参见作者原研究论文

本实验方案简略版
Jun 2019

本文章节


 

Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids
利用甲基化敏感扩增多态性分析野生马铃薯杂交种   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Keywords: MSAP (甲基敏感扩增多态性), DNA Methylation Pattern Inheritance (DNA甲基化模式遗传), Epigenetic changes (表观遗传变化), Solanum (茄属植物), Interspecific hybridization (种间杂交), Epialleles (等位基因), R Script (R脚本)

Background

Nucleotide sequences are not the only form of genomic information: DNA methylation, histone proteins, enzymes that modify histones and nucleotide residues on DNA and even RNAs, influence gene activity and provide another layer of instructions to the cell. Epigenetic changes, also called epimutations, can be inherited and have important phenotypic consequences. In plants, methylation reactions modify cytosine residues into 5-methylcytosines. This epigenetic mechanism is essential to maintain the genomic integrity and contributes to regulate gene expression in developmental processes and in response to biotic and abiotic stresses. In addition, changes in DNA methylation are triggered by genomic shocks like hybridization and polyploidization, two vital phenomena in plant evolution (Cara et al., 2019).

There are different alternatives to study changes in DNA methylation. Global cytosine methylation can be assessed by using high-performance liquid chromatography (HPLC), analytical methods that allow to quantify cytosines and 5-methylcytosines and to calculate the percentage of methylated residues in the genome. For studying the DNA methylation at specific positions on the genome two alternatives can be mentioned. One is to use isoschizomers with different sensitivities to methylation at cytosines of the restriction site. For example, Methylation-Sensitive Amplification Polymorphism (MSAP) markers characterize the methylation pattern at anonymous 5′-CCGG sequences from random genomic DNA. This is an adaptation of the original AFLP protocol (Vos et al., 1995) substituting the frequent cutter enzyme MseI by HpaII and MspI. These enzymes recognize the same tetranucleotide restriction site (5′-CCGG), but HpaII is sensitive to full methylation (both strands methylated) and cleaves the hemi-methylated external cytosine, whereas MspI is sensitive only to methylation of the external cytosines of the restriction site. Another possibility to study site-specific methylation status is by using a bisulphite sequencing methodology. Bisulphite treatments on DNA convert cytosines into uracils while 5-methylcytosines remain unchanged. Then, by sequencing amplicons (i.e., a target gene or promoter) of bisulphited and control DNA, it is possible to distinguish between methylated and unmethylated cytosines. With the development of next generation sequencing technologies, whole-genome bisulphite sequencing, or WGBS, can be implemented to infer the position for all 5-methylcytosines in a genome. However, this approach requires a high-quality reference genome to perform epigenetic analyses. Although the increasing number of draft genomes and the reduction in sequencing costs offer possibilities to implement massive methylation analyses in non-model species, the use of MSAP markers continues to be a valuable tool in many laboratories.

Wild potatoes (Solanum, section Petota) are a group of species related to the cultivated potato Solanum tuberosum L. Internal breeding barriers can be incomplete, thus, interspecific hybridization occurs in areas of sympatry (Camadro et al., 2012). Epigenetic changes in response to interspecific hybridization have been documented in synthetic and natural hybrids of wild potato species (Cara et al., 2019). Solanum x rechei H. & H. is a hybrid species that grows in sympatry with its wild progenitors, Solanum kurtzianum B. & W. and Solanum microdontum B. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in hybrids and their parental genotypes. Using an R script, fragments present in the synthetic hybrids are categorized as S. microdontum or S. kurtzianum species-specific if they are present on the parental genotypes, S. x rechei species-specific, if they are present in at least one of the S. x rechei evaluated genotypes or as novel if they are only observed in the synthetic hybrids.

Materials and Reagents

  1. Consumables
    1. Microfuge tubes (DELTALAB, catalog numbers: 4095.5N , 4095.9N )
    2. Pipette tips (DELTALAB, catalog numbers: 200072 , 200016 )
    3. PCR microplate 96-well (Axygen, catalog number: 32165051 )
    4. Plastic pestle (Sigma, catalog number: Z359947 )

  2. Chemicals
    1. Tris base (BIOPACK, catalog number: 200016 6800)
    2. Ethylenediaminetetraacetic acid (EDTA) (BIOPACK, catalog number: 2000964500 )
    3. Sodium Chloride (BIOPACK, catalog number: 200016 4606)
    4. Cetyltrimethyl ammonium bromide (CTAB) (Bio Basic INC, catalog number: DB0108 )
    5. β-mercaptoethanol (BIOPACK, catalog number: 2000954500 )
    6. Chloroform (BIOPACK, catalog number: 200016 5100)
    7. Isoamyl alcohol (BIOPACK, catalog number: 2000972500 )
    8. Ethyl alcohol (BIOPACK, catalog number: 200016 5400)
    9. Sodium acetate (BIOPACK, catalog number: 200016 8000)
    10. RNase A (Thermo Fisher Scientific, catalog number: EN0531 )
    11. Lambda DNA/EcoRI+HindIII Marker (Promega, catalog number: G1731 )
    12. Glycerol (BIOPACK, catalog number: 200016 2000)
    13. Bromophenol blue (BIOPACK, catalog number: 2000962200 )
    14. Xylene cyanol FF (SIGMA, catalog number: X4126 )
    15. Boric acid (BIOPACK, catalog number: 2000935900 )
    16. Agarose (TransGen, catalog number: GS201 )
    17. UltraPureTM Ethidium Bromide (Thermo Fisher Scientific, catalog number: 15585011 )
    18. EcoRI (New England Biolabs, catalog number: R0101S )
    19. Bovine Serum Albumin (BSA) (Promega, catalog number: R3961 )
    20. HpaII (New England Biolabs, catalog number: R0171S )
    21. MspI (New England Biolabs, catalog number: R0106 )
    22. T4 DNA Ligase (Promega, catalog number: M1801 )
    23. Oligonucleotides (Table 1) (IDT, Integrated DNA Technologies, Inc., Iowa, USA)

      Table 1. Sequences of adaptors and primers used


    24. Taq DNA Polymerase (Thermo Fisher Scientific, catalog number: 11615044 )
    25. MgCl2 (Thermo Fisher Scientific, catalog number: 11615044 )
    26. dNTP Set (100 mM) (Thermo Fisher Scientific, catalog number: 10297018 )
    27. Hi-Di formamide (Thermo Fisher Scientific, catalog number: 4311320 )
    28. GeneScan 500HD Rox size standard (Thermo Fisher Scientific, catalog number: 401734 )
    29. ddH2O (sterile)
    30. Extraction buffer (see Recipes)
    31. TBE (Tris-Borate-EDTA) buffer (see Recipes)
    32. 6x DNA loading buffer (see Recipes)

