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High-throughput Method for Determination of Seed Paternity by Microsatellite Markers
利用微卫星标记法高通量鉴定种子亲权   

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Abstract

In this protocol, determination of seed paternity by microsatellite markers in Nicotiana attenuata is described. However, this does not include a protocol for the novel marker selection/identification, but rather exploits the markers generated for a closely related species N. tabacum (Bindler et al., 2007). This is a high-throughput protocol optimized and streamlined for one skilled person to process 384 (96 x 4) seeds in 5 days, from DNA isolation (from seedlings) to paternity assessment by microsatellite genotype data.

Keywords: Microsatellite genotyping(微卫星基因分型), Multiplex PCR(多重PCR), High-throughput seed paternity test(高通量种子亲子鉴定试验), Nicotiana attenuata(野生烟草)

Materials and Reagents

  1. Agencourt Chloropure Kit (this kit is discontinued by Beckman Coulter but can be ordered) or Quiagen MagAttract 96 DNA plant core kit (QIAGEN, catalog number: 67163 )
  2. Qiagen Type-it Multiplex PCR kit (QIAGEN, catalog number: 206243 )
  3. QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
  4. Liquid nitrogen
  5. 100% Isopropanol, ultrapure
  6. Freshly prepared 70% ethanol made with nuclease free water
    Note: 70% ethanol is hygroscopic, always make fresh for optimal result.
  7. Agarose
  8. Ethydium bromide solution
  9. Nuclease free water
  10. Pure molecular biology grade ethanol (96– 100%)
    Note: Calculate the volume of ethanol needed before you start. A new kit of 24 plates needs about 5 L of ethanol.
  11. GENSCAN 500 ROX (Applied Biosystems, catalog number: 401734 )
  12. 1x TAE (Tris-Acetate-EDTA) buffer (see Recipes)

Equipment

  1. 2.2 ml Ritter Deep-well plates (ABGene, catalog number: AB-6661 )
  2. Adhesive plate film (ABGene, catalog number: AB-0558 , AB-626 , AB-662 )
  3. 4 mm steel balls (SPEX SamplePrep, catalog number: 2150 )
  4. Pipette tips
  5. Processsing Plate: Deep-well titer plate with a 96-well format (SPEX SamplePrep, catalog number: 2210 )
  6. Destination Plate: 300 μl round bottom microtiter plate (96 well, 300 μl well capacity, round bottom) (Corning, Costar®, catalog number: 07-200-105 )
  7. Desktop centrifuge for 96 well plates
  8. Reagent reservoir
  9. Genogrinder (SPEX SamplePrep, catalog number: 2010 )
  10. Cooling block (SPEX SamplePrep, catalog number: 2665 )
  11. Steel ball dispenser (SPEX SamplePrep, catalog number: 2100 )
  12. Multi-channel pipettes (0.1-10, 10-100, 30-300 μl)
  13. Agencourt Supermagnet magnetic Plate (Agencourt SPRIPlate 96R-Ring Magnet Plate, catalog number: A29164 )
  14. BioRad Gel casting tray, running tray, power pack etc (Bio-Rad Laboratories)
  15. Nanodrop spectrophotometer (Nanodrop)
  16. Pipette (0.2-2 μl), pipette tips , soft tissue paper
  17. ABI 3100 Genetic Analyzer (Applied Biosysyems)

Procedure

  1. Sample grinding (Day 1)
    1. Take up to 40 mg fresh tissue sample (leaf punches/seedling) in 2.2 ml plate. Take care to group similar samples/treatments/experimental designs together for easier data handling during sequencing and genotype analysis.
    2. Dispense 2 steel balls in each well with dispenser.
    3. Balance two plates exactly for grinding in Genogrinder.
    4. Seal the plate(s) with adhesive plate film.
    5. Cool the plate(s) in liquid nitrogen.
    6. Cool the cooling racks along with the plates.
    7. Grind in Genogrinder at Speed 250, 1x, for 1 min. If not crushed completely, again for 1 min.

