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Determination of the Developmental Origin of Seeds Containing Endosperm Using Flow Cytometric Analysis
流式细胞仪检测含胚乳的种子的发育起源   

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Abstract

Seeds derived from a diploid, sexual plant typically contain a 2n embryo (n+n) and 3n endosperm, a ratio characteristic for most flowering plants. However, this ratio is altered in apomictic species, which reproduce asexually through seeds (Koltunow and Grossniklaus, 2003). Apomixis is usually a facultative trait and encompasses several developmental steps: (1) apomeiosis (avoidance of meiosis), (2) parthenogenesis (embryo development without fertilization), and (3) functional endosperm formation (autonomous without fertilization or pseudogamous requiring fertilization). If all three steps occur, this process results in maternal offspring (2n+0), which is genetically identical to the mother plant (clonal). Moreover, sexual and apomictic pathways can occur in the same plant and sometimes they cross over, producing polyhaploid offspring (n+0; resulting from meiosis and parthenogenesis) or BIII hybrids (2n+n; resulting from apomeiosis and fertilization) (Rutishauser, 1947). The different types of offspring can be determined in a flow cytometric seed screen (FCSS), in which the relative chromatin content of stained nuclei is determined by measuring their fluorescence intensity. This allows a comparison of the ploidy of the endosperm to the ploidy of the embryo and, thus, an inference of the pathway by which a seed was formed (Matzk et al., 2000). This method is particularly useful to characterize the developmental origin of seeds in apomictic plants or reproductive mutants of sexual species.
Here, we present the protocol for an FCSS in Brassicaceae that has specifically been adapted to plants of the genus Boechera. However, in principle this protocol can be applied to any species producing seeds that contain endosperm.

Keywords: Flow cytometry(流式细胞仪), Seed development(种子发育), Endosperm(胚乳), Flow cytometric seed screen(流式细胞仪的种子筛), Apomixis(无融合生殖)

Materials and Reagents

  1. Citric acid monohydrate (Sigma-Aldrich, catalog number: 33114 )
  2. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  3. Na2HPO4.2H2O (Merck KGaA, catalog number: 1.06580.1000 )
  4. 4’,6-Diamidin-2-phenylindol (DAPI) (Life Technologies, InvitrogenTM, catalog number: D1306 )
  5. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  6. Sheath fluid; Iso-Diluent
 (Beckman Coulter, catalog number: NPE 629967 )
  7. Cleaning solution (Beckman Coulter, catalog number: 629969 )
  8. Shutdown solution (Beckman Coulter, catalog number: 629968 )
  9. Stock solutions (see Recipes)
  10. Otto 1 solution 0.01% (see Recipes)
  11. Otto 1 solution 1% (see Recipes)
  12. Otto 1 solution 0.5% (see Recipes)
  13. Otto 2 solution (see Recipes)

Equipment

  1. 1.2 ml Cluster tube and rack (sterile) (Thermo Fischer Scientific, catalog number: 07-200-320 )
  2. Storage plate cap strips (Thermo Fisher Scientific, catalog number: AB-0981 )
  3. 3 mm stainless steel beads (Schieritz & Hauenstein, catalog number: 22.455.0011 )
  4. Nunc Fritted deep well plates (Thermo Fisher Scientific, catalog number: 278011 )
  5. 96-well V-bottom plates (SARSTEDT AG, catalog number: 82.1583.001 )
  6. 200 μl tips (SARSTEDT AG, catalog number: 70.760.002 )
  7. 1 ml tips (Rainin/Mettler Toledo, catalog number: RC-1000/10 )
  8. Single channel pipettes (Gilson, catalog number: P10 , P1000 )
  9. Multichannel pipette 10-100 µl (Thermo Fisher Scientific, catalog number 4661130 )
  10. Multichannel pipette 30-300 µl (Thermo Fisher Scientific, catalog number 4661140 )
  11. Mixer mill (Retsch, model: MM300 )
  12. Centrifuge (Eppendorf, model: 5810R )

