搜索

Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains
Dictyochloropsis绿藻菌株的基因组DNA提取和基因分型   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Dictyochloropsis is an ecologically important genus of free-living and symbiotic green algae. Representatives of this genus are horizontally transmitted among several fungi of the family Lobariaceae, thus forming photobiont-mediated guilds. This protocol is suitable for extracting DNA from algal cultures and lichen samples and for genotyping seven unlinked Dictyochloropsis reticulata microsatellite markers in a single PCR multiplex.



Figure 1. Schematic representation of the analysis pipeline

Materials and Reagents

  1. Qiagen Type-it Microsatellite PCR kit (QIAGEN, catalog number: 206243 )
  2. 1x TE buffer (10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA)
  3. GelRed (Biotium, catalog number: 41003 )
  4. 1 kb DNA ladder (GeneRuler 1 kb DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0313 )
  5. Nuclease-free water
  6. Pure molecular biology grade ethanol (96–100%)
  7. 2 ml tubes
  8. DNeasy 96 Plant Kit (QIAGEN, catalog number: 69181 )
  9. Hi-Di™ formamide (Life Technologies, catalog number: 4311320 )
  10. GeneScan 500 LIZ size standard (Life Technologies, catalog number: 4322682 )
  11. Ice
  12. Glucose (BD Biosciences, catalog number: 215510 )
  13. Proteose pepton (BD Biosciences, catalog number: 212230 )
  14. Agar (BD Biosciences, catalog number: 214883 )
  15. 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Life Technologies, Gibco®, catalog number: 11330032 )
  16. Laboratory wipes (e.g., KimWipes, Kimberly-Clark, catalog number: TW31KWPBX )
  17. 4 mm steel balls (e.g., Spex Certiprep, catalog number: 12145950 )
  18. Pipette tips
  19. Pipette (0.1-2 μl), pipette tips
  20. Processing plate: 96-well plate (e.g., MicroAmp Optical 96-Well Reaction Plate) and 96-well plate (Septa)
  21. 0.22 µm sterile polyethersulfon syringe filter (Merck Millipore, catalog number: P/N SLMP025SS )
  22. Algal medium (see Recipes)
  23. Algal culture medium preparation for growing Dictyochloropsis and other trebouxiophycean algal strains (see Recipes)
  24. Vitamin solution (see Recipes)

Equipment

  1. Multi-channel pipettes (0.1-10, 10-100, 100-1000 μl), with extended tips
  2. Microcentrifuge with rotor for 2 ml tubes
  3. Centrifuge for 96-well plates
  4. BioRad Gel casting tray, running tray, power pack etc. (Bio-Rad Laboratories, catalog number: 164-0305 )
  5. Incubator (65 °C)
  6. Freezer or cold room at -20 °C
  7. PCR Thermal cycler
  8. ABI3130/ 3130xl/3730/3730xl DNA Analyzer (Applied Biosystems)
  9. Ball Mill MM 400 (Retsch®, catalog number: 20.745.0001 )
  10. Lyophilizer (e.g., FreeZone 4.5 Liter Benchtop Freeze Dry System, Labconco, catalog number: 7750021 )

Software

  1. GeneMapper® (Software v4.1, Applied Biosystems, catalog number: 4366925)

Procedure

  1. Sample preparation (Day 1)
    1. Take up to 10 mg fresh tissue sample (approximate wet weight) or 5 mm diameter
in size of algal culture (Beck et al., 1998) / lichen thallus in 2 ml tube (see Figure 2).
    2. Add 2 steel balls in each tube.
    3. Cover the open tubes with soft tissue paper.
    4. Cool samples in -20 °C freezer for at least 6 h.
    5. Fit the tubes in the lyophilizer and lyophilize overnight.


      Figure 2. Example of samples used for DNA extraction

  2. Sample grinding and DNA isolation (Day 2)
    1. Close the tubes.
    2. Crash the lyophilized samples using the ball mill MM 400 at 30 Hz for 30 sec without buffer at ambient temperature.
    3. Spin down for a few seconds the crashed sample before proceeding with DNA isolation.
    4. For DNA isolation follow DNeasy 96 Plant Kit, Qiagen protocol (Plant Handbook 10/2012, http://www.qiagen.com, pages 31-34).
      Notes:
      1. For buffers AW1 and AW2, before using for the first time, add the appropriate amount of ethanol as indicated on the bottle to obtain a working solution.
      2. Preheat Buffer AP1 to 65 °C.
    5. Elute the purified DNA from the DNeasy spin column using Buffer AE twice (2 x 50 μl).
      Note: Smaller or larger elution volumes can be used for more or less concentrated products. To ensure complete elution, 40 μl should be the minimum elution volume.