  2. DNA isolation (Day 1)
    Assembly in step B-1 is performed once for each new Agencourt Chloropure kit. If you have already made the following preparations for a previous experiment, please skip ahead to step B-2. Standard RNase treatment can be included either at the step B-5 with the homogenized lysate (requires large amount of enzyme, but convenient for high-throughput) or selectively after determination of the degree of RNA contamination at "quality check".
    1. Add 80 ml of 100% Isopropanol to the wash buffer bottle provided with the kit. After addition of Isopropanol invert the bottle to mix. Once the solution has been thoroughly mixed, store at room temperature.
    2. Prepare bind buffer: Combine 6 μl bind buffer with 150 μl of 100% isopropanol for each individual isolation in a nuclease free vessel of suitable size (for example: for 10 isolations, add 60 μl of bind buffer to 1.5 ml of 100% isopropanol in a 15 ml conical tube). Vortex bind buffer bottle thoroughly before combining. Unused combined solution should be discarded.
    3. Homogenize each sample in 300 μl of lysis buffer, provided with the kit. (sample input should not exceed three 6 mm lyophilized leaf punches or 40 mg ground seeds or fresh leaf material.)
    4. Centrifuge lysate for 10 min at 1,100 RCF at room temp to pellet debris.
    5. Transfer 150 μl of homogenized lysate to 1.2 ml processing plate.
    6. Pipette 150 μl bind buffer (prepared in step B-2), mix by slowly pipetting 5 times and incubate at room temperature for 5 min.
    7. Move the plate onto the Agencourt Supermagnet and separate for 2-4 min. Wait for the solution to clear before proceeding to the next step.
    8. Slowly aspirate the cleared solution from the plate and discard.
      Note: Aspirate from the top down to avoid disturbing the pellet. This step must be performed while the plate is situated on the magnet. Do not disturb the separated magnetic beads. If beads are drawn out, leave a few microliters of supernatant behind.
    9. Remove the plate from the magnet and add 300 μl of Wash buffer. Pipette mix 10 times and incubate for 1 min at RT.
    10. Return plate to the magnet and separate for 2-4 min. Wait for the solution to clear before proceeding to the next step.
    11. Slowly aspirate the cleared solution from the plate and discard. This step must be performed while the plate is situated on the magnet.
    12. Remove the plate from the magnet and add 300 μl of 70% ethanol. Pipette mix 10 times to re-suspend the beads.
    13. Return plate to the magnet and separate for 2 min. Wait for the solution to clear before proceeding to the next step.
    14. Slowly aspirate the cleared solution from the plate and discard.
    15. Repeat steps l-n for a total of 2 ethanol washes.
    16. Let the plate air dry for 5 min at room temperature. The plate should air-dry until the last visible traces of ethanol evaporate. Over-drying the sample may result in a lower recovery.
    17. Remove the plate from the magnet and add 50 μl of nuclease-free water. Re-suspend the beads by pipette mixing 10 times.
      Note: Smaller or larger elution volumes can be used for more or less concentrated product; however the minimum elution volume should be 40 μl to ensure complete elution. Optimal elution volumes need to be experimentally determined.
      1. DNA quality/quantity check
        1) Quality
        a) Prepare 0.8% agarose gel with 1x TAE buffer. Add 2 μl Ethydium Bromide before casting.
        b) Load 2 μl DNA+1 μl loading dye
        c) Add 1 kb or 100 bp ladder for reference
        d) Run at constant 100 V for 30 min
        e) Check DNA for quality, presence of RNA and quantity in gel doc system and record.



        Figure 1. DNA quality check on 0.8% agarose gel. Lane 1-6, 8-13 represent extracted DNA samples, lane 7 represents DNA marker.
        2) Quantity
        a) Follow the procedure to measure concentration of DNA in Nanodrop.
        b) Export to excel file for reference.
      2. Dilution of DNA sample
        a)Dilute the samples to 50 ng/μl as working stock.
        b)Dispense them in 3 PCR plates (for 3 multiplex PCR reactions) similarly and maintaining the sample organization of working DNA plate. Make a record of the samples.

  3. PCR amplification (Day 2)
    1. Multiplex groups
      Primer groups are selected based on primer sequences using software MultiPLX version 2.0 (available at http://bioinfo.ut.ee/?page_id=167, Ref: doi: 10.1093/bioinformatics/bti219) to select multiplex grouping. Medium stringency was chosen to select groups.
      Example of 3 multiplex groups of 8 primer pairs:

      Table 1. Example of 3 multiplex groups of 8 primer pairs:


    2. 10x Primer mix
      1. First make 100 μM primer stock (if using 50 μM primer stock, adjust the volume of the table below accordingly).
      2. Mix all primers according to the table below to get 3 primer-mix groups (sufficient for 1x PCR plate i.e. 96 samples).

        Table 2. Schematic representation of preparation of 3 primer mix groups:



    3. PCR reaction mix
      1. Thaw the 2x Type-it Multiplex PCR Master Mix (MM) (if stored at -20 °C), template DNA (already dispensed in PCR plates), RNase-free water, and the primer mix. Mix the solutions completely before use.
        Note: It is important to mix the solutions completely before use to avoid localized concentrations of salts.
      2. Prepare a reaction mix according to the table below. Note: The reaction mix typically contains all the components required for multiplex PCR except the template DNA. Prepare a volume of reaction mix 10% greater than that required for the total number of reactions to be performed. For reaction volumes less than 25 μl, the 1:1 ratio of Type-it Multiplex PCR Master Mix to primer mix and template should be maintained.
        Note: Starting with an initial Mg2+ concentration of 3 mM is recommended as provided by the 2x Type-it Multiplex PCR MM.