Equipment

  1. Pipettes (BOECO, Wheaton SOCOREX and Finnpipette)
  2. Vortex (IKA, model: MS1 )
  3. UltraCentrifuge (Eppendorf, model: 5804R )
  4. Thermostatic bath (Vicking, model: Masson )
  5. Agarose gel electrophoresis system (Bio-Rad Laboratories, model: 1645056 )
  6. Microwave oven (HITPLUS, model: CM203M )
  7. Spectrophotometer (AmpliQuant, model: AQ-07
  8. Gel Imager (Bio-Rad Laboratories, model: Gel Doc 1000 )
  9. Incubator (SANYO, model: MIR 262 )
  10. Thermocycler Veriti 96 well (Applied Biosystems, model: 9902 )
  11. Genetic Analyzer (Invitrogen, model: ABI PRISM 3130 )
  12. Ultra-low Freezer -80 °C (Forma Scientific, model: 8270 )

Software

  1. GeneMapper v3.7. (Applied Biosystems, Foster City, CA, USA)
  2. R v2.15.1. (R Foundation for Statistical Computing, Vienna, Austria)
  3. RStudio v1.1.442 (RStudio Inc., Boston, MA, USA)

Procedure

  1. DNA extraction procedure
    1. Collect 50-100 mg of leaf tissue in a 1.5 ml centrifuge microtube. Flash-freeze the tissue in liquid nitrogen and grind it with a plastic pestle.
    2. Add 300 µl of extraction buffer and mix it vigorously by vortexing (5-10 s) to homogenize the tissue. Incubate the homogenate at 65 °C for 30 min in a thermostatic bath.
    3. Add 100 µl of chloroform:isoamyl alcohol (24:1) and mix the sample gently by inversion.
    4. Centrifuge the homogenate at 20,800 x g at 4 °C for 5 min.
    5. By using a 200 µl micropipette, carefully pipette the aqueous phase and transfer it to a new microtube.
    6. Add 500 µl of ice-cold ethanol:acetate (24:1) and mix the sample by inverting the microtube 2-3 times to precipitate the DNA, and let it settle at -20 °C for 30 min.
    7. Centrifuge the sample at 20,800 x g at 4 °C for 10 min to form a DNA pellet at the bottom of the microtube.
    8. Discard the supernatant and clean the pellet with 100 µl of 70% ethanol. Vortex the tube for 1 min to wash the pellet.
    9. Centrifuge the sample at 15,300 x g at room temperature for 5 min and let the pellet dry by inverting the microtube onto a paper towel for 30 min until the ethanol evaporates completely or by placing it in a drying chamber for 15 min.
    10. Resuspend the DNA in 50 µl of ultrapure water with RNase A (50 μg·ml-1). Incubate at room temperature for 30 min.
    11. Store the DNA sample at 4 °C until use. If the DNA will not be used for a long time, store it at -20 °C.
    12. Check the integrity and purity of the extraction by loading 2 μl of the DNA solution on a 0.8% agarose gel and running it at 90 V for 30 min. It should show a thick band of high molecular weight, above 21 Kbp, with no smear (Figure 1).
    13. Measure the concentration of DNA samples in a spectrophotometer and prepare working solutions of ~100 ng·μl-1.


      Figure 1. Representative image of DNA samples visualized in a 0.8% agarose gel

  2. Digestion with EcoRI
    1. Calculate the volume of the sample that corresponds to 700 ng of DNA, according to its concentration. However, consider that DNA concentration should be higher than 40.5 ng·μl-1, otherwise it will exceed the reaction’s final volume.
    2. Calculate the amount of each reagent needed for the total number of samples based on Table 2. To compensate pipetting errors, it is convenient to consider an additional sample to the total number of analyzed samples.

      Table 2. Master mix for the enzymatic digestion with EcoRI


    3. Prepare the Master mix, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    4. Distribute the Master mix in individual tubes and add the DNA. Homogenize gently by inversion or by flicking the tubes (do not vortex) and centrifuge for 10 s.
    5. Incubate at 37 °C overnight in an incubator.
    6. Place the tubes on ice if you continue with the next step. Otherwise, store them in freezer at -20 °C until use.

  3. Digestion with HpaII and MspI
    1. For each tube from the previous reaction (B), distribute the content into two tubes (10 μl in each tube) and label the new tubes with the sample code, adding the letter "H" to one and the letter "M" to the other. From now on you will work with two tubes for each analyzed sample.
    2. Two Master mixes will be prepared, only differing on the restriction enzyme, one with HpaII and the other with MspI. For the two reactions, calculate the required amount of each reagent for the total number of samples plus one, based on Table 3.

      Table 3. Master mixes for enzymatic digestions with HpaII y MspI


    3. Prepare the Master mixes, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    4. Distribute the Master mix H in the tubes labeled with the letter "H" and the Master mix M in the tubes labeled with the letter "M". From now on you will have two tubes per sample. Homogenize gently by inversion or by flicking the tubes (do not vortex) and centrifuge for 10 s.
    5. Incubate at 37 °C overnight in an incubator.
    6. (Optional) Examine the efficiency of both digestions by running 6 μl of the restriction products on a 1.2% agarose gel, at 90 V for 30 min. It should show a subtle smear from 5,100 to 100 bp (Figure 2).


      Figure 2. Representative image of restriction products visualized in a 1.2% agarose gel

    7. Keep the tubes on ice if you will continue with the next step. Otherwise, store them in the freezer at -20 °C until use.

  4. Adaptors ligation
    1. Prepare the double stranded EcoRI and MspI/HpaII adaptors from the single strand oligonucleotides by adding equal amounts of each oligo in a tube (Table 1). Place the tubes in the thermocycler and run the ADAPTORS program (Table 4) with a hot lid.

      Table 4. PCR programs


    2. Based on Table 5, calculate the amount of each reagent for the total number of samples plus one.

      Table 5. Master mix for the ligation reaction


    3. Prepare the Master mix, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    4. Distribute the Master mix in individual tubes and add 10 μl of digestions. Homogenize gently by inversion or by flicking the tubes (do not vortex) and centrifuge for 10 s.
    5. Incubate at room temperature for 3 h.
    6. Keep the tubes on ice if you will continue with the next step. Otherwise, store them in a freezer at -20 °C until use.

  5. First amplification (Preamplification)
    For this PCR reaction, oligonucleotides with the same sequences as the adaptors plus 1 selective nucleotide are used. These oligonucleotides are identified as Primer EcoRI +1 and Primer H/M +1 (Table 1).
    1. Calculate the necessary amount of each reagent needed for the total samples based on Table 6, considering an additional sample to compensate for pipetting errors.

