Population Epigenetics: Methods and Protocols

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Methods and Protocols

Zhang, M. Genomics 37, 1— Wibowo, A. Dubin, M. Steward, N. Wada, Y. Genomics , — Choi, C. Secco, D. Verhoeven, K. New Phytol. Silvertown, J. Plant Sci. Evolution 68, — Bossdorf, O. Kronholm, I.

Alligator weed, Alternanthera philoxeroides , is an invasive clonal plant and has been considered as an ideal species for ecological epigenetics, in which epigenetic variation can be studied independently from genetic variation. Richardson, Melbourne, Australia. Wang, R. Weed Sci. Sainty, G. Sosa, A. International, Wallingford, UK. Geng, Y. Invasions 9, — Julien, M. Longstreth, D. Plant Cell Tissue Organ Cult. Naqvi, S.

Eberbach, P. Plant Manag. In this study, we used alligator weed as a model to investigate epigenetic variation in response to salinity stress. Both seedlings and mature plants of different genotypes were exposed to high salinity to evaluate epigenetic modification in response to environmental stress. Amplified fragment length polymorphism AFLP and methylation-sensitive amplification polymorphism MSAP markers were used to assess genetic variation and methylation variation, respectively. Three key questions of this study are as follows.

First, can salinity stress induce significant DNA methylation changes? Second, if so, are these changes consistent across different genotypes?

Epigenetics

Third, how are epigenetic changes affected by stress duration and plant developmental stage i. Alligator weed is a clonal plant species and most individuals in China share one single multi-locus genotype. However, there is considerable genetic diversity within natural populations in other areas e.

The aim of this study was to examine the effect of salinity stress on epigenetic variation in different genotypes of alligator weed. We also wanted to know whether any such effect depends on plant developmental stage. Thus, we used a factorial design with three factors: genotype six genotypes , salinity stress treatment vs. There were five replications for each treatment combination, totally including plant individuals. The pots were labeled and then watered with tap water every two days. Next, when the young seedlings were established and had produced two or three pairs of leaves, 20 healthy individuals for each genotype individuals in total were randomly assigned to four different treatment groups i.

The plants in Control groups were watered as above with tap water throughout the experiment, while the plants in the Stress-Young group were exposed to salinity stress from the very beginning of the experiment by adding ml NaCl solution 0. In contrast, the plants in the Stress-Mature group were grown under control conditions for one month i.

The salinity stress lasted for 30 days, and then all plants were switched to control conditions for one week. Newly produced and fully expanded leaves were collected for epigenetic analysis. After sampling, all plants were harvested at the end of the experiment. For genetic background analysis, two leaves were collected and dried in sealed plastic bags with silica gel. For epigenetic analyses, leaves were collected from the Young groups and Mature groups under different schedules. Specifically, for the Young groups, epigenetic samples were collected from newly produced leaves after 30 days of stress duration.

For the Mature groups, epigenetic samples were collected at two time points i. There were DNA samples in total.

The AFLP technique was used to determine the genetic background information for each genotype. Nucleic Acids Res. Gao, L. Plant Cell Environ. MSAP fingerprinting was used to examine the epigenetic variation of alligator weed in response to salinity stress. Schulz, B. To assess reproducibility and minimize experimental error, MSAP reactions were repeated twice for each sample using independent DNA isolations.

Visually poor-quality samples were excluded from scoring and only reproducible fragments were scored. All scoring was performed by the same person in the absence of information on sample identities. The AFLP results were scored as a binary matrix following a conventional protocol. The MSAP results were somewhat more complicated. The first three conditions represent different methylation status while the last indicates a complicated state that could reflect either methylation variation or DNA sequence mutation.

Here, we used clonal offspring as the plant material, and presumed that there was no genetic variation within each genotype.

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Accordingly, we included the last fragments status epi-loci type: 00 as part of the data sets and considered these fragments as methylated. Population genetic software for teaching and research-an update. Bioinformatics 28, — The biomass data were log-transformed if necessary to meet assumptions of homoscedasticity, and all data were analyzed with R statistical software version 3. A total of AFLP bands were scored from the nine primer combinations. The AFLP results showed that each genotype had a unique multi-locus profile. In contrast, for each genotype, the five clonal offspring were genetically uniform, as revealed by AFLP markers.