        Table 3. Reaction Mix preparation for one 96 well plate samples
        (modified after manufacturer's instruction)


      1. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or plates. Note: Mix gently, for example, by pipetting the reaction mix up and down a few times. Due to the hot start, it is not necessary to keep samples on ice during reaction setup.
    4. PCR condition

      Table 4. PCR condition (according to manufacturer's instruction)


    5. PCR programme:
      Ctrl. Tube
      Lid- 105 °C
      (1) 95 °C-05:00 min
      (2) 95 °C-00:30 min
      (3) 60°C- 01:30 min
      (4) 72 °C-00:30 min
      (5) Go to step (2), repeat 29
      (6) 60°C-30:00 min
      (7) 20 °C-
      (8) End
      (9)Store PCR products in -20 °C, till further processing.
    6. PCR check
      Check PCR products randomly on 1.8% agarose gel (this is just to check if everything is working before proceeding to next steps. So choose random samples). Load 4 μl product and 100 bp ladder.
      Note: Use only Xylene Cyanol loading dye as Bromophenol Blue dye interfere with the amplified band range.
  4. PCR purification (Day 3)
    1. Add 75 μl nuclease free water to each well of PCR plate (containing 25 μl PCR product) to make it 100 μl.
    2. In a 96 well plate (at-least 1 ml capacity) dispense 300 μl Buffer PM. Add 100 μl PCR sample and mix by pipetteing.
    3. Apply the samples to the wells of the QIAquick plate while it is fitted with the collecting deepwell plate.
    4. Centrifuge at 1,100 RCF (g) for 2 min.
    5. Discard flow-through.
      Note: If the sample volume is more than 600 μl apply the remaining samples to the wells of the QIAquick plate and repeat step 4.
    6. Wash wells of QIAquick plate by adding 900 μl of Buffer PE to each well and centrifuge at 1,100 RCF (g) for 2 min.
    7. Repeat step 4-f.
    8. Discard flow-through and gently soak any buffer remaining on the tip of column with soft tissue.
    9. Centrifuge at 1,500 RCF (g) for 4 min.
      Important: This step removes residual Buffer PE from the membrane.
    10. Let the plate dry for 5 min or until all the ethanol (of Buffer PE) evaporates. Place it under fume hood for 10 min is sufficient.
    11. To elute, place the plate on collection tubes (provided), add 50 μl of RNase-free water (provided) to the center of each well of the QIAquick 96 plate, incubate for 2 min, and centrifuge at 1,500 RCF (g) for 2 min.
      Important:
      Ensure that the elution buffer is dispensed directly onto the center of QIAquick membrane for complete elution of bound DNA. Please note that the average eluate volume is 60 μl from 80 μl elution buffer volume, and 40 μl from 60 μl elution buffer volume.
      1. Quality check
        Check Purified PCR product by nanodrop or 1.8% agarose gel.
        Note: For nanodrop analysis, centrifuge the samples at 1,500 RCF for 2 min and estimate from the upper layer (to precipitate any remaining silica particles from the column, which sometimes interfere with sensitive nanodrop estimation). Imporant: It is better to check randomly the purified product on 1.8% agarose gel to be sure of quality. As this is a multiplex PCR, do not expect a clear band. The gel will appear as smear of many bands.


        Figure 2. PCR amplification check on 1.8% agarose gel. Lanes 1-15 represent PCR products (with a couple of failed amplification on lane 7 and 14), lane 16 represents 100bp DNA marker.

      2. Dilution
        It is not needed to further dilute the samples if you start with 50 ng DNA sample and followed the procedure exactly. At this step the concentration of samples should be around 250 ng/μl.
        For the size sequencing set up, it is recommended to dilute samples in this step to facilitate sample handling.
        Add 200 μl nuclease free water to each purified sample tube (1/5th dilution). So the concentration becomes 50 ng/μl.
  5. Size sequencing (Day 4)
    1. Sequencing mix
      The size sequence mix for each sample contains
      1. 5 μl purified PCR product (250 ng total)
      2. 4.5 μl nuclease free water
      3. 0.5 μl GENSCAN 500 ROX marker
        For a single sequence plate (prepare for 105 samples), set up MM as below:
      1. Mix 472.5 μl nuclease free water with 52.5 μl of ROX marker.
      2. Dispense 5 μl of above MM to each of sequencing plate.
      3. Mix in 5 μl purified PCR samples.
      4. Do not leave any sample well blank. Fill it with Nuclease free water.
      5. Cover the plate with adhesive seal mat.
      6. Centrifuge the plates briefly at 1,500 RCF.
    2. Prepare sequencing table of samples.
      Prepare the sample list conveniently for recognition later. e.g. Sample name_primer mix group_Sample number_any other info.

      Table 5. Sample list example



  1. Analysis (Day 5)
    Before starting analysis, import the data files generated by the sequencer to the computer with GeneMapper software. It is recommended to read the manual if you want to modify and fine-tune the marker settings and allele size binning for genotyping.
    1. Genotype table
      1. Genotyping in GeneMapper
        1) Go to 'file' menu and create new project. Name it conveniently.
        2) Import your samples that belong to one primer mix group.
        3) Create marker panel and bin set for each multiplex primer groups.
        Refer to the manual for modifying or creating a new marker panel for new set of markers/primer mix in microsatellite. This involves:
        a) Marker set creation
        b) Autobinning of alleles
        c) Manual binning of alleles
        d) Create new marker panel and bin set.
        4) Use predefined marker panel for respective samples
      2. Analysis parameters
        1) Select the table setting as ‘Microsatellite default’
        2) Select the analysis method as ‘Microsatellite analysis method’
        3) Set the predefined panel as EV1 for primer mix 1 and so on.
        4) Set size standard as GEN500.
        5) Analyze
        6) If some samples fail to match size standards, go to edit size standards and override size. If it does not improve the sizing, then there was some problem with the sample.