  13. UV lamp (part of 13. flow cytometer)
  14. Flow cytometer (Beckman Coulter, Cell Lab Quanta SC, serial number: AN020010 )
  15. Flow cytometer robotics (Beckman Coulter, MPL, Cell Lab Quanta SC, serial number: AN90022 )

Procedure

  1. Isolation of nuclei by bead-beating (3-4 h)
    1. Place a single 3 mm stainless steal bead into each cluster tube (Figure 1A).
    2. Place cluster tubes into 96-wellrack (Figure 1B).
    3. Label the tubes and mark positions of samples (as schematic drawing in the lab book).
    4.  Fix the silique on a sticky tape and slit it open with a dissection or hypodermic needle (Figure 1C-D). Add a single mature but still green seed (Figure 1C-D) into each cluster tube. In Boechera spp. endosperm degrades upon seed maturation. If a fully mature seed of the species under investigation contains endosperm, the mature seed can be used. This can be checked by microscopic analyses of cleared seeds at various stages. The method of clearing depends on the species and should be chosen and adapted based on published protocols.


      Figure 1. A. A single 3 mm stainless steel bead in a cluster tube. B. Cluster tubes containing single beads in the appropriate 96-well rack. C. Optimal seed stage for Boechera spp. seeds. The top silique has seeds that are too young to be used (too few cells), and the bottom silique has seeds at an optimal stage. D. The top silique has seeds that are too old (they start to turn brown, coinciding with degradation of the endosperm). The lower silique has seeds at an optimal stage. E. Close-up of C. F. Close-up of D. Lines indicate that part of the picture was removed in the close-up.

    5. Add 80 μl of Otto 1 (0.01%) to each tube.
    6. Close the tubes using the storage plate cap strips.
    7. Crush the seeds by shaking the samples for 3 min at 30 Hz using the mixer mill.
    8. Turn plates 180° and repeat step A6. The distal part of the arm of the mixer mill has a larger amplitude than the proximal part. Turning the plates compensates for the different amplitudes of the mixer mill.
    9. Control whether the seeds are crushed by visual inspection. Seeds are crushed if the solution turned milky (Figure 2).


      Figure 2. Crushed and non-crushed seeds. The green arrows indicate successfully crushed seeds and a milky solution. The white arrow points to a non-crushed seed. The red arrow shows a tube without a seed. If the solution is clear and the seed is visible in the cluster tube, it was not crushed and cannot be measured.

    10. If not all seeds are crushed repeat steps A7-9 until all seeds are crushed.
    11. Shake down the solution to remove drops from the lid, either by gently knocking the plate flat against the table, or by a quick spin (5 sec) in the centrifuge.
    12. Add 80 μl of Otto 1 (1%) to each tube. Be careful to not touch the tube or the foam with the tip to not cross-contaminate the samples.
    13. Close the tubes and mix by inverting 40x manually and avoid generating foam by fast movements. Now the volume is 160 μl and the concentrations are 0.1 M citric acid and 0.5% Triton X-100.
    14. Place a filter plate onto a clean 96-well V-bottom plate (Figure 3A).
    15. Label the plates.
    16. Transfer the solution with the extracted nuclei into the fritted deep well plates, either by using a multichannel pipette or by pouring. Be careful to keep the order of the samples.
    17. Centrifuge 5 min at 150 x g at room temperature (20 °C) to pellet the nuclei.
    18. Carefully remove the plates from the centrifuge. The pelleted nuclei are very loose.
    19. Remove the fritted deep well plates. If they are thoroughly washed with tap water and rinsed with deionized water, they can be reused.
    20. Remove the supernatant with a pipette tip placed at the edge of the V (Figure 3B).
    21. Do not pipette fast; otherwise nuclei will be sucked up.

    22. The pellet should be of white to light brown appearance.

    23. Add 30 μl of Otto 1 (0.5%) to resuspend the nuclei. Adding the buffer by fast pipetting is sufficient to resuspend the nuclei.
    24. Samples can now be stored for up to 72 h at 4 °C until used.