  3. DNA check
    1. Prepare 1% agarose gel with 1x TAE buffer. Add 0.5 μl GelRed for 100 ml gel.
    2. Load 2 μl DNA.
    3. Add 1 kb or 100 bp ladder for reference.
    4. Run the gel electrophoresis.
    5. Check DNA for quality and quantity in gel doc system (Figure 3).


    Figure 3. Example of good quality genomic DNA of Dictyochloropsis reticulata run on a 1% w/v agarose gel

  4. PCR reactions and preparation of the genotyping plate (Day 3)
    7x Primer mix
    1. First prepare 100 μM primer stocks.
    2. Mix all primers according to Tables 1 and 2 to get 1ml primer-mix (sufficient for 10x 96-sample PCR plate).


    Table 1. Primer sequences and labeling of the microsatellite Multiplex 1 specific to Dictyochloropsis reticulata (Dal Grande et al., 2009; Dal Grande et al., 2014)

    Algal Loci
    Locus GenBank
    Primer Sequence (5'-3')
    Label




    LPh1
    FJ754261
    F: GTCTCAGGTGACCACTTGATTG
    VIC


    R: GCAATGGATATGATGCTTGTTC

    LPh2
    FJ754262
    F: GACAGCTGTTCCAGTGCATC



    R: GCAGAGGAAGTGCATGACG
    FAM
    LPh3
    FJ754263
    F: TGCAGTAGGTGTCATATGTGT
    NED


    R: GAAGGCGCATCTTGATATAC

    LPh4
    FJ754264
    F: GTGGTGGTACAACATGCTCA
    NED


    R: ACGACCACGTGGGATATCTA

    LPh5
    FJ754265
    F: TGGTGTTAGTAAGAATCGGCATC
    PET


    R: GTGTATGTCGGCCCCAATAA

    LPh6
    FJ754266
    F: GAATCCTGCCTGCCTACAAG
    FAM


    R: AGCAACCCATTTCAACCAAC

    LPh7
    FJ754267
    F: TGTGACAGGTGAAACACCAA
    VIC


    R: TATGGTCCCTCATGGCAAAT


    Table 2. 7x primer mix preparation
    Concentration of normalized primer stocks: 100 μM (100 pmol/μl)
    Each primer 20 μl (30 μl for LPh7)  
    TE buffer 700 μl
    Total volume 1 ml

  5. PCR reaction mix
    1. Thaw template DNA, RNase-free water, the primer mix and the 2x Type-it Microsatellite PCR Master Mix, if stored at -20 °C.
    2. Mix the solutions completely before use.
    3. Prepare a reaction mix according to Table 3.
      Note: The reaction mix contains all the components required for multiplex PCR except the template DNA. Prepare a volume of reaction mix 10% greater than that required volume for the total number of reactions to be performed.
    4. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or plates.
      Note: Mix gently by pipetting the reaction mix up and down a few times. It is not necessary to keep samples on ice during reaction setup.

    Table 3. PCR components of the microsatellite multiplex (total volume = 10 μl)
    PCR component
    Multiplex 1 (10 μl)
    Multiplex PCR Master Mixa
    5 μl (1x)
    Primer mix
    1 μl (0.2 μM of each primer, 0.3 μM for LPh7)
    DNA template
    1 μl (1–10 ng)
    RNase-free water
    3 μl 

  6. PCR program
    1. Step 1: 95 °C for 5 min (initial activation)
    2. Step 2: 95 °C for 30 sec (denaturation)
    3. Step 3: 62 °C for 90 sec (annealing)
    4. Step 4: 72 °C for 60 sec (extension)
    5. Step 5: go to step F2, repeat 24 times
    6. Step 6: 60 °C for 30 min (final extension)
    7. Store PCR products in -20 °C, until further processing.

  7. Preparation of the plate for genotyping (Day 4)
    1. Dilute the PCR products 1: 10 with RNAse-free water.
    2. Combine 1 μl of diluted PCR product with a buffer containing 9 μl of a denaturing agent (Hi-Di™ Formamide) and 0.5 μl LIZ500 size standard.
    3. Note: For one 96 well plate prepare buffer for 105 samples.
    4. Centrifuge the plates briefly (5 sec) at 1,500 RCF.
    5. Prepare table of samples.
      Note: Fill any blank sample-well with nuclease-free water.
    6. Analyze on an ABI3730xl.