      Table 6. Master mix for the pre-amplification


    2. Prepare the Master mix, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    3. Distribute 18 μl of the Master mix in each tube and add 2 μl of the ligations. Add 2 μl of water to the remaining mix to use it as a negative reaction control. Homogenize gently by inversion or flicking (do not vortex) and centrifuge for 10 s.
    4. Place the samples plus the negative control in the thermocycler and run the PREAMPLIFICATION program (Table 4) with a hot lid.
    5. Check the efficiency of the reaction by loading 5 μl of the pre-amplifications on a 1.2% agarose gel and running at 90 V for 30 min. It should show an intense smear with few or some diffuse bands from 1,500 to 100 bp (Figure 3).


      Figure 3. Representative image of pre-amplification products visualized in a 1.2% agarose gel

    6. Dilute the pre-amplifications 1:3. Keep on ice if you continue with the next step or store in a freezer at -20 °C. Preamplifications can be safely stored in a freezer for several months.

  6. Second amplification (Selective amplification)
    For this PCR reaction, oligonucleotides with the same sequences as the adaptors plus 3 selective nucleotides are used. These oligonucleotides are identified as Primer EcoRI +3 and Primer H/M +3 (Table 1).
    1. Calculate the necessary amount of each reagent needed for the total number of samples plus one, based on Table 7.

      Table 7. Master mix for the amplification reaction


    2. Prepare the Master mix, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    3. Distribute 19 μl of the Master mix in each tube and add 1 μl of 1:3 diluted preamplifications. To the remaining mix add 1 μl of water to use it as a negative reaction control. Homogenize gently by inversion or flicking (do not vortex) and centrifuge for 10 s.
    4. Place in the thermocycler and run the AMPLIFICATION program (Table 4) with a hot lid.
    5. Check the efficiency of the reaction by loading 5 μl of the amplification products on a 1.2% agarose gel and running at 90 V for 30 min. Depending of the primer combination, it should show a smear with diffuse bands from 700 to 100 bp (Figure 4).


      Figure 4. Representative image of amplification products visualized in a 1.2% agarose gel

    6. Keep the tubes on ice if you continue with the next step or store them in a freezer at -20 °C. Fluorescence-labeled amplifications can be safely stored at least for 6 months at -20 °C maintaining their light emission.

  7. Electrophoresis
    1. Calculate the necessary amount of each reagent needed for the total samples based on Table 8, considering an extra sample to compensate for pipetting errors.

      Table 8. Master mix for the amplification reaction


    2. Prepare the Master mix, homogenize by vortexing (2-3 s) and centrifuge for 10 s.
    3. Distribute 9 μl of the Master mix in each well of the 96-well plate and add 1 μl of amplification products.
    4. Place the plate in the thermocycler and run the program DENATURATION (Table 4) with a hot lid.
    5. Take the plate out of the thermocycler and put it on ice.
    6. Place the plate in the ABI Prism 3,130 DNA sequencer (Applied Biosystems) and run.

Data analysis

  1. Allele calling and matrix generation
    1. Semi-automated scoring was performed on the resulting electronic profiles using GeneMapper v3.7 (Applied Biosystems). The length range of analyzed fragments was from 100 to 500 bp. Full specifications of the parameters used in GeneMapper are summarized in Cara et al. (2014). GeneMapper’s Report Manager tool was used to retrieve only sample names and allele columns into a Comma Separated Values (.csv) file.
    2. Patterns of presence/absence between the EcoRI/HpaII and the EcoRI/MspI digests were codified from 0 to 3; then, this codification was converted into a binary matrix for either presence (1) or absence (0) of patterns 3, 2 or 1 (Figure 5). Pattern 0 was not codified in the binary matrix because the absence of fragments in EcoRI/HpaII and EcoRI/MspI digests might be due to either methylation of external cytosines or variations in the nucleotide sequence. The following R script was developed to perform this analysis.

      R Script for generating an MSAP binary matrix:
      ##### R Commands used to generate an MSAP binary matrix from a Genemapper csv file

      ## The following command makes sure that the working memory is clear
      rm(list = ls(all = TRUE))

      ## Set working directory (PATH will be specific to where data are)
      setwd("C:/...")

      ## Read in dataset. Input file should contain Samples by rows and Loci by columns, including header (loci names) and sample names in the first column
      ## Each sample digested by HpaII and MspI is identified as 'SampleName_H' and 'SampleName_M', one below the other.
      Data = read.csv("MSAP_Genemapper.csv", header=T, row.names = 1, check.names=F)

      ## Create objects
      out <- c()
      MSAP_patterns <- matrix(nrow = dim(Data)[2], ncol = 0)

      ## This is a double loop for j hybrids and i loci. It generates a matrix of MSAP patterns (0, 1, 2 or 3).
      for (j in 1:dim(Data)[1]) if (j %% 2 ==1)
      {
      for (i in 1:dim(Data)[2])
      {
      out <- append(out, ifelse(Data[j,i]+Data[j+1,i]==0, '0',
      ifelse(Data[j,i]+Data[j+1,i]==2, '1',
      ifelse(Data[j,i]==1, '2', '3'))))
      }
      MSAP_patterns <- cbind(MSAP_patterns, matrix(out, dimnames = list(colnames(Data), substr(row.names(Data)[j], 1, nchar(as.character(row.names(Data)[j]))-2))))
      out <- c()
      }

      ##This is a loop that creates a binary matrix of presence/absence of each pattern (1, 2 or 3) for i loci.
      dummies <- matrix(nrow = dim(MSAP_patterns)[2], ncol = 0)
      for (i in 1:dim(MSAP_patterns)[1])
      {
      dummies <- cbind(dummies, matrix(as.numeric(MSAP_patterns[i,] == 1), dimnames = list(colnames(MSAP_patterns), paste(row.names(MSAP_patterns)[i], "1", sep = "_"))), matrix(as.numeric(MSAP_patterns[i,] == 2), dimnames = list(colnames(MSAP_patterns), paste(row.names(MSAP_patterns)[i], "2", sep = "_"))), matrix(as.numeric(MSAP_patterns[i,] == 3), dimnames = list(colnames(MSAP_patterns), paste(row.names(MSAP_patterns)[i], "3", sep = "_"))))
      }
      binary_matrix <- dummies[,colSums(dummies != 0) != 0]

      ## Save output to working directory
      write.csv(binary_matrix, file = "MSAP_binary_matrix.csv")


      Figure 5. Methylation-sensitive Amplified Polymorphism (MSAP) pattern analysis. A. Inference of methylation status of CCGG restriction sites from the fragment amplification profiles and DNA methylation pattern codification. Black C represents methylated cytosines. B. Binary matrix obtained by codifying the presence (1) or absence (0) of the particular methylation pattern.