      Figure 3. A. A fritted deep well plate on a 96-well V-bottom plate. B. Cartoon representing the placement of the tip to remove the supernatant.

  2. Measuring DNA-content (ploidy) (ca. 7-8 min per sample, depending on the concentration of the nuclei)
    1. Start the flow cytometer and the UV lamp. Please refer to the user manual of the flow cytometer or get an introduction from the person in charge.
    2. While the UV lamp heats up (30 min), place the samples at room temperature to equilibrate (see Notes).
    3. Load or change the detection settings so that the diploid peak is at channel 200 (relative fluorescent units) on a linear scale (see Notes).
    4. Add 160 μl of Otto 2 solution either manually or by using the flow cytometer’s robotics.
    5. Measure the sample and record the data. Stop collecting data either after a total count of 6,000 (this takes about 7-8 min) or after a certain time period (e.g. 5 min).
    6. Treat all additional samples accordingly.
    7. Manually annotate the peaks (see Figures 4-6). Discard the samples in which the endosperm does not give a clear peak! It is essential to see the peak of the endosperm (see Notes).
    8. Save your data in a table that provides at minimum the sample name, mean, median and coefficient of variance.

  3. Analyzing the data
    1. Compute the ratio of endosperm to embryo.
    2. If the ploidy of the mother plant is available, compute the ratio of embryo to mother plant.

Representative data



Figure 4. Sexual offspring (n + n) of diploid Boechera stricta.
 This histogram shows the fluorescence intensity on the x-axis and the number of counts for each intensity on the y-axis. The peaks are manually annotated and labeled according to ploidy inferred from the internal or external standard. Green represents the nuclei of the embryo. The 4x and 8x peaks are from endoreduplicated embryonic nuclei (autopolyploidization). Brown represents the
endosperm nuclei. The ratio of the 3x:2x peaks is 275.99/187.4 = 1.47, indicating sexual development. The ratio of the 6x:4x peaks is 1.48. Using the autopolyploid peaks for ratio calculations provides an internal control.


Figure 5. Apomictic offspring (2n+0) of triploid Boechera gunnisoniana.
 Green represents the nuclei of the embryo. The 3x, 6x, and 12x peaks represent autopolyploidization. As apomictic Boechera spp. are typically pseudogamous (Rushworth et al., 2011), we expect a fertilized endosperm (Table 1). In this case, the endosperm is 9x (9x:3x ratio is 2.86), indicating fertilization by an unreduced sperm cell.


Figure 6. BIII hybrid offspring (2n+2n) of triploid Boechera gunnisoniana.
 This histogram is almost identical to that shown in Figure 6. However, the red peak (3x) is much smaller than the 6x peak, indicating that this is contamination from maternal sporophytic tissues, due to scraping the seeds from the silique. As B. gunnisoniana produces both unreduced female and male gametes, the 2n+2n embryo peak (6x) will be double the size of that from maternal nuclei (3x). These data were published by Schmidt and colleagues (2014).