        a) Open the genotype Tab. Do not bother about the GQ (genotype quality) column as it is set to a very high stringency.
        b) Go to tools-panel manager-microsatellite kit-attenuata panel-EV1 binset. Select ‘show project alleles’. If you see any ‘*’ out of the grey bars (defines the range of allele bins), then either modify the range of existing bins (click on the grey bar and you’ll get handles for modification), or add a new bin (click on the blank space near your odd allele (that is not binned) and name it. Save the panel. Again analyze the samples with the modified binset.
        c) If you want to see data for only two alleles and want to modify the way you want your data, modify the table output style. However, manual inspection of allele size, height etc is sometimes necessary to ensure optimal result than automation.
        d) Go to file and export the genotype table in .csv or other excel compatible format.
    2. Paternity analysis in COLONY
      For paternity/maternity analysis, i.e. to determine the parent of an offspring the software COLONY (http://www.zsl.org/science/research/software/colony,1154,AR.html).
      COLONY is a Fortran program written by Jinliang Wang . It implements a maximum likelihood method to assign sibship and parentage jointly, using individual multilocus genotypes at a number of codominant or dominant marker loci. (Jones and Wang, 2010).
      1. Formatting of genotype table.
        1) Keep only the sample name, two allele sizes for each marker for all the marker.
        2) Compile data for all the marker sets into a single datasheet for all offspring and all marker.
      2. Marker table (save as tab delimited .txt file and rename 'marker').

        Table 6. Marker details


      3. Parent table (save as tab delimited .txt file and rename 'father/mother')

        Table 7. Paternal allele list


        Table 8. Maternal allele list


      4. Offspring table (save as tab delimited .txt file and rename 'offspring')

        Table 9. Offspring allele list


      5. Analysis parameters
        Feed all the information step by step as asked by the software and brose for the files to upload.
        A typical parameter is as follows:
        Number of loci:16
        Number of offspring in the sample:
        Number of male candidates:0
        Number of female candidates:
        Number of known paternal sibships:0
        Number of known maternal sibships:0
        Number of offspring with excluded fathers:0
        Number of offspring with excluded mothers:0
        Male mating system:Polygamy/monogamy
        Female mating system:Polygamy/monogamy
        Number of threads : 1
        Number of Excluded Paternal Sibships:0
        Number of Excluded Maternal Sibships:0
        Seed for random number generator:1,234
        Allele frequency:No updating by accounting for the inferred relationship
        Species:Diploid/haploid
        Sibship size prior:No
        Known population allele frequency:No
        Number of run:1
        Length of run : short/medium/long
        Monitor intermiediate results by:Every 1 second
        Prob. a mum is included in the female candidates:0.5
        Project data input produced:
        NOTE to the Project:
      6. Analysis outputs
        1) Select Show result-'best configuration'.
        2) Copy the table of assigned father ID/mother ID to each sample.
        3) Sort parents and count offsprings sired by them in excel.
        Population analysis in GenAlEx
    3. Population analysis in GenAlEx
      When comparing populations for genotypes, or simply want to look at the genotypic diversity within a population, apart from parental analysis, it is better to use the excel addon 'GenAlEx' (http://biology.anu.edu.au/GenAlEx/Welcome.html). (Ref: Peakall R. and Smouse P.E. 2006. Genalex 6: Genetic analysis in Excel. Population genetic software for teaching and research, Molecular Ecology Notes, Volume 6, Issue 1, pages 288–295).
      This can analyze:
      1. Heterozygosity, F-statistics and Polymorphism
      2. Allelic Patterns
      3. Allele List
      4. Allele Frequencies and Principal Component Analysis (PCA).

Recipes

  1. 1x TAE (Tris-Acetate-EDTA) buffer
    1. Prepare a stock solution of EDTA (ethylenediamine tetraacetic acid)
      For a 500 ml stock solution of 0.5 M EDTA, dissolve 93.05 g EDTA disodium salt (FW = 372.2) in 400 ml deionized water and adjust the pH with NaOH to 8.0. Make up the final volume to 500 ml.
    2. Prepare a 50x Stock Solution of TAE
      Dissolve 242 g Tris base (FW = 121.14) in approximately 750 ml deionized water. Add 57.1 ml glacial acid and 100 ml of 0.5 M EDTA (pH 8.0) and adjust the solution to a final volume of 1 L. This stock solution can be stored at room temperature for a long time. The pH of this buffer needs no further re-adjustment and remains about 8.5.
    3. Prepare a Working Solution of 1x TAE
      Dilute the 50X stock solution by 1:50 with deionized water. Final solute concentrations are 40 mM Tris acetate and 1mM EDTA.

Acknowledgments

This work was supported by the Max Planck Gesellschaft. The protocol was adapted from the publication: Kessler et al. (2012).

References

  1. Bindler, G., van der Hoeven, R., Gunduz, I., Plieske, J., Ganal, M., Rossi, L., Gadani, F. and Donini, P. (2007). A microsatellite marker based linkage map of tobacco. Theor Appl Genet 114(2): 341-349.
  2. Jones, O. R. and Wang, J. (2010). COLONY: a program for parentage and sibship inference from multilocus genotype data. Mol Ecol Resour 10(3): 551-555.
  3. Kessler, D., Bhattacharya, S., Diezel, C., Rothe, E., Gase, K., Schöttner, M. and Baldwin, I. T. (2012) Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata. Plant J 71(4): 529-538.