  2. Determination of species-specific and novel fragments
    1. The amplification patterns observed in the evaluated natural hybrids were compared with the patterns obtained in the synthetic hybrids and their parental genotypes. Fragments present in the synthetic hybrids were categorized as S. microdontum or S. kurtzianum species-specifics. In addition, the presence of novel amplification fragments in the synthetic hybrids (that is present in the hybrids but absent in the genotypes of the parental species S. microdontum and S. kurtzianum) was assessed in the natural hybrids. Novel fragments were classified as S. x rechei species-specific if they were also present in at least one of the S. x rechei evaluated genotypes or as novel if they were only observed in the synthetic hybrids. The following R script was developed to perform this analysis.

      R Script for analyzing MSAP epiloci inheritance in hybrids:
      #### R Commands used to analyze MSAP epiloci inheritance in Hybrids

      ## The following command makes sure that the working memory is clear
      rm(list = ls(all = TRUE))

      ## Set working directory (PATH will be specific to where data are)
      setwd("C:/...")

      ## Read in dataset. Data is organized as Samples by rows and Loci by columns
      Data = read.csv("MSAP_binary_matrix.csv", header=T, row.names = 1, check.names=F)

      ## Create objects
      out <- c()
      results <- matrix(nrow = 4, ncol = 0)

      ## This is a double loop for j hybrids and i loci. Rows are as follows: 1-5 Hybrids, 6 S. kurtzianum (Parental1), 7 S. microdontum (Parental2), 8-15 S. x rechei (Natural hybrid). Any modifications in these numbers should be applied to the following commands.
      for (j in 1:5)
      {
      for (i in 1:dim(Data)[2])
      {
      out <- append(out, ifelse(Data[j,i]>0 & Data[6,i]>0 & Data[7,i]==0 & sum(Data[8:15,i])==0, 'A', ## Fragments shared only by hybrids and S. kurtzianum
      ifelse(Data[j,i]>0 & Data[6,i]==0 & Data[7,i]>0 & sum(Data[8:15,i])==0, 'B', ## Fragments shared only by hybrids and S. microdontum
      ifelse(Data[j,i]>0 & Data[6,i]==0 & Data[7,i]==0 & sum(Data[8:15,i])>0, 'C', ## Fragments shared only by hybrids and S. x rechei
      ifelse(Data[j,i]>0 & Data[6,i]==0 & Data[7,i]==0 & sum(Data[8:15,i])==0, 'D', 'E'))))) ## Fragments specific to hybrids
      }

      results <- cbind(results, matrix(table(out, exclude = "E"),dimnames = list(c("Kurtzianum", "Microdontum", "Rechei", "Hybrid"), row.names(Data)[j])))
      out <- c()
      }

      ## Transform counts by hybrid into percentages
      percentages <- t(results)/colSums(results)*100

      ## Save output to working directory
      write.csv(percentages, file = "MSAP_Hybrids.csv")

      ## A basic graphical representation
      barplot(t(percentages), horiz = TRUE, las=1, col = c("red", "blue", "green", "yellow"))

Recipes

  1. Extraction buffer
    100 mM Tris-HCl (pH 8.0)
    20 mM EDTA (pH 8.0)
    1.4 M NaCl
    0.4% (v/v) β-mercaptoethanol
    2% (w/v) CTAB
  2. 1x TBE (Tris-Borate-EDTA) gel running buffer
    89 mM Tris base
    89 mM Boric acid
    2 mM EDTA (pH 8.0)
  3. 6x DNA loading buffer
    30% (v/v) Glycerol
    0.25% (w/v) Bromophenol blue
    0.25% (w/v) Xylene cyanol FF

Notes

Prepare all Master mixes by first adding the larger volumes and then the smaller ones, leaving the enzymes to the end.

Acknowledgments

We thank Dr María Virginia Sanchez Puerta for critical reading of the draft manuscript. This work was supported by Agencia Nacional de Promoción Científica y Tecnológica and Universidad Nacional de Cuyo, Argentina PICT 1243. This protocol was adapted from our previous work (Cara et al., 2019).

Competing interests

The authors have not competing interest.

References

  1. Camadro, E. L., Erazzu, L. E., Maune, J. F. and Bedogni, M. C. (2012). A genetic approach to the species problem in wild potato. Plant Biol (Stuttg) 14(4): 543-554.
  2. Cara, N., Ferrer, M. S., Masuelli, R. W., Camadro, E. L. and Marfil, C. F. (2019). Epigenetic consequences of interploidal hybridisation in synthetic and natural interspecific potato hybrids. New Phytol 222(4): 1981-1993.
  3. Cara, N., Marfil, C. F., García Lampasona, S. C., Masuelli, R. W. (2014). Comparison of two detection systems to reveal AFLP markers in plants. Botany 92: 607-610.
  4. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and et al. (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23(21): 4407-4414.

简介

[摘要]甲基化敏感扩增多态性(MSAP)是一种用于分析非模型物种DNA甲基化模式的多功能标记。这种技术的实施不需要参考基因组,并且可以确定分布在整个基因组中的数百个匿名位点的甲基化状态。此外,特定甲基化模式的遗传可以被研究。在这里,我们提出了通过MSAP标记分析马铃薯种间杂交种及其亲本基因型的DNA甲基化模式的协议。