  8. Analysis (Day 5)
    Import the raw data files generated by the sequencer to the computer with GeneMapper software.
    Genotyping in GeneMapper
    1. Select ‘Create new project’ from the 'File' menu.
    2. Import your samples.
    3. Create marker panel and bin set.
      Analysis parameters
    4. Choose ‘Microsatellite default’ as table setting and ‘Microsatellite analysis method’ as the analysis method.
    5. Set the panel for the 7x-primer mix.
    6. Set size standard as LIZ500 and exclude the 35- and 250-bp peaks.
    7. Analyze (see Figure 4).
      Note: If some samples fail to match size standards, go to edit size standards and override size. If this does not solve the issue, there might be some problem with the sample or the analysis run.


    Figure 4. Example of the 7x-multiplex microsatellite electropherogram for one sample of Dictyochloropsis reticulata

Representative data

  1. A simple, representative example of data that indicates what type of results to expect (Table 4).

    Table 4. Example of a 7x-multiplex microsatellite allele table
    Sample
    Population
    LPh1
    LPh2
    LPh3
    LPh4
    LPh5
    LPh6
    LPh7
    1
    Pop1
    262  
    157
    108
    191
    143
    121
    170
    2
    Pop2
    260
    151
    138
    176
    156
    121
    190
    3
    PopN
    148
    159
    122
    181
    140
    116
    152
    Note: Numbers indicate the relative size of each microsatellite allele in base pairs.

Recipes

  1. Algal medium
    Ingredients for the preparation of algal culture medium
    1. Macronutrients (g/400 ml)
      NaCl                     1                 (42.7 mM)
      CaCl2.2H2O          1                 (17.0 mM)
      KNO3                   20               (495 mM)
      MgSO4.7H2O       3                 (30.4 mM)
      (NH4)2.HPO4       10                (189 mM)
    2. Micronutrients (g/500 ml)
      KOH                      15             (535 mM)
      EDTA:                    25             (171 mM)
      FeSO4.7H2O         2.49           (17.9 mM)
      H3BO3                  5.52           (179 mM)
      ZnSO4.7H2O         4.41           (30.7 mM)
      MnCl2.4H2O          0.72           (7.28 mM)
      NaMoO4                0.36           (3.50 mM)
      CuSO4.5H2O         0.79           (6.33 mM)
      Co(NO3)2.6H2O      0.25          (1.72 mM)
  2. Algal culture medium preparation for growing Dictyochloropsis and other trebouxiophycean algal strains
    1. For one liter of medium, take 10 ml of macronutrients and 1 ml of micronutrients, add 0.715 g HEPES buffer (final concentration 3 mM) and fill up to 1,000 ml with distilled water.
    2. Adjust pH to 5.5 with HCl.
    3. 20 g agar may be added.
    4. Autoclave and add 1 ml sterile vitamin solution after cooling down to at least 60 °C. Sterilize the vitamin solution by filtration using a 0.22 µm sterile polyethersulfon syringe filter.
      Note: To receive higher amounts of algal cells 1.5% glucose and 1% proteose pepton can be added.
  3. Vitamin solution
    Thiamine               0.1 g/100 ml               (3.77 mM)
    Biotin                    2.5 mg/100 ml            (0.10 mM)
    Vitamin B12           1.5 mg/100 ml             (9.50 µM)

Acknowledgments

This study was supported by ‘LOEWE, Landes-Offensive zur Entwicklung Wissenschaftlich-oekonomischer Exzellenz’ of Hesse’s Ministry of Higher Education, Research, and the Arts, by the Swiss National Science Foundation (projects 31003A-105830 and 31003A-127346 to C.S.), and by the German National Science Foundation (project BE3825/4-1 to A.B.).

References

  1. Beck, A., Friedl, T. and Rambold, G. (1998). Selectivity of photobiont choice in a defined lichen community: inferences from cultural and molecular studies. New Phytologist 139(4): 709-720.
  2. Dal Grande, F., Beck, A., Cornejo, C., Singh, G., Cheenacharoen, S., Nelsen, M. P. and Scheidegger, C. (2014). Molecular phylogeny and symbiotic selectivity of the green algal genus Dictyochloropsis s.l. (Trebouxiophyceae): a polyphyletic and widespread group forming photobiont-mediated guilds in the lichen family Lobariaceae. New Phytol 202(2): 455-470.
  3. Dal Grande, F., Widmer, I., Beck, A. and Scheidegger, C. (2010). Microsatellite markers for Dictyochloropsis reticulata (Trebouxiophyceae), the symbiotic alga of the lichen Lobaria pulmonaria (L.). Conservation Genetics 11(3): 1147-1149.