Notes

  1. Colder samples show higher fluorescence intensity.
  2. The settings are changed in distinct ways using different software. The software is usually provided together with the flow cytometer. Therefore, we do not provide details about the software used. However, adapting the protocol to a new species or new tissue always requires some optimization first. Please refer to the user manual and/or obtain training from the person in charge of the instrument.
  3. Staining intensity changes over time after the Otto 2 solution has been added. It has to be ensured that the time interval from adding the Otto 2 solution to measuring the sample is constant, or that the measuring takes place once the staining intensity is stable. Here we describe the procedure using sampling robotics, which ensures that the time interval from staining to measuring is constant.
  4. The volume ratio of Otto 2 to Otto 1 solutions should be about 4:1.
  5. In our experience, 25-35% of the data has to be discarded due to unidentifiable endosperm peaks (Figure 7). This can go up to 45% in material from natural populations.
  6. The results show higher reproducibility if the median is used for ratio calculations (4 independent measurements, CV < 0.01).
  7. Use internal standards whenever possible. Internal standards are mixed to every single sample (Figure 8). For Arabidopsis thaliana we have successfully used Solanum lycopersicum var. San Marzano, and for Hieracium pilosella we have used Bellis perennis.
  8. Choosing internal standards: The DNA content of an internal standard should differ enough from the DNA content of the sample species to avoid any overlapping ploidy peaks between the standard and the sample species. Values can be found in literature. Ideally, an internal standard has a lower DNA content than the sample (always the very left peak in the histogram), or a higher DNA content than any expected ploidy peak of the sample species (always the very right peak of the histogram).
  9. If internal standards are unavailable, use external standards (plants of known ploidy). The external standard should be the first and the last sample measured in a set of samples on one plate. From the position of the external standard of the known ploidy (median of the peak), the ploidy of the unknown sample can be calculated.


    Figure 7. Unidentifiable endosperm peak


    Figure 8. Diploid (A) and triploid (B) Boechera plants with internal standard Solanum.
    Red marks the internal standard having a higher DNA content than the sample. It is therefore on the right hand side of the histogram.

Recipes

  1. Stock solutions
    1 mol/L citric acid

    Saturated (0.54 mol/L at 20 °C) Na2HPO4.2H2O solution
    2 mg/ml DAPI in water (ultrapure water, conductivity > 18 MΩ-1 cm-1)
  2. Otto 1 solution 0.01% (for 100 ml)
    0.1 mol/L citric acid

    10 ml of 1 mol/L
    0.01% (v/v) Triton X-100
    10 μl
    Fill up with water (conductivity > 18 MΩ-1 cm-1) to a total volume of 100 ml
  3. Otto 1 solution 1% (for 100 ml)
    0.1 mol/L citric acid

    10 ml of 1 mol/L
    1% (v/v) Triton X-100
    1,000 μl
    Fill up with water (conductivity > 18 MΩ-1 cm-1) to a total volume of 100 ml
  4. Otto 1 solution 0.5% (for 100 ml)
    0.1 mol/L citric acid
    10 ml of 1 mol/L
    0.5 % (v/v) Triton X-100
    500 μl
    Fill up with water (conductivity > 18 MΩ-1 cm-1) to a total volume of 100 ml
  5. Otto 2 solution (for 100 ml)
    0.4 mol/L Na2HPO4.2H2O
    74.1 ml saturated solution
    0.4 μg/ml DAPI
    200 μl of 2 mg/mL stock solution
    0.2 μl/ml β-mercaptoethanol
    200 μl
    Fill up with water (conductivity > 18 MΩ-1 cm-1) to a total volume of 100 ml

Acknowledgments

This protocol is based on previously published work (Matzk et al., 2000; Schmidt et al., 2014). Our research in this area was supported by the University of Zürich, the Marie Curie project IDEAGENA, a Syngenta Ph.D. Project of the Zurich-Basel Plant Science Center, and a grant from the “Staatssekretariat für Bildung und Forschung” in the framework of the COST Action FA0903. We thank Manuel Waller for help with flow cytometry, and Anja Herrmann and Margarida Sofia Nobre for careful reading of this protocol.

References

  1. Koltunow, A. M. and Grossniklaus, U. (2003). Apomixis: a developmental perspective. Annu Rev Plant Biol 54: 547-574.
  2. Matzk, F., Meister, A. and Schubert, I. (2000). An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J 21(1): 97-108.
  3. Rutishauser, A. (1947). Untersuchungen über die Genetik der Aposporie bei pseudogamen Potentillen. Experientia 3(5): 204-205.
  4. Rushworth, C. A., Song, B. H., Lee, C. R. and Mitchell-Olds, T. (2011). Boechera, a model system for ecological genomics. Mol Ecol 20(23): 4843-4857.
  5. Schmidt, A., Schmid, M. W., Klostermeier, U. C., Qi, W., Guthorl, D., Sailer, C., Waller, M., Rosenstiel, P. and Grossniklaus, U. (2014). Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation. PLoS Genet 10(7): e1004476.