[背景]核苷酸序列并不是基因组信息的唯一形式,DNA甲基化、组蛋白、修改DNA上的组蛋白和核苷酸残基的酶,甚至RNA,都会影响基因的活动,并为细胞提供另一层指令。DNA甲基化、组蛋白、修改组蛋白的酶和DNA上的核苷酸残基,甚至RNA,都会影响基因活动,并为细胞提供另一层指令。表观遗传学变化,也称为表观遗传,可以遗传,并具有重要的表型后果。在植物中,甲基化反应将胞嘧啶残基修饰成5-甲基胞嘧啶。这种表观遗传机制对于维持基因组的完整性是至关重要的,并有助于调节基因在发育过程中的表达以及对生物和非生物胁迫的反应。此外,DNA甲基化的变化是由杂交和多倍体化等基因组冲击引发的,这是植物进化过程中的两个重要现象(Cara et al., 2019)。
有各种不同的方法来研究DNA甲基化的变化。可以使用高效液相色谱法(HPLC)评估全局性的胞嘧啶甲基化,这种分析方法可以量化胞嘧啶和5-甲基胞嘧啶,并计算基因组中甲基化残基的百分比。对于研究基因组上特定位置的DNA甲基化,可以提到两种选择。一种是使用对限制性位点胞嘧啶甲基化具有不同敏感性的异构体。例如,甲基化敏感扩增多态性(MSAP)标记表征来自随机基因组DNA的匿名5′-CCGG序列处的甲基化模式。这是对原始AFLP协议的改编(Voset al., 1995)用HpaII和MspI代替频繁的切割酶MseI。这些酶识别相同的四核苷酸限制性位点(5′-CCGG),但HpaII对完全甲基化(两股甲基化)敏感,并切割半甲基化的外胞嘧啶,而MspI仅对限制性位点的外胞嘧啶的甲基化敏感。另一种研究位点特异性甲基化状态的可能性是使用亚硫酸氢盐测序方法。亚硫酸氢盐对DNA的处理可将胞嘧啶转化为尿嘧啶,而5-甲基胞嘧啶保持不变。然后,通过对双亚硫酸盐和对照DNA的扩增子(即 ,目标基因或启动子)进行测序,就可以区分甲基化和非甲基化的胞嘧啶。随着新一代测序技术的发展,全基因组双亚硫酸盐测序,或WGBS,可以实现推断基因组中所有5-甲基胞嘧啶的位置。然而,这种方法需要一个高质量的参考基因组来进行表观遗传学分析。尽管草案基因组数量的增加和测序成本的降低为在非模式物种中实施大规模甲基化分析提供了可能,但MSAP标记的使用仍然是许多实验室的宝贵工具。
野生马铃薯(Solanum,section Petota)是一组与栽培马铃薯Solanum tuberosum L相关的物种,内部的繁殖障碍可能是不完整的,因此,种间杂交发生在交感区(Camadro et al., 2012).在野生马铃薯物种的合成和天然杂交中,已经记录了响应于种间杂交的表观遗传变化(Cara et al., 2019).Solanum x rechei H. & H.是一个杂交种,与它的野生祖先Solanum kurtzianum B. & W.和Solanum microdontum B.在这里,我们提出了一个协议,通过MSAP标记在杂交种及其亲本基因型中分析DNA甲基化模式。使用R脚本,存在于合成杂交种中的片段被归类为S.microdontum或S.kurtzianum物种特异性,如果它们存在于亲本基因型上,S.x rechei物种特异性,如果它们存在于至少一个S.x rechei评估的基因型中,或者作为新颖的,如果它们仅在合成杂交种中观察到。

关键字:甲基敏感扩增多态性, DNA甲基化模式遗传, 表观遗传变化, 茄属植物, 种间杂交, 等位基因, R脚本

材料和试剂


A. 消耗品
1. 显微镜管(DELTALAB,目录号:4095.5N,4095.9N)。
2. 移液器吸头(DELTALAB,目录号:200072,200016)。
3. PCR微孔板96孔(Axygen,目录号:32165051)
4. 塑料杵(Sigma,目录号:Z359947)


B. 化工产品
1. Tris底座(BIOPACK,目录号:2000166800)。
2. 乙二胺四乙酸(EDTA)(BIOPACK,目录号:2000964500)。
3. 氯化钠(BIOPACK,目录号:2000164606)
4. 十六烷基三甲基溴化铵(CTAB)(Bio Basic INC,目录号:DB0108);
5. β-巯基乙醇(BIOPACK,目录号:2000954500)
6. 氯仿(BIOPACK,目录号:2000165100)
7. 异戊醇(BIOPACK,目录号:2000972500)
8. 乙醇(BIOPACK,目录号:2000165400)
9. 醋酸钠(BIOPACK,目录号:2000168000)
10. RNase A (赛默飞世尔科技公司,目录号:EN0531)
11. 羔羊DNA/EcoRI+HindIII标记(Promega,目录号:G1731)。
12. 甘油(BIOPACK,目录号:2000162000)
13. 溴酚蓝(BIOPACK,目录号:2000962200)
14. 二甲苯氰醇FF(SIGMA,目录号:X4126)。
15. 硼酸(BIOPACK,目录号:2000935900)
16. 琼脂糖(TransGen公司,目录号:GS201)
17. UltraPureTM 溴化乙锭(赛默飞世尔科技公司,目录号:15585011)
18. EcoRI(新英格兰生物实验室,目录号:R0101S)
19. 牛血清白蛋白(BSA)(Promega,目录号:R3961)。
20. HpaII(新英格兰生物实验室,目录号:R0171S)
21. MspI(新英格兰生物实验室,目录号:R0106)
22. T4 DNA连接酶(Promega,目录号:M1801)
23. 寡核苷酸(表1)(IDT,Integrated DNA Technologies,Inc.,Iowa,USA)


表1.所用适配器和引物的序列。
寡核苷酸 序列(5′-3′)
适配器
生态研究所 CTCGTAGACTGCGTACC
AATTGGTACGCAGTCTAC。
HpaII/MspI GACGATGAGTCTCGAT
CGATCGAGACTCATC
预扩增引物
生态研究所+0 GACTGCGTACCAATTC
HpaII/MspI +1 ATGAGTCTCGATCGGA
扩增引物
生态研究所+3 6-FAM-GACTGCGTACCAATTC +3
HpaII/MspI +3 ATGAGTCTCGATCGGA +3。


24. Taq DNA聚合酶(赛默飞世尔科技公司,目录号:11615044)。
25. 氯化镁(赛默飞世尔科技公司,目录号:11615044)
26. dNTP套装(100mM)(赛默飞世尔科技公司,目录号:10297018)。
27. 高地甲酰胺(赛默飞世尔科技公司,目录号:4311320)。
28. GeneScan 500HD Rox尺寸标准(赛默飞世尔科技公司,目录号:401734)。
29. ddH2O(无菌)
30. 萃取缓冲液(见配方
31. TBE(Tris-Borate-EDTA)缓冲液(见配方)。
32. 6倍的DNA装载缓冲液(见配方)。


装备


1. 移液器(BOECO, Wheaton SOCOREX和Finnpipette)
2. Vortex(IKA,型号:MS1)
3. 超速离心机(Eppendorf,型号:5804R)
4. 恒温浴缸(Vicking,型号:Masson)。
5. 琼脂糖凝胶电泳系统(Bio-Rad实验室,型号:1645056)。
6. 微波炉(HITPLUS,型号:CM203M)
7. 分光光度计(AmpliQuant,型号:AQ-07) 
8. 凝胶成像仪(Bio-Rad实验室,型号:Gel Doc 1000)
9. 孵化器(三洋,型号:MIR 262)
10. 热循环器Veriti 96孔(Applied Biosystems,型号:9902)。
11. 基因分析仪(Invitrogen,型号:ABI PRISM 3130)。
12. 超低温冷冻机-80 °C (Forma Scientific, 型号:8270)