简介

Dictyochloropsis是一种生态上重要的自由生活和共生绿藻类。 这个属的代表在家庭龙眼科的几种真菌之间水平传播,从而形成photobiont介导的公会。 该方案适用于从藻类培养物和地衣样品中提取DNA,以及用于在单个PCR多重中对七个未连接的微卫星标记进行基因分型。



图1.分析管道的示意图

材料和试剂

  1. Qiagen Type-it Microsatellite PCR试剂盒(QIAGEN,目录号:206243)
  2. 1×TE缓冲液(10mM Tris,用HCl,1mM EDTA调至pH8.0)
  3. GelRed(Biotium,目录号:41003)
  4. 1kb DNA梯(GeneRuler 1kb DNA Ladder)(Thermo Fisher Scientific,目录号:SM0313)
  5. 无核酸酶水溶液
  6. 纯分子生物学级乙醇(96-100%)
  7. 2 ml管
  8. DNeasy 96 Plant Kit(QIAGEN,目录号:69181)
  9. Hi-Di TM甲酰胺(Life Technologies,目录号:4311320)
  10. GeneScan 500 LIZ尺寸标准(Life Technologies,目录号:4322682)
  11. 冰块
  12. 葡萄糖(BD Biosciences,目录号:215510)
  13. 蛋白pepton(BD Biosciences,目录号:212230)
  14. 琼脂(BD Biosciences,目录号:214883)
  15. 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)(Life Technologies,Gibco ,目录号:11330032)
  16. 实验室抹布(例如,KimWipes,Kimberly-Clark,目录号:TW31KWPBX)
  17. 4毫米钢球(例如,Spex Certiprep,目录号:12145950)
  18. 移液器提示
  19. 移液管(0.1-2μl),移液器吸头
  20. 处理板:96孔板(例如,MicroAmp光学96孔反应板)和96孔板(Septa)
  21. 0.22μm无菌聚醚砜注射器过滤器(Merck Millipore,目录号:P/N SLMP025SS)
  22. 藻类培养基(见配方)
  23. 藻类培养基准备用于培养微小绿藻和其他trebouxiophycean藻类菌株(参见配方)
  24. 维生素溶液(见配方)

设备

  1. 多通道移液器(0.1-10,10-100,100-1000μl),具有延长的提示
  2. 带有转子的微量离心机,用于2 ml管
  3. 96孔板离心机
  4. Bio/Rad实验室,目录号:164-0305)。
  5. 孵育器(65℃)
  6. 冷冻室或冷藏室-20°C
  7. PCR热循环仪
  8. ABI3130/3130xl/3730/3730xl DNA分析仪(Applied Biosystems)
  9. 球磨机MM 400(Retsch ,目录号:20.745.0001)
  10. 冻干器(例如,FreeZone 4.5升Benchtop Freeze Dry System,Labconco,目录号:7750021)

软件

  1. GeneMapper (Software v4.1,Applied Biosystems,目录号:4366925)

程序

  1. 样品制备(第1天)
    1. 取最多10毫克新鲜组织样品(近似湿重)或5毫米   藻类培养物的大小(Beck等人,1998)/地衣地衣 在2ml管中(见图2)
    2. 在每个管中添加2个钢球
    3. 用软薄纸覆盖开口管。
    4. 将样品在-20°C冷冻至少6小时
    5. 将管子安装在冻干机中并冻干过夜

      图2.用于DNA提取的样品示例

  2. 样品研磨和DNA分离(第2天)
    1. 关闭管。
    2. 使用球磨机MM 400在30Hz下,在环境温度下,无缓冲液,冻干冻干样品30秒
    3. 在进行DNA分离之前,将粉碎的样品旋转几秒钟
    4. 对于DNA分离,遵循DNeasy 96 Plant Kit,Qiagen方案(Plant Handbook 10/2012,http://www.qiagen.com,第31-34页)。
      注意:
      1. 对于缓冲区AW1和AW2,在第一次使用之前,请添加 适量的乙醇,按瓶上所示取得a 工作解决方案。
      2. 将AP1预热至65°C。
    5. 使用缓冲液AE从DNeasy离心柱洗脱纯化的DNA两次(2×50μl)。
      注意:   更小或更大的洗脱体积可以或多或少地使用 浓缩产品。 为了确保完全洗脱,40μl应该 最小洗脱体积。