简介

在该方案中,描述了通过微卫星标记在烟草中确定种子亲子。 然而,这不包括新标记选择/鉴定的方案,而是利用为密切相关物种N产生的标记。 tabacum (Bindler ,2007)。 这是一种高通量方案,对于技术人员来说,优化和流线化以在5天内从微生物分离(从幼苗)到通过微卫星基因型数据的亲子鉴定来处理384(96×4)种子。

关键字:微卫星基因分型, 多重PCR, 高通量种子亲子鉴定试验, 野生烟草

材料和试剂

  1. Agencourt Chloropure Kit(该试剂盒由Beckman Coulter停产,但可订购)或Quiagen MagAttract 96 DNA植物核心试剂盒(QIAGEN,目录号:67163)
  2. Qiagen Type-it Multiplex PCR试剂盒(QIAGEN,目录号:206243)
  3. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  4. 液氮
  5. 100%异丙醇,超纯
  6. 新鲜制备的70%乙醇用无核酸酶水制备
    注意:70%乙醇是吸湿的,总是新鲜的最佳结果。
  7. 琼脂糖
  8. 溴化镓溶液
  9. 无核酸酶水
  10. 纯分子生物学级乙醇(96-100%)
    注意:计算开始之前所需的乙醇体积。 24个板的新试剂盒需要大约5升乙醇。 。
  11. GENSCAN 500 ROX(Applied Biosystems,目录号:401734)
  12. 1x TAE(Tris-醋酸-EDTA)缓冲液(参见配方)

设备

  1. 2.2ml Ritter深孔板(ABGene,目录号:AB-6661)
  2. 粘合片膜(ABGene,目录号:AB-0558,AB-626,AB-662)
  3. 4mm钢球(SPEX SamplePrep,目录号:2150)
  4. 移液器提示
  5. 处理板:96孔格式的深孔滴定板(SPEX SamplePrep,目录号:2210)
  6. 目标板:300μl圆底微量滴定板(96孔,300μl孔容,圆底)(Corning,Costar ,目录号:07-200-105)
  7. 96孔板的台式离心机
  8. 试剂库
  9. Genogrinder(SPEX SamplePrep,目录号:2010)
  10. 冷却块(SPEX SamplePrep,目录号:2665)
  11. 钢球分配器(SPEX SamplePrep,目录号:2100)
  12. 多通道移液器(0.1-10,10-100,30-300μl)
  13. Agencourt超磁磁板(Agencourt SPRIPlate 96R-Ring磁铁板,目录号:A29164)
  14. BioRad凝胶浇铸托盘,运行托盘,电源包等(Bio-Rad Laboratories)
  15. Nanodrop分光光度计(Nanodrop)
  16. 移液管(0.2-2μl),移液管吸头,软组织纸
  17. ABI 3100遗传分析仪(Applied Biosysyems)

程序

  1. 样品研磨(第1天)
    1. 在2.2ml板中收集至多40mg新鲜组织样品(叶冲/幼苗)。 注意将类似的样品/处理/实验设计组合在一起,以便在测序和基因型分析期间更容易处理数据
    2. 在每个井中用分配器分配2个钢球。
    3. 平衡两个平板,用于在Genogrinder中磨削
    4. 用粘性板膜密封板。
    5. 在液氮中冷却板。
    6. 将冷却架与板一起冷却。
    7. 研磨在Genogrinder在速度250,1x,1分钟。 如果没有完全粉碎,再次1分钟。