軟件


1. GeneMapper v3.7.(Applied Biosystems, Foster City, CA, USA)
2. R v2.15.1。(R统计计算基金会,奥地利维也纳)
3. RStudio v1.1.442 (RStudio Inc., Boston, MA, USA)


流程


A. DNA提取程序
1. 收集50-100毫克的叶组织在1.5毫升离心微管。将组织在液氮中闪冻,并用塑料杵研磨。
2. 加入300微升提取缓冲液,并通过涡旋(5-10秒)大力混合,使组织匀浆。孵育匀浆在65℃下30分钟,在恒温浴中。
3. 加入100微升氯仿:异戊醇(24:1),通过倒置轻轻混合样品。
4. 将匀浆液在4℃下以20,800×g离心5分钟。
5. 通过使用200微升的微量吸管,小心地吸出水相,并将其转移到一个新的微管。
6. 加入500微升冰冷的乙醇:醋酸(24:1),倒置微管2-3次混合样品,沉淀DNA,在-20℃下静置30分钟。
7. 离心样品在20,800 xg,在4℃下离心10分钟,以形成一个DNA颗粒在微管的底部。
8. 弃去上清液,用100微升70%的乙醇清洗颗粒。涡旋管1分钟以清洗颗粒。
9. 将样品在室温下以15,300×g离心5分钟,并将微管倒置在纸巾上30分钟,直到乙醇完全蒸发,或将其置于干燥室中15分钟,让颗粒干燥。
10. 将DNA用RNase A(50 μg-ml-1)重悬于50 µl超纯水中。在室温下孵育30分钟。
11. 将DNA样品储存在4℃,直到使用。如果DNA长时间不使用,请将其保存在-20℃。
12. 通过在0.8%琼脂糖凝胶上装载2μl的DNA溶液,并在90 V下运行30分钟,检查提取的完整性和纯度。它应该显示一个高分子量的厚带,超过21Kbp,没有涂抹( 图1)。
13. 在分光光度计中测量DNA样品的浓度,并制备约100 ng-μl-1的工作溶液。




图1.在0.8%琼脂糖凝胶中可视化的在0.8%琼脂糖凝胶中可视化的DNA样品的代表图像。


B. 用EcoRI消化
1. 根据样品的浓度,计算出对应700 ng DNA的样品体积。但要考虑到DNA浓度应高于40.5 ng-μl-1,否则会超过反应的最终体积。
2. 根据表2计算出样品总数所需的每种试剂的量。为了补偿移液误差,可以考虑在分析样品总数的基础上增加一个样品,这很方便。

表2.用EcoRI酶解的母液用EcoRI进行酶解的主混合物
试剂 每个样本的微升
DNA(700纳克) X
缓冲区10倍 2
EcoRI 20 U/μl (NEB) 0.5
BSA 100x 0.2
超纯水 17.3 – x
最后一卷 20


3. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
4. 分布在各个管中的主混合,并加入DNA。通过倒置或轻弹管子(不要涡流)和离心机10秒,轻轻地均质。
5. 在37℃的培养箱中孵育过夜。
6. 如果您继续进行下一步,请将管子放在冰上。否则,将它们存储在冷冻室在-20℃,直到使用。


C. 用HpaII和MspI消化。
1. 对于上一个反应(B)的每支管子,将内容分配到两支管子中(每支管子中10μl),并在新管子上标上样品代码,在一支管子上加字母"H",在另一支管子上加字母"M"。从现在开始,您将为每个分析样品使用两支试管。
2. 将准备两个母液混合液,只是在限制性酶上有所不同,一个用HpaII,另一个用MspI。对于这两个反应,根据表3计算出样品总数加一个的每个试剂的所需量。

表3.用HpaII和MspI进行酶解的母液。用HpaII和MspI进行酶解的母液。
母带H
试剂 每个样本的微升
EcoRI消化系统 10
缓冲区1 10x 2
HpaII 10 U/μl (NEB) 1
BSA 100x 0.2
超纯水 6.8
最后一卷 20
主混音M
试剂 每个样本的微升
EcoRI消化系统 10
缓冲区4 10x 2
MspI 20 U/μl (NEB) 0.5
BSA 100x 0.2
超纯水 7.3
最后一卷 20


3. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
4. 将母液H分配到标有字母"H"的试管中,将母液M分配到标有字母"M"的试管中。从现在起,每个样品将有两支试管。通过倒置或轻弹试管(不要涡流)轻轻地均质,并离心10秒。
5. 在37℃的培养箱中孵育过夜。
6. (可选)通过运行6μl的限制性产物在1.2%琼脂糖凝胶上,在90V下30分钟,检查这两个消化的效率。它应该显示一个微妙的涂抹从5100到100 bp( 图2)。


 
图2.在1.2%琼脂糖凝胶中可视化的限制性产物的代表图像。在1.2%琼脂糖凝胶中可视化的限制性产物的代表图像。


7. 保持管在冰上,如果你将继续与下一步。否则,将它们存储在冷冻室在-20℃,直到使用。
D. 适配器结扎
1. 准备双链EcoRI和MspI/HpaII适配器从单链寡核苷酸通过添加等量的每个寡核苷酸在管(表1)。将管子放在热循环器中,并用热盖子运行ADAPTORS程序(表4)。


 


表4.PCR程序
适配器 前期工作 扩增 废止
步骤 时间 °T 步骤 时间 °T 步骤 时间 °T 步骤 时间 °T
1 5分钟 95 °C 1 30 s 94 °C 1 30 s 94 °C 1 3分钟 90 °C
2 1分钟 94 °C 2 1分钟 56 °C 2 30 s 65 °C 2 4 °C
3 转到步骤2 x69,每周期降低1℃。 3 1分钟 72 °C 3 1分钟 72 °C
4 转到步骤1x19 4 转到步骤1 x13,每周期降低0.7℃。

4 4 °C 5 4 °C
5 30 s 94 °C
6 30 s 56 °C
7 1分钟 72 °C  
8 转到步骤5 x22
9 4 °C




 
2. 根据表5,计算出样品总数加1的每种试剂的用量。


表5.结扎反应的母液结扎反应的母液
试剂 每个样本的微升
消化 10
适配器 EcoRI 1.25
适配器MspI/HpaII 1.25
10倍连接酶缓冲液 2
T4连接酶3 U/μl (Promega) 0.25
超纯水 5.25
最后一卷 20


3. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
4. 分布在各个管中的主混合,并加入10μl的消化。通过倒置或轻弹管子(不要涡流)和离心机10秒轻轻均质。
5. 室温下孵育3小时。
6. 保持管在冰上,如果你将继续与下一步。否则,将它们存储在冷冻室在-20℃,直到使用。