简介

源自二倍体,有性植物的种子通常含有2n胚胎(n + n)和3n胚乳,这是大多数开花植物的比率特征。然而,这种比例在无性生殖物种中改变,其通过种子无性繁殖(Koltunow和Grossniklaus,2003)。单性生殖通常是兼性性状,包括几个发育步骤:(1)蚜虫病(避免减数分裂),(2)孤雌生殖(没有受精的胚发育)和(3)功能性胚乳形成(无需受精的自主性或需要受精的假性乳房)。如果发生所有三个步骤,该过程导致母体后代(2n + 0),其与母本植物(克隆)在遗传上相同。此外,性和无融合途径可以在同一植物中发生,并且有时它们交叉,产生多倍体倍生(n + 0;由减数分裂和孤雌生殖产生)或BIII杂种(2n + n;由蚜虫和受精产生)(Rutishauser,1947 )。可以在流式细胞计数种子筛选(FCSS)中测定不同类型的后代,其中染色的核的相对染色质含量通过测量它们的荧光强度来确定。这允许将胚乳的倍性与胚胎的倍性进行比较,并且因此推断形成种子的途径(Matzk等人,2000)。这种方法特别用于表征无形生殖植物中的种子或有性生殖的繁殖突变体的发育起源。
这里,我们提出了在十字花科中的FCSS的方案,植物的 Boechera 。然而,原则上,该方案可以应用于产生含有胚乳的种子的任何物种。

关键字:流式细胞仪, 种子发育, 胚乳, 流式细胞仪的种子筛, 无融合生殖

材料和试剂

  1. 柠檬酸一水合物(Sigma-Aldrich,目录号:33114)
  2. Triton X-100(Sigma-Aldrich,目录号:X100)
  3. (Merck KGaA,目录号:1.06580.1000)。
  4. 4',6-二脒基-2-苯基吲哚(DAPI)(Life Technologies,Invitrogen TM,目录号:D1306)
  5. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  6. 鞘液; Iso-Diluent(Beckman Coulter,目录号:NPE 629967)
  7. 清洁溶液(Beckman Coulter,目录号:629969)
  8. 关闭溶液(Beckman Coulter,目录号:629968)
  9. 库存解决方案(参见配方)
  10. Otto 1溶液0.01%(参见配方)
  11. Otto 1溶液1%(见配方)
  12. Otto 1溶液0.5%(参见配方)
  13. Otto 2解决方案(参见配方)

设备

  1. 1.2ml团簇管和架(无菌)(Thermo Fischer Scientific,目录号:07-200-320)
  2. 存储板盖条(Thermo Fisher Scientific,目录号:AB-0981)
  3. 3mm不锈钢珠(Schieritz& Hauenstein,目录号:22.455.0011)
  4. Nunc多孔深孔板(Thermo Fisher Scientific,目录号:278011)
  5. 96孔V型底板(SARSTEDT AG,目录号:82.1583.001)
  6. 200μl尖端(SARSTEDT AG,目录号:70.760.002)
  7. 1ml吸头(Rainin/Mettler Toledo,目录号:RC-1000/10)
  8. 单通道移液器(Gilson,目录号:P10,P1000)
  9. 多通道移液器10-100μl(Thermo Fisher Scientific,目录号4661130)
  10. 多通道移液器30-300μl(Thermo Fisher Scientific,目录号4661140)
  11. 混合机(Retsch,型号:MM300)
  12. 离心机(Eppendorf,型号:5810R)
  13. UV灯(13.流式细胞仪的一部分)
  14. 流式细胞仪(Beckman Coulter,Cell Lab Quanta SC,序列号:AN020010)
  15. 流式细胞仪机器人(Beckman Coulter,MPL,Cell Lab Quanta SC,序列号:AN90022)