  2. DNA分离(第1天)
    对于每个新的Agencourt Chloropure试剂盒,在步骤B-1中进行一次装配。如果您已经为之前的实验进行了以下准备工作,请跳到步骤B-2。标准RNase处理可以包括在步骤B-5与均质化的溶胞产物(需要大量的酶,但是对于高通量方便),或者在"质量检查"测定RNA污染程度后选择性地。 >
    1. 向试剂盒提供的洗涤缓冲液瓶中加入80ml 100%异丙醇。加入异丙醇后倒瓶混合。一旦溶液彻底混合,在室温下储存。
    2. 制备结合缓冲液:在适当大小的无核酸酶的容器中,将6μl结合缓冲液与150μl100%异丙醇混合,每次单独分离(例如:10分钟,将60μl结合缓冲液加入到1.5ml 100%异丙醇15ml锥形管)。在结合前彻底涡旋结合缓冲液瓶。未使用的组合溶液应该被丢弃。
    3. 将每个样品在300μl裂解缓冲液(试剂盒提供)中匀浆。 (样品输入不应超过三个6mm冻干叶刺或40mg研磨种子或新鲜叶材料)
    4. 在室温下以1,100RCF离心裂解物10分钟以沉淀碎片
    5. 转移150微升匀浆裂解物到1.2毫升处理板
    6. 移液器150μl结合缓冲液(在步骤B-2中制备),通过缓慢移液5次混合,并在室温下孵育5分钟。
    7. 将板移动到Agencourt超级磁铁并分开2-4分钟。 等待解决方案清除,然后继续下一步。
    8. 从板中慢慢吸出清除的溶液并丢弃。
      注意:从顶部向下吸,以避免打扰沉淀。 该步骤必须在板位于磁体上时执行。 不要打扰分离的磁珠。 如果取出珠子,留下几微升的上清液。
    9. 从磁铁上取下板,加入300μl洗涤缓冲液。 移液器混合10次,并在室温下孵育1分钟
    10. 将板返回磁铁并分开2-4分钟。 等待解决方案清除,然后再进行下一步骤。
    11. 从板中慢慢吸出清除的溶液并丢弃。 该步骤必须在板位于磁体上时执行。
    12. 从磁铁上取下板,加入300μl的70%乙醇。 移液器混合10次以重新悬浮珠子
    13. 将板放回磁铁并分开2分钟。 等待解决方案清除,然后再进行下一步骤。
    14. 从板中慢慢吸出清除的溶液并丢弃。
    15. 重复步骤1-n,总共进行2次乙醇洗涤
    16. 让板在室温下空气干燥5分钟。 板应该空气干燥,直到最后可见痕量的乙醇蒸发。 过度干燥样品可能会导致回收率降低。
    17. 从磁铁中取出板,加入50μl无核酸酶的水。 通过吸移管混合10次重悬浮珠。
      注意:较小或较大的洗脱体积可用于或多或少浓缩的产物; 但最小洗脱体积应为40μl,以确保完全洗脱。 最佳洗脱体积需要通过实验确定。
      1. DNA质量/数量检查
        1) 质量
        一个) 用1x TAE缓冲液制备0.8%琼脂糖凝胶。 在浇铸前加入2μl溴化乙锭。
        b) 加载2微升DNA + 1微升加载染料
        C) 加入1 kb或100 bp梯度作为参考
        d) 在恒定100V下运行30分钟
        e) 检查DNA的质量,RNA和数量在凝胶文件系统和记录的存在。



        图1.在0.8%琼脂糖凝胶上的DNA质量检查泳道1-6,8-13代表提取的DNA样品,泳道7代表DNA标记。
        2) 数量
        一个) 按照程序测量Nanodrop中的DNA浓度
        b) 导出为excel文件以供参考。
      2. 稀释DNA样品
        a)将样品稀释至50 ng /μl作为工作母液 b)类似地将它们分配在3个PCR板(用于3个多重PCR反应)中,并保持工作DNA板的样品组织。 记录样品。

  3. PCR扩增(第2天)
    1. 多重组
      使用软件MultiPLX版本2.0(可从http://bioinfo.ut.ee/?page_id=167,Reference:doi:10.1093/bioinformatics/bti219获得)基于引物序列选择引物组以选择多重组。选择中等严格性以选择组。
      3个8个引物对的多重组的实施例:

      表1. 8个引物对的3个复用组示例:


    2. 10x引物混合物
      1. 首先制备100μM引物储备液(如果使用50μM引物储液,则相应地调整下表的体积)。
      2. 根据下表混合所有引物,得到3个引物混合组(足以用于1×PCR板,即96个样品)。

        表2.制备3个引物混合物组的示意图:


    3. PCR反应混合物
      1. 解冻2x型多重PCR主混合物(MM)(如果储存在-20℃),模板DNA(已经分配在PCR板中),无RNA酶的水和引物混合物。在使用前将溶液完全混合。
        注意:重要的是在使用前完全混合溶液,以避免局部浓度的盐。
      2. 根据下表制备反应混合物。注意:反应混合物通常包含多重PCR所需的所有组分,除了模板DNA。制备比待进行的反应总数所需的体积大10%的反应混合物体积。对于小于25μl的反应体积,应保持1:1比例的Type-it多重PCR主混合物与引物混合物和模板。
        注意:从初始Mg 2 + 浓度为3 mM开始,建议使用2x Type-it Multiplex PCR MM 。

        表3.一个96孔板样品的反应混合物制备
        (根据制造商的说明修改)


      1. 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:轻轻混合,例如,通过向上和向下移动反应混合物几次。 由于热启动,在反应设置期间不必将样品保持在冰上。
    4. PCR条件

      表4. PCR条件 (根据制造商的说明)