E. 第一次放大(前置放大)
对于该PCR反应,使用与适配器相同序列的寡核苷酸加1个选择性核苷酸。这些寡核苷酸被确定为引物EcoRI+1和引物H/M+1(表1)。
1. 根据表6计算总样品所需的每个试剂的必要量,考虑到一个额外的样品,以补偿移液误差。


表6.用于预放大的主混音
试剂 每个样本的微升
结扎 2
缓冲区10倍 2
MgCl2 50 mM 0.6
dNTPs 2 mM 1
Primer EcoRI +0 20 μM 0.2
底漆 H/M +1 20 μM 0.2
Taq聚合酶 5 U/μl (Invitrogen) 0.2
超纯水 13.8
最后一卷 20


2. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
3. 分布18微升的主混合在每个管中,并添加2微升的结扎。添加2微升的水到剩余的混合使用它作为阴性反应控制。通过倒置或轻弹(不要涡流)和离心机10秒轻轻均质。
4. 将样品加上阴性对照放入热循环器中,用热盖运行PREAMPLIFICATION程序(表4)。
5. 检查反应的效率,通过加载5μl的预扩增在1.2%琼脂糖凝胶和运行在90 V下30分钟。它应该显示一个强烈的涂片,从1500到100 bp的少数或一些弥漫带( 图3)。




图3.预扩增产品的代表图像在1.2%琼脂糖凝胶中可视化的预扩增产品的代表图像。


6. 稀释预扩增1:3。如果继续进行下一步,请在冰上保存,或在-20℃的冷冻室中保存。预增剂可以安全地保存在冷冻室中几个月。


F. 第二次扩增(选择性扩增)。
对于该PCR反应,使用与适配器相同序列的寡核苷酸加上3个选择性核苷酸。这些寡核苷酸被确定为引物EcoRI +3和引物H/M +3(表1)。
1. 根据表7,计算出样品总数加1所需的每种试剂的必要量。

表7.扩增反应的母液扩增反应的母液
试剂 每个样本的微升
预放大1:3 1
缓冲区10倍 1
MgCl2 50 mM 0.3
dNTPs 2 mM 0.5
Primer EcoRI +3 4 μM 0.5
底漆 H/M +3 20 μM 0.1
Taq聚合酶5U/μl
(Invitrogen) 0.05
超纯水 6.55
最后一卷 10


2. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
3. 在每个管中分配19微升的主混合液,并加入1微升的1:3稀释的预放大。对剩余的混合加入1微升的水,将其作为阴性反应对照。通过倒置或轻弹(不要涡流)和离心机10秒轻轻均质。
4. 放置在热循环器和运行AMPLIFICATION程序(表4)与热盖。
5. 检查反应的效率,通过加载5μl的扩增产物在1.2%琼脂糖凝胶和运行在90 V 30分钟。根据引物组合,它应该显示一个涂片与扩散带从700到100 bp( 图4)。




图4。在1.2%琼脂糖凝胶中可视化的扩增产物的代表图像。


6. 保持管在冰上,如果你继续与下一步,或存储在冷冻室在-20℃。荧光标记的扩增可以安全地存储在-20℃下至少6个月,保持其光发射。

G. 电泳
1. 根据表8计算总样品所需的每个试剂的必要量,考虑一个额外的样品,以补偿移液误差。


表8.扩增反应的母液。扩增反应的母液
试剂 每个样本的微升
扩增 1
Hi-Di 甲酰胺 8.5
GeneScan 500HD ROX (Applied Biosystems) 0.5
最后一卷 10


2. 准备母液混合,通过涡旋(2-3秒)和离心机10秒均质。
3. 分布在96孔板的每个孔中的9微升的主混合,并加入1微升的扩增产物。
4. 将板子放在热循环器中,用热盖子运行程序DENATURATION(表4)。
5. 将盘子从热循环器中取出,放在冰上。
6. 将板放在ABI Prism 3,130 DNA测序仪(Applied Biosystems)中并运行。


数据分析


A. 等位基因调用和矩阵生成
1. 使用GeneMapper v3.7(Applied Biosystems)对所得电子图谱进行半自动评分。分析片段的长度范围为100~500 bp。GeneMapper中使用的参数的完整规格总结在(Cara et al., 2014)中。GeneMapper的报告管理器工具被用来只检索样品名称和等位基因列到逗号分隔值(.csv)文件中。
2. EcoRI/HpaII和EcoRI/MspI消化液之间存在/不存在的模式被编码为0至3;然后,将此编码转换为二进制矩阵,以显示模式3、2或1的存在(1)或不存在(0)(图5)。模式0没有在二元矩阵中编码,因为EcoRI/HpaII和EcoRI/MspI消化液中没有片段可能是由于外部胞嘧啶的甲基化或核苷酸序列的变化。下面的R脚本是为了进行这种分析而开发的。


用于生成MSAP二进制矩阵的R脚本。
##### R命令用于从Genemapper csv文件生成MSAP二进制矩阵。


## 下面的命令可以确保工作内存被清除。
rm(list = ls(all = TRUE))


## 设置工作目录(PATH将具体到数据所在的位置)。
setwd("C:/...")


## 读取数据集。输入文件应包含按行排列的样本和按列排列的位点,包括标题(位点名称)和第一列的样本名称。
## ##每个被HpaII和MspI消化的样品都被标识为"SampleName_H"和"SampleName_M",一前一后。 
Data = read.csv("MSAP_Genemapper.csv", header=T, row.names = 1, check.names=F)


## 创建对象
out <- c()
MSAP_patterns <- matrix(nrow = dim(Data)[2], ncol = 0)


## 这是一个针对j个杂种和i个位点的双循环。它生成MSAP模式矩阵(0、1、2或3)。
for (j in 1:dim(Data)[1]) if (j %% 2 ==1)

for (i in 1:dim(Data)[2])
{
    out <- append(out, ifelse(Data[j,i]+Data[j+1,i]==0, '0',
ifelse(Data[j,i]+Data[j+1,i]==2,'1'。
ifelse(Data[j,i]==1, '2', '3')))
}
MSAP_patterns <- cbind(MSAP_patterns, matrix(out, dimnames = list(colnames(Data), substr(row.names(Data)[j], 1, nchar(as.char.character(row.names(Data)[j]))-2)))
out <- c()
}