  3. DNA检查
    1. 用1X TAE缓冲液制备1%琼脂糖凝胶。 加入0.5μlGelRed用于100 ml凝胶
    2. 加载2μlDNA
    3. 添加1 kb或100 bp梯形图作为参考
    4. 运行凝胶电泳。
    5. 在凝胶文件系统中检查DNA的质量和数量(图3)


    图3.在1%w/v琼脂糖凝胶上运行的 Dictyochloropsis reticulata的优质基因组DNA的实施例

  4. PCR反应和基因分型板(第3天)的制备
    7x 底漆组合
    1. 首先制备100μM引物储液
    2. 根据表1和2混合所有引物,得到1ml引物混合物(对于10x 96-样品PCR板足够)。


    表1.微卫星的引物序列和标记(Dal Grande等人,2009; Dal Grande等人,2009)。 ,2014)

    Algal Loci
    轨迹GenBank
    引物序列(5'-3')
    标签




    LPh1
    FJ754261
    F:GTCTCAGGTGACCACTTGATTG
    VIC


    R:GCAATGGATATGATGCTTGTTC

    LPh2
    FJ754262
    F:GACAGCTGTTCCAGTGCATC



    R:GCAGAGGAAGTGCATGACG
    FAM
    LPh3
    FJ754263
    F:TGCAGTAGGTGTCATATGTGT
    NED


    R:GAAGGCGCATCTTGATATAC

    LPh4
    FJ754264
    F:GTGGTGGTACAACATGCTCA
    NED


    R:ACGACCACGTGGGATATCTA

    LPh5
    FJ754265
    F:TGGTGTTAGTAAGAATCGGCATC
    PET


    R:GTGTATGTCGGCCCCAATAA

    LPh6
    FJ754266
    F:GAATCCTGCCTGCCTACAAG
    FAM


    R:AGCAACCCATTTCAACCAAC

    LPh7
    FJ754267
    F:TGTGACAGGTGAAACACCAA
    VIC


    R:TATGGTCCCTCATGGCAAAT


    表2. 7x底漆混合液制备
    标准化引物储备液的浓度:100μM(100pmol /μl)
    每个引物20μl(30μl为LPh7)
    TE缓冲液700μl
    总体积为1 ml

  5. PCR反应混合物
    1. 解冻模板DNA,无RNA酶的水,引物混合物和2x Type-it Microsatellite PCR Master Mix,如果储存在-20°C。
    2. 在使用前将溶液完全混合。
    3. 按照表3准备反应混合物。
      注意:反应混合物包含所需的所有组分 除了模板DNA以外的多重PCR。 准备一定体积的反应混合物 10%大于反应总数所需的体积
    4. 充分混合反应混合物并将适当体积分配到PCR管或板中。
      注意:轻轻地混合上下反应混合物几次。   在反应设置期间不必将样品保持在冰上。

    表3.微卫星多重PCR的PCR组分(总体积=10μl)
    PCR分量
    多重1(10μl)
    Multiplex PCR Master Mix a
    5μl(1x)
    底漆混合物
    1μl(每种引物0.2μM,LPh7为0.3μM)
    DNA模板
    1μl(1-10 ng)
    无RNase水
    3μl 

  6. PCR程序
    1. 步骤1:95℃5分钟(初始活化)
    2. 步骤2:95℃30秒(变性)
    3. 步骤3:62℃90秒(退火)
    4. 步骤4:72℃60秒(延伸)
    5. 步骤5:转到步骤F2,重复24次
    6. 步骤6:60℃30分钟(最终延伸)
    7. 将PCR产物储存在-20°C,直到进一步处理。

  7. 制备用于基因分型的平板(第4天)
    1. 用无RNA酶的水稀释PCR产物1:10
    2. 合并1   μl稀释的PCR产物与含有9μl变性的缓冲液混合   试剂(Hi-Di TM甲酰胺)和0.5μlLIZ500大小 标准。
    3. 注意:对于一个96孔板准备105个样品的缓冲液。
    4. 以1500 RCF短暂离心板(5秒)。
    5. 准备样品表。
      注意:用无核酸酶的水填充任何空白样品孔。
    6. 在ABI3730xl上分析。