    5. PCR程序:
      控制。 管
      盖子-105℃
      (1)95℃-05:00min
      (2)95℃-00:30分钟
      (3)60℃-01:30分钟
      (4)72℃-00:30分钟
      (5)转到步骤(2),重复29
      (6)60℃-30:00min
      (7)20°C-
      (8)结束
      (9)将PCR产物储存在-20℃,直到进一步处理
    6. PCR检查
      在1.8%琼脂糖凝胶上随机检查PCR产物(这只是为了检查一切是否正常,然后再进行下一步骤,因此选择随机样品)。 加载4μl产品和100 bp梯。
      注意:仅使用二甲苯Cyanol载色染料作为溴酚蓝染料干扰放大的带范围。
  4. PCR纯化(第3天)
    1. 向PCR板的每个孔(含有25μlPCR产物)中加入75μl无核酸酶水,使其达到100μl。
    2. 在96孔板(至少1ml容量)中分配300μl缓冲液PM。 加入100μlPCR样品并通过移液器混合
    3. 将样品应用于QIAquick板的孔中,同时装有收集深孔板。
    4. 在1100RPM( g )离心2分钟。
    5. 丢弃流通。
      注意:如果样品体积超过600μl,将剩余的样品加入QIAquick板的孔中,并重复步骤4.
    6. 通过向每个孔中加入900μl缓冲液PE并在1100RPM( g )离心2分钟来洗涤QIAquick板的孔。
    7. 重复步骤4-f。
    8. 弃去流出物,用软组织轻轻浸泡留在色谱柱顶端的任何缓冲液
    9. 以1500RCF离心4分钟( 重要:此步骤从膜上除去残留的缓冲液PE。
    10. 让板干燥5分钟或直到所有乙醇(缓冲液PE)蒸发。 将其放在通风橱下10分钟即可。
    11. 为了洗脱,将板置于收集管(提供)上,向QIAquick 96板的每个孔的中心加入50μl无RNA酶的水(提供),孵育2分钟,并在1500RCF离心g )2分钟。
      重要提示: 确认洗脱缓冲液直接分配到QIAquick膜的中心,以完全洗脱结合的DNA。请注意,平均洗脱液体积为80μl洗脱缓冲液体积的60μl,和60μl洗脱缓冲液体积的40μl。
      1. 质量检查
        通过nanodrop或1.8%琼脂糖凝胶检查纯化的PCR产物 注意:对于纳米滴分析,将样品在1500RCF离心2分钟并从上层估计(以沉淀来自柱的任何剩余的二氧化硅颗粒,其有时干扰敏感的纳米推测)。注意:最好在1.8%琼脂糖凝胶上随机检查纯化的产物,以确保质量。由于这是一个多重PCR,不要期望一个清晰的频带。凝胶将显示为许多条带的涂片。


        图2.在1.8%琼脂糖凝胶上的PCR 扩增检查 。泳道1-15代表PCR产物(在泳道7上有几个失败的扩增和14),泳道16代表100bp DNA标记
      2. 稀释
        如果您从50 ng DNA样品开始,并且完全遵循程序,则不需要进一步稀释样品。 在这一步,样品的浓度应该在250ng /μl左右 对于大小测序设置,建议在此步骤中稀释样品以方便样品处理。
        向每个纯化的样品管中加入200μl无核酸酶的水(1/5稀释)。 所以浓度变为50 ng /μl
  5. 大小测序(第4天)
    1. 排序混合
      每个样本的大小序列混合包含
      1. 5μl纯化的PCR产物(总共250 ng)
      2. 4.5μl无核酸酶水
      3. 0.5μlGENSCAN 500 ROX标记
        对于单个序列板(准备105个样品),设置MM如下:
      1. 混合472.5μl无核酸酶水和52.5μlROX标记
      2. 将5μl上述MM分配到每个测序板。
      3. 混合在5μl纯化的PCR样品中
      4. 不要将任何样品留空。 用无核酸酶的水填充。
      5. 用粘性密封垫覆盖板。
      6. 以1500 RCF短暂离心板。
    2. 准备样品的测序表。
      方便地准备样品列表以便以后识别。 例如示例name_primer mix group_Sample number_any其他信息。

      表5.示例列表示例


  1. 分析(第5天)
    在开始分析之前,使用GeneMapper软件将序列生成器生成的数据文件导入计算机。 如果要修改和微调基因分型的标记设置和等位基因大小分级,建议阅读手册。
    1. 基因型表
      1. 基因分型在GeneMapper
        1) 转到"文件"菜单并创建新项目。 方便地命名。
        2) 导入属于一个引物混合组的样品。
        3) 为每个多重引物组创建标记面板和仓集。
        参考手册修改或创建一个新的标记面板新标记/引物混合在微卫星中。 这包括:
        一个) 标记集创建
        b) 等位基因的自动合并
        C) 手动装箱等位基因
        d) 创建新的标记面板和bin设置。
        4) 对各个样品使用预定义标记面板
        <! -
        1. 转到"文件"菜单并创建新项目。 方便地命名。
        2. 导入属于一个引物混合组的样品。
        3. 为每个多重引物组
          创建标记面板和仓集
        4. 参考手册修改或创建一个新的标记面板新标记/引物混合在微卫星中。 这包括:
          1. 标记集创建
          2. 等位基因的自动合并
          3. 手动装箱等位基因
          4. 创建新的标记面板和bin设置。
        5. 对各个样品使用预定义标记面板
        - >
      2. 分析参数
        1) 选择表格设置为'Microsatellite default'
        2) 选择分析方法为'微卫星分析方法'
        3) 将预定义面板设置为引物混合1的 EV1 ,依此类推。
        4) 将大小标准设为 GEN500 。
        5) 分析
        6) 如果一些样品不符合大小标准,请转到编辑大小标准并覆盖大小。 如果它没有提高大小,那么样本有一些问题。