程序

  1. 通过珠磨(3-4小时)分离细胞核
    1. 将单个3毫米不锈钢珠放入每个簇管(图1A)。
    2. 将集群管放入96孔架(图1B)。
    3. 标记试管并标记试样的位置(如实验室书中的示意图)。
    4.  将长角果固定在胶带上,并用夹层将其缝开 或皮下注射针(图1C-D)。 添加一个成熟但仍为绿色 种子(图1C-D)。 在 boechera 胚乳 种子成熟后降解。如果一个完全成熟的种子的种 正在调查含有胚乳,可以使用成熟种子。 这可以通过显微镜分析在各种清除的种子 阶段。清除的方法取决于物种,应该是 根据已公布的协议进行选择和调整

      图1。 A.在簇管中有一个3毫米不锈钢珠。 B.在合适的96孔架中含有单珠的簇管。 C.最佳种子阶段为Boechera spp。种子。顶部长角果具有太年轻不能使用的种子(太少的细胞),并且底部长角果在最佳阶段具有种子。 D.顶部长角果具有太老的种子(它们开始变成棕色,与胚乳的降解一致)。下部长角果在最佳阶段具有种子。 E. C.特写镜头D.特写镜头D.线条表示照片的一部分在特写镜头被删除。

    5. 每管加入80μl的Otto 1(0.01%)。
    6. 使用存储板盖条关闭管。
    7. 通过使用混合机在30Hz下振动样品3分钟来粉碎种子。
    8. 将板旋转180°并重复步骤A6。 手臂的远端部分 混合器粉碎机具有比近端部分更大的振幅。 车削 板补偿了混合器磨机的不同振幅。
    9. 控制种子是否通过目视检查破碎。 如果溶液变成乳状,则压碎种子(图2)。


      图2.粉碎和未粉碎的种子。绿色箭头表示 成功破碎的种子和乳状溶液。 白色箭头点 到非压碎种子。 红色箭头显示没有种子的管。 如果   溶液是澄清的,种子在簇管中可见,它是 未压碎,无法测量。

    10. 如果不是所有的种子都被压碎,重复步骤A7-9,直到所有的种子被压碎。
    11. 摇动溶液以从盖子上除去液滴,通过 轻轻地将板平放在桌子上,或通过快速旋转(5 秒)。
    12. 每管加入80μl的Otto 1(1%)。 小心不要触摸管或泡沫与尖端不 交叉污染样品。
    13. 关闭管和混合 手动倒转40x,避免快速运动产生泡沫。 现在 体积为160μl,浓度为0.1M柠檬酸和 0.5%Triton X-100
    14. 将过滤板放在干净的96孔V型底板上(图3A)
    15. 标记板。
    16. 将提取的核的溶液转移到烧结的 深孔板,通过使用多通道移液管或通过倾倒。 小心保持样品的顺序。
    17. 在室温(20℃)下以150×g离心5分钟以沉淀细胞核。
    18. 小心地从离心机中取出板。 颗粒核非常松散
    19. 取出多孔深孔板。 如果他们彻底洗涤 用自来水和去离子水冲洗,可以重复使用
    20. 用移液管吸头放置在V的边缘(图3B)除去上清液。
    21. 不要快速吸移; 否则核将被吸出。
    22. 颗粒应该是白色至浅棕色的外观。
    23. 加入30μl的Otto 1(0.5%),以重悬细胞核。 添加 缓冲液通过快速移液足以重悬细胞核
    24. 样品现在可以在4°C储存72小时,直到使用

