddH 2 O(溶剂)
    注意:在4°C下存储1年。
  2. PI解决方案
    2.04mg / ml碘化丙啶
    ddH 2 O(溶剂)
    注意:请在黑暗中存放4°C长时间存放。

致谢

Kuo等人访问并构建了提取无核维管植物孢子核的珠子漩涡条件。 (2017)。感谢李菲菲和三位匿名评论员对本手稿草案提出意见。

参考

  1. Doležel,J.,Greilhuber,J.and Suda,J。(2007)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/17853881”目标=“_ blank”>使用流式细胞术估计植物中的核DNA含量。 Nat Protoc 2(9):2233-2244。
  2. Ebihara,A.,Ishikawa,H.,Matsumoto,S.,Lin,SJ,Iwatsuki,K.,Takamiya,M.,Watano,Y.and Ito,M。(2005)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/21646171“target =”_ blank“>核DNA,叶绿体DNA和倍性分析阐明了Vandenboschia radicans的生物学复杂性< / em>> in。。。。。。。。。。。。>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
  3. Greilhuber,J.,Temsch,EM和Loureiro,JCM(2007)。&lt; a class =“ke-insertfile”href =“http://onlinelibrary.wiley.com/doi/10.1002/9783527610921.ch4/summary”目标=“_ blank”>核DNA内容测量。在Doleel,J.,Greilhuber,J.和Suda,J.(Eds。)。流式细胞术与植物细胞:分析基因,染色体和基因组。 Wiley ,pp:67-102。
  4. Johnston,J.,Bennett,M.,Rayburn,A.,Galbraith,D。和Price,H。(1999)。&lt; a class =“ke-insertfile”href =“https://www.ncbi。 nlm.nih.gov/pubmed/10330063“target =”_ blank“>用于测定植物核的DNA含量的参考标准。Am J Bot 86(5):609-613。
  5. (a)=“ke-insertfile”href =“http://onlinelibrary.wiley.com/doi/” 10.1111 / nph.14291 / epdf?r3_referer = wol&tracking_action = preview_click&show_checkout = 1&purchase_referrer = xueshu.baidu.com&purchase_site_license = LICENSE_DENIED“target =”_ blank“>评估蕨类植物和lycophytes的孢子基因组大小:流式细胞术方法。 em>新的Phytol 213(4):1974-1983。
  6. Loureiro,J.,Rodriguez,E.,Dolezel,J.and Santos,C。(2007)。&nbsp; 用于植物DNA流式细胞术的两种新的核分离缓冲液:具有37种物种的测试。 Ann Bot 100(4):875-888。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
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