        一个) 打开基因型Tab。 不要打扰GQ(基因型质量)列,因为它设置为非常高的严格性。
        b) 转到tools-panel manager-microsatellite kit-attenuata panel-EV1 binset。选择"显示项目等位基因"。如果你看到灰色条中的任何'*'(定义等位基因箱的范围),那么修改现有箱的范围(点击灰色栏,你会得到修改的句柄),或者添加一个新箱(点击您的奇数等位基因附近的空白区域(不分箱),并命名它。保存面板。再次使用修改的binset分析样品。
        C) 如果希望仅查看两个等位基因的数据,并希望修改您希望数据的方式,请修改表输出样式。然而,有时需要手动检查等位基因大小,高度等以确保比自动化优化的结果。
        d) 转到文件并以.csv或其他excel兼容格式导出基因型表。
    2. COLONY中的父子分析
      对于父亲/母性分析,即以确定后代的父母软件COLONY( http://www.zsl.org/science/research/software/colony,1154,AR.html )。
      COLONY是由Jinliang Wang编写的Fortran程序。 它实施最大似然法以在多个共显性或显性标记基因座处使用个体多基因座基因型联合分配同胞和亲本。 (Jones和 Wang,2010)。
      1. 基因型表格的格式化。
        1) 仅保留样品名称,所有标记的每个标记的两个等位基因大小。
        2) 将所有标记集的数据编译为所有后代和所有标记的单个数据表。
      2. 标记表(另存为制表符分隔的.txt文件并重命名'marker')。

        表6.标记详情


      3. 父表(另存为制表符分隔的.txt文件并重命名为"父/母")

        表7.父系等位基因列表


        表8.母体等位基因列表


      4. 后代表(保存为制表符分隔的.txt文件和重命名'后代')

        表9.后代等位基因列表


      5. 分析参数
        根据软件的要求逐步提供所有信息,并分析要上传的文件。
        典型的参数如下:
        位点数:16
        样本中的后代数:
        男性候选人数:0
        女性候选人人数:
        已知父亲兄弟姐妹数:0
        已知母亲兄弟姐妹数:0
        排除父亲的后代数:0
        排除母亲的后代数:0
        男性交配系统:一夫多妻/一夫一妻制
        雌性交配系统:一夫多妻/一夫一妻制
        线程数:1
        排除的父系兄弟人数:0
        排除的母亲家庭数:0
        随机数生成器种子:1,234
        等位基因频率:不考虑推断的关系而更新
        物种:二倍体/单倍体
        先前团体规模:无
        已知的群体等位基因频率:无
        运行次数:1
        运行时间:短/中/长
        监视中间结果:每1秒
        概率。 女性候选人包括母亲:0.5
        生成的项目数据输入:
        项目注意事项:
      6. 分析输出
        1) 选择显示result-'best configuration'。
        2) 将分配的父亲ID /母亲ID的表格复制到每个样品
        3) 排序父母和计数他们在excel中的子女。
        GenAlEx中的群体分析
    3. GenAlEx中的群体分析
      当比较人口 基因型,或只是想看一下基因型的多样性 人口,除了父母分析,最好使用excel addon'GenAlEx'( http://biology.anu.edu.au/GenAlEx/Welcome。 html )。 (参考: Peakall R.和Smouse P.E. Genenex 6:Excel中的遗传分析。 人口基因软件的教学和研究,分子生态学   Notes ,第6卷,第1期,第288-295页)。
      这可以分析:
      1. 杂合性,F统计学和多态性
      2. 等位基因模式
      3. 等位基因列表
      4. 等位基因频率和主成分分析(PCA)。

食谱

  1. 1x TAE(Tris-乙酸酯-EDTA)缓冲液
    1. 制备EDTA(乙二胺四乙酸)的储备溶液 对于500ml 0.5M EDTA储备溶液,在400ml去离子水中溶解93.05g EDTA二钠盐(FW = 372.2),并用NaOH调节pH至8.0。 最终体积为500 ml。
    2. 准备TAX的50x库存解决方案
      在约750ml去离子水中溶解242g Tris碱(FW = 121.14)。 加入57.1ml冰醋酸和100ml 0.5M EDTA(pH8.0),并将溶液调节至1L的最终体积。该储备溶液可以在室温下长时间储存。 该缓冲液的pH值不需要进一步重新调节,并且保持在约8.5
    3. 准备1x TAE的工作解决方案
      用去离子水稀释50倍储备溶液1:50。 最终溶质浓度为40mM Tris乙酸盐和1mM EDTA。

致谢

这项工作得到了马克斯普朗克协会的支持。 该方案改编自出版物:Kessler等人(2012)。

参考文献

  1. Bindler,G.,van der Hoeven,R.,Gunduz,I.,Plieske,J.,Ganal,M.,Rossi,L.,Gadani,F.and Donini,P.(2007)。 基于微卫星标记的烟草连锁图。 Theor Appl Genet 114(2):341-349。
  2. Jones,O.R。和Wang,J。(2010)。 COLONY:用于多基因座基因型数据的家长和同胞推断的程序。 Mol Ecol Resour 10(3):551-555。
  3. Kessler,D.,Bhattacharya,S.,Diezel,C.,Rothe,E.,Gase,K.,Schöttner,M.and Baldwin,IT(2012)花蜜尼古丁的不可预测性促进了蜂鸟在烟草中的突变。。 71 71(4):529-538。 >
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bhattacharya, S. and Baldwin, I. . (2013). High-throughput Method for Determination of Seed Paternity by Microsatellite Markers. Bio-protocol 3(8): e452. DOI: 10.21769/BioProtoc.452.
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