      图3。 A.在96孔V型底板上的烧结深孔板。 乙。 卡通代表尖端的位置,以去除上清液。

  2. 测量DNA含量(倍性)(每个样品约7-8分钟,取决于细胞核的浓度)
    1. 启动流式细胞仪和紫外灯。 请参考用户 手动流式细胞仪或从中获得介绍 充电。
    2. 当UV灯加热(30分钟)时,将样品置于室温下以平衡(见注释)。
    3. 加载或更改检测设置,使二倍体峰为 在通道200(相对荧光单位)在线性刻度(见 注释)。
    4. 手动或使用流式细胞仪的机器人添加160μl的Otto 2溶液
    5. 测量样品并记录数据。 停止收集数据   总计6,000(这需要大约7-8分钟)或之后 一定时间(例如 5分钟)。
    6. 相应处理所有其他样品。
    7. 手动   注释峰(见图4-6)。 丢弃其中的样品 胚乳不能给出清晰的峰! 这是必要的看到的高峰   胚乳(见注释)。
    8. 将数据保存在至少提供样品名称,平均值,中值和方差系数的表中。

  3. 分析数据
    1. 计算胚乳与胚胎的比例。
    2. 如果母本的倍性可用,计算胚胎与母本的比率。

代表数据



图4.二倍体boechera stricta的性后代(n + n)。此直方图显示x轴上的荧光强度和每个强度的计数数y轴。峰根据从内部或外部标准推断的倍性手动注释和标记。绿色代表胚胎的核。 4x和8x峰来自核心重复的胚胎核(自体多倍体化)。棕色代表
胚乳核。 3x:2x峰的比率为275.99/187.4 = 1.47,表明性发育。 6x:4x峰的比率为1.48。使用自动多倍体峰用于比率计算提供了内部控制

图5.三倍体Boechera gunnisoniana的单性生殖后代(2n + 0)。绿色代表胚胎的核。 3x,6x和12x峰表示自体多倍体化。作为apomictic Boechera 通常是假性的(Rushworth等人,2011),我们预期受精胚乳(表1)。在这种情况下,胚乳为9x(9x:3x比率为2.86),表明未还原的精子细胞受精。


图6.三倍体boechera gunnisoniana 的B 混合后代(2n + 2n)。 此直方图几乎与图6中所示的直方图相同。然而,红色峰(3x)远小于6x峰,这表明这是来自母体孢子体组织的污染,这是由于从长角果上刮下种子。作为 B。 gunnisoniana 产生未减数的雌性和雄性配子,2n + 2n胚胎峰(6x)将是来自母体核(3x)的大小的两倍。这些数据由Schmidt及其同事(2014)发表。

笔记

  1. 较冷的样品显示较高的荧光强度。
  2. 使用不同的软件以不同的方式更改设置。软件通常与流式细胞仪一起提供。因此,我们不提供有关所使用软件的详细信息。然而,将该方案适应于新物种或新组织总是首先需要一些优化。请参阅用户手册和/或从仪器负责人那里获得培训。
  3. 在添加Otto 2溶液后,染色强度随时间而变化。必须确保从加入Otto 2溶液到测量样品的时间间隔是恒定的,或者一旦染色强度稳定就进行测量。在这里我们描述使用采样机器人的程序,确保从染色到测量的时间间隔是恒定的。
  4. Otto 2与Otto 1溶液的体积比应为约4:1。
  5. 根据我们的经验,由于无法鉴别的胚乳峰,25-35%的数据必须被丢弃(图7)。自然人口的材料可以达到45%。
  6. 如果将中值用于比率计算(4个独立测量,CV <0.01),则结果显示更高的再现性。
  7. 尽可能使用内部标准。内部标准混合到每个单一样品(图8)。对于拟南芥 ,我们已经成功使用了番茄。圣马尔扎诺,以及 Hieracium pilosella ,我们使用了贝利斯perennis。
  8. 选择内标:内标的DNA含量应与样品物种的DNA含量充分不同,以避免标准样品和样品物质之间的重叠倍数峰。值可以在文献中找到。理想地,内标具有比样品更低的DNA含量(总是直方图中非常左的峰),或者比样品物种的任何预期的倍性峰更高的DNA含量(总是直方图的非常正确的峰) br />
  9. 如果内部标准不可用,使用外部标准(已知倍性的植物)。外部标准应该是在一个板上的一组样品中测量的第一个和最后一个样品。从已知倍数的外标的位置(峰的中值),可以计算未知样品的倍性。