  8. 分析(第5天)
    使用GeneMapper软件将序列生成器生成的原始数据文件导入计算机。
    GeneMapper中的基因分型
    1. 从"文件"菜单中选择"创建新项目"。
    2. 导入样品。
    3. 创建标记面板和面元集。
      分析参数
    4. 选择"微卫星默认值"作为表设置,"微卫星分析方法"作为分析方法
    5. 设置7x底漆混合物的面板。
    6. 将大小标准设置为LIZ500,并排除35和250 bp峰
    7. 分析(见图4)。
      注意:如果一些样本不符合大小标准,请转到编辑大小 标准和覆盖尺寸。 如果这不解决问题,那里 可能是与示例或运行分析 有关的一些问题。


    图4.示例性网叶螨的一个样品的7x多重微卫星电泳图的例子

代表数据

  1. 一个简单,有代表性的数据示例,指示期望的结果类型(表4)。

    表4. 7x多重微卫星等位基因表格示例
    示例
    人口
    LPh1
    LPh2
    LPh3
    LPh4
    LPh5
    LPh6
    LPh7
    1
    Pop1
    262  
    157
    108
    191
    143
    121
    170
    2
    Pop2
    260
    151
    138
    176
    156
    121
    190
    3
    PopN
    148
    159
    122
    181
    140
    116
    152
    注意: 数字表示每个微卫星等位基因碱基对的相对大小。

食谱

  1. 藻类培养基
    用于制备藻类培养基的成分
    1. 大量营养素(g/400 ml)
      NaCl                       1                  (42.7mM) CaCl 2 2H 2 O        1                 (17.0 mM) KNO 3                   20               (495mM) MgSO 4 4 7H O      3                 (30.4 mM) (NH 4) 2 .HPO 4      10                 (189mM)
    2. 微量营养素(g/500 ml)
      KOH                      15             (535mM)
      EDTA:                 25             (171mM) FeSO 4 7H 2       2.49          (17.9mM) H 3 BO 3                 5.52           (179mM) ZnSO 4 4H 2       0.72           (7.28mM) NaMoO 4                0.36          (3.50mM) CuSO 4 5H       0.79          (6.33mM) Co(NO 3 2 6H 2 O    0.25         (1.72mM)
  2. 用于培养微小绿藻和其他trebouxiophycean藻类菌株的藻培养基准备
    1. 对于一升培养基,取10ml大量营养素和1ml 微量营养素,加入0.715g HEPES缓冲液(终浓度3mM) 用蒸馏水填充至1000ml
    2. 用HCl调节pH至5.5。
    3. 可以加入20g琼脂
    4. 高压灭菌并加入1毫升无菌维生素溶液冷却后 至少60℃。 通过过滤使用a消毒维生素溶液 0.22μm无菌聚醚砜注射器过滤器。
      ote:要接收更多的藻类细胞,可以添加1.5%葡萄糖和1%蛋白胨。
  3. 维生素溶液
    硫胺素            0.1 g/100 ml             (3.77mM) 生物素                2.5 mg/100 ml         (0.10mM) 维生素B12         1.5 mg/100 ml           (9.50μM)

致谢

这项研究由瑞士国家科学基金会(项目31003A-105830和31003A-127346到CS)的Hesse高等教育,研究和艺术部的"LOEWE,Landes-Offensive zur Entwicklung Wissenschaftlich-oekonomischer Exzellenz"和德国国家科学基金会(项目BE3825/4-1至AB)。

参考文献

  1. Beck,A.,Friedl,T。和Rambold,G。(1998)。 定义的地衣群落中光生物选择的选择性:来自文化的推断和新分子研究。新植物学家 139(4):709-720。
  2. Dal Grande,F.,Beck,A.,Cornejo,C.,Singh,G.,Cheenacharoen,S.,Nelsen,M.P.and Scheidegger,C。 绿色藻类属的分子系统发育和共生选择性 sl (Trebouxiophyceae):一种多形性和广泛的群体形成photobiont介导的地衣在地衣家庭Lobariaceae。新植物 202(2):455-470。
  3. Dal Grande,F.,Widmer,I.,Beck,A。和Scheidegger,C。(2010)。 网球藻属的微卫星标记(Trebouxiophyceae),地衣的共生藻类(L.)。 保护遗传学 11(3):1147-1149。
  • English
  • 中文翻译
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:Grande, F. D., Cornejo, C., Scheidegger, C. and Beck, A. (2015). Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains. Bio-protocol 5(15): e1545. DOI: 10.21769/BioProtoc.1545.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。