##这是一个循环,为i个位点创建一个二进制矩阵,显示每个模式的存在/不存在(1、2或3)。
dummies <- matrix(nrow = dim(MSAP_patterns)[2], ncol = 0)
for (i in 1:dim(MSAP_patterns)[1])
{
dummies <- cbind(dummies, matrix(as.numeric(MSAP_patterns[i,] == 1), dimnames = list(colnames(MSAP_patterns), paste(row.names(MSAP_patterns)[i], "1", sep = "_")), matrix(as.numeric(MSAP_patterns[i,]== 2), dimnames = list(colnames(MSAP_patterns), paste(row.名称(MSAP_patterns)[i], "2", sep = "_")), matrix(as.numeric(MSAP_patterns[i,] == 3), dimnames = list(colnames(MSAP_patterns)), paste(row.names(MSAP_patterns)[i], "3", sep = "_")))
}
binary_matrix <- dummies[,colSums(dummies !=0) !=0] 。


## 将输出保存到工作目录
write.csv(binary_matrix, file = "MSAP_binary_matrix.csv") 




图5.甲基化敏感扩增多态性(MSAP)模式分析。甲基化敏感扩增多态性(MSAP)模式分析。A.从片段扩增图谱和DNA甲基化模式编码推断CCGG限制性位点的甲基化状态。黑色C代表甲基化的胞嘧啶。B.通过对特定甲基化模式的存在(1)或不存在(0)进行编码得到的二元矩阵。

B. 确定特定物种和新型碎片
1. 将在评估的天然杂交种中观察到的扩增模式与合成杂交种及其亲本基因型中获得的模式进行比较。合成杂交种中存在的片段被归类为S. microdontum或S. kurtzianum物种特异性。此外,在天然杂交种中评估了合成杂交种中是否存在新的扩增片段(即存在于杂交种中但不存在于亲本物种S. microdontum和S. kurtzianum的基因型中)。如果新的片段也存在于至少一个被评估的S. x rechei基因型中,则被归类为S. x rechei物种特异性,如果它们仅在合成杂交种中被观察到,则被归类为新颖性。为了进行这一分析,开发了以下R脚本。


用于分析杂交种中MSAP表位遗传的R脚本。
#### R命令用于分析Hybrids中MSAP表位继承。


## 下面的命令可以确保工作内存被清除。
rm(list = ls(all = TRUE))


## 设置工作目录(PATH将具体到数据所在的位置)。
setwd("C:/...")


## 读取数据集。数据的组织形式为:按行排列的样本和按列排列的位点。
Data = read.csv("MSAP_binary_matrix.csv", header=T, row.names = 1, check.names=F)


## 创建对象
out <- c()
results <- matrix(nrow = 4, ncol = 0)


## ##这是一个双循环的j杂交和i位点。行数如下:1-5杂交种,6 S. kurtzianum (Parental1),7 S. microdontum (Parental2),7 S. microdontum (Parental2)。1-5个杂交种,6个S. kurtzianum (Parental1),7个S. microdontum (Parental2),8-15个S. x rechei (自然杂交种)。对这些数字的任何修改应适用于以下命令。
对于(j在1:5中)
{
for (i in 1:dim(Data)[2])
{
    out <- append(out, ifelse(Data[j,i]>0 & Data[6,i]>0 & Data[7,i]==0 & sum(Data[8:15,i])==0, 'A', ## 片段    仅由杂交种和S. kurtzianum共享
ifelse(Data[j,i]>0 & Data[6,i]=0 & Data[7,i]>0 & sum(Data[8:15,i])==0, 'B', ## 片段仅由杂交种和S. microdontum    共享
ifelse(Data[j,i]>0 & Data[6,i]==0 & Data[7,i]==0 & sum(Data[8:15,i])>0, 'C', ## 片段仅由杂交种和S. x rechei    共享
ifelse(Data[j,i]>0 & Data[6,i]==0 & Data[7,i]==0 & sum(Data[8:15,i])==0, 'D', 'E'))))##     ##混血儿特有的碎片
}
 
results <- cbind(results, matrix(table(out, exclude = "E"),dimnames = list(c("Kurtzianum", "Microdontum", "Rechei", "Hybrid"), row.names(Data)[j]))
out <- c()



##将混合数转化为百分比 
百分比 <- t(results)/colSums(results)*100


## 将输出保存到工作目录
write.csv(percentages, file = "MSAP_Hybrids.csv")


## 一个基本的图形表示
barplot(t(百分比), horiz = TRUE, las=1, col = c("红", "蓝", "绿", "黄"))


食谱


1. 萃取缓冲液
100mM Tris-HCl (pH 8.0)
20mM EDTA (pH 8.0)
1.4M氯化钠
0.4% (v/v) β-巯基乙醇。
2%(w/v)CTAB
2. 1x TBE(Tris-Borate-EDTA)凝胶运行缓冲液
89mM Tris碱
89 mM 硼酸
2mM EDTA (pH 8.0)
3. 6x DNA装载缓冲液
30% (v/v) 甘油
0.25% (w/v) 溴酚蓝 (Bromophenol blue)
0.25% (w/v) 二甲苯氰醇 FF


笔记


准备所有的母液,先加入体积较大的,再加入体积较小的,将酶留在最后。


鸣谢


我们感谢 María Virginia Sanchez Puerta 博士对手稿的批判性阅读。这项工作是由Agencia Nacional de Promoción Científica y Tecnológica和阿根廷库约国立大学PICT 1243支持。这个协议是从我们以前的工作(卡拉 et al., 2019)。


竞争性利益


作者没有竞争利益。


参考文献


1. Camadro,E. L.、Erazzu,L. E.、Maune,J. F.和Bedogni,M. C.(2012年)。野生马铃薯物种问题的遗传方法。植物生物学(斯图加特)14(4):543-554。
2. Cara,N.,Ferrer,M. S.,Masuelli,R. W.,Camadro,E. L. and Marfil,C. F.(2019).Epigenetic consequences of interploidal hybridisation in synthetic and natural interspecific potato hybrids.New Phytol 222(4):1981-1993.
3. Cara, N., Marfil, C. F., García Lampasona, S. C., Masuelli, R. W. (2014)。比较两种检测系统以揭示植物中的AFLP标记。Botany 92: 607-610.
4. Vos, P.、Hogers, R.、Bleeker, M.、Reijans, M.、van de Lee, T.、Hornes, M.、Frijters, A.、Pot, J.、Peleman, J.、Kuiper, M.等人(1995年)。AFLP:DNA指纹的新技术。Nucleic Acids Res 23(21): 4407-4414。

登录/注册账号可免费阅读全文
  • 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: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cara, N., Marfil, C. F., Bertoldi, M. V. and Masuelli, R. W. (2020). Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids. Bio-protocol 10(13): e3671. DOI: 10.21769/BioProtoc.3671.
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的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。