    图7.无法鉴别的胚乳峰


    图8.具有内标Solanum的二倍体(A)和三倍体(B)Boechera植物。。 红色标记具有比样品更高的DNA含量的内标。 因此它在直方图的右手边。

食谱

  1. 库存解决方案
    1mol/L柠檬酸
    饱和的(0.54mol/L,在20℃)Na 2 HPO 4溶液
    2 H 2 O溶液
    2mg/ml DAPI的水溶液(超纯水,电导率>18MΩ cm <-1> ),
  2. Otto 1溶液0.01%(对于100ml)
    0.1mol/L柠檬酸
    10ml 1mol/L
    0.01%(v/v)Triton X-100 10微升
    用水(电导率>18MΩ cm -1 )填充至总体积为100ml。
  3. Otto 1溶液1%(对于100ml)
    0.1mol/L柠檬酸
    10ml 1mol/L
    1%(v/v)Triton X-100 1000微升
    用水(电导率>18MΩ< - > cm <-1> -1 )填充至总体积为100ml。
  4. Otto 1溶液0.5%(对于100ml)
    0.1mol/L柠檬酸
    10ml 1mol/L
    0.5%(v/v)Triton X-100 500微升
    用水(电导率>18MΩ cm -1 )填充至总体积为100ml。
  5. Otto 2溶液(对于100ml)
    0.4mol/L Na 2 HPO 4 sub。 2H 2 O 74.1ml饱和溶液
    0.4μg/ml DAPI
    200μl2mg/mL储备液
    0.2μl/mlβ-巯基乙醇 200μl
    用水(电导率>18MΩ< - > cm <-1> -1 )填充至总体积为100ml。

致谢

该协议基于以前发表的工作(Matzk等人,2000; Schmidt等人,2014)。 我们在这方面的研究由苏黎世大学,Marie居里项目IDEAGENA,先正达博士支持。 苏黎世 - 巴塞尔植物科学中心的项目,以及"COST行动FA0903"框架下的"StaatssekretariatfürBildung und Forschung"的赠款。 我们感谢Manuel Waller帮助流式细胞术,Anja Herrmann和Margarida Sofia Nobre仔细阅读这个协议。

参考文献

  1. Koltunow,A.M.和Grossniklaus,U。(2003)。 Apomixis:发展观点 54:547-574。
  2. Matzk,F.,Meister,A。和Schubert,I。(2000)。 使用单子叶植物和双子叶植物的成熟种子进行繁殖途径的有效筛选 Plant J 21(1):97-108。
  3. Rutishauser,A。(1947)。 Untersuchungenüberdie Genetik der Aposporie bei pseudogamen Potentillen。 Experientia 3(5):204-205。
  4. Rushworth,CA,Song,BH,Lee,CRand Mitchell-Olds,T。(2011)。 Boechera 一个生态基因组学模型系统。 Mol Ecol 20(23):4843-4857。
  5. Schmidt,A.,Schmid,M. W.,Klostermeier,U. C.,Qi,W.,Guthorl,D.,Sailer,C.,Waller,M.,Rosenstiel,P.and Grossniklaus, 单性生殖和性生殖发育在细胞周期,转录,激素和表观遗传调节方面不同。 a> PLoS Genet 10(7):e1004476。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sailer, C., Schmidt, A. and Grossniklaus, U. (2015). Determination of the Developmental Origin of Seeds Containing Endosperm Using Flow Cytometric Analysis. Bio-protocol 5(11): e1484. DOI: 10.21769/BioProtoc.1484.
  2. Schmidt, A., Schmid, M. W., Klostermeier, U. C., Qi, W., Guthorl, D., Sailer, C., Waller, M., Rosenstiel, P. and Grossniklaus, U. (2014). Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation. PLoS Genet 10(7): e1004476.
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