These data indicated that the clonal offspring of each genotype were genetically homogeneous and that all MSAP polymorphisms within genotypes can be interpreted as true epigenetic variation rather than genetic mutation. Salinity stress had a significant effect on plant growth i. Above- and below-ground biomass of young plants and mature plants. Different superscript letters indicate means that are significantly different at P Fig. Methylation status change after salinity stress in mature plants treated for 30 days. The color code indicates the different methylation states epi-loci type of MSAP profile.

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Note that the numbers of loci were variable in each genotype. We found that epigenetic modification in young plants was less sensitive to salinity stress than that in mature plants. In this study, we used six different genotypes to examine epigenetic change in response to salinity stress. We did find some evidence that different genotypes had contrasting sensitivity to salinity stress in plant growth and epigenetic modification. Different superscript letters indicate means that are significantly different at P Numbers and percentage of changed and stable epi-loci after salinity treatment in mature plants treated for 30 days.

Once the tissue cells are in suspension, fluorescent markers that attach only to the cell or cells of choice can be used to isolate them. Drilling down to the single-cell level is tricky, however, and may require more advanced microfluidics, especially with smaller numbers of cells.

Disassociating cells into suspension loses information about where they are positioned in tissue samples.

Epigenetics

With these tools, researchers scan across thinly sliced tissues, locate the cell of interest, and punch it out of its surroundings. The cell can be lifted out with a sticky film and dropped onto a slide or into a tube by gravity or by using a pulse from the laser to catapult it to the correct spot. One drawback is the risk of nicking your cell and losing cytoplasm containing RNA, or bisecting the cell during the slicing process and cutting away part of the nucleus, Voet cautions.

The rarest cells, however, can only be harvested manually using a capillary tube and pipetting. Once the cell has been isolated, the epigenetic marks can be assessed. Thus far, single-cell epigenetics allows only one type of assay at a time.


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You will need to decide if you are looking at marks attached to single nucleic acid residues, at histone modifications, or for changes at the chromatin level. Such marks usually indicate regions of the genome that have been silenced during early development as cells become specifically differentiated. Researchers have long been able to tease out which bases are methylated using bisulfite sequencing, a rather harsh chemical method that swaps each unmethylated cytosine for a uracil.

After PCR enrichment, the changes are then easily detected using next-generation sequencing. In the past few years, several groups have scaled that technology down to analyze methylation in single cells. First they had to overcome the large loss of genetic material caused by the harsh bisulfite treatment. Instead of fragmenting DNA first, as is typical, they did the bisulfite conversion on single-cell lysates and then performed an amplification step five times to generate multiple copies of the DNA fragments created during treatment Nat Methods , , The method is fairly hands-on, but not much harder than regular bisulfite sequencing.

The main thing to consider is that each step along the way is a potential spot for something to go wrong. Run plenty of negative controls, Kelsey says. This will also help you narrow in on problem points when you are troubleshooting your technique. The rest of the DNA is discarded.

Plant epigenetics : methods and protocols - Ghent University Library

That also means the assay produces results faster, making it suitable for IVF embryo screening, for example. It could also be applied to looking at other rare cells, such as adult stem cells in intestinal crypts. Every effort should be made to keep your DNA in good shape. For the finer points of the protocol, watch out for an updated methods paper from his group, currently in press in Nature Protocols.

Still, with practice, the marks are easy to detect under the microscope, and the technique can quickly produce data from a few hundred cells. Hiroyuki Sasaki of Kyushu University in Japan and his colleagues developed a way to see if specific DNA sequences were methlyated or not in single cells Nucleic Acids Res , e, Their methlyation-specific in situ hybridization MeFISH binds fluorescent probes to methylated cytosines. Notably, it can distinguish between 5-methylcytosine, which just carries a methyl group, and 5-hydroxymethylcytosine, another important type of epigenetic mark. The bisulfite-sequencing—based methods described above cannot distinguish between the two.

In addition, all the regulatory proteins that ramp up or tamp down gene transcription are physically located on the DNA. In the single-cell technique, these reactions take place in intact nuclei as opposed to the normal procedure, which uses cell lysates. Further characterization is then performed using nuclei isolated from individual cells.

To study chromatin-level changes, researchers use chromosome-conformation capture techniques in cell populations, but these methods only give an average 3-D shape. Laue is part of a group that has successfully developed a way to quantify the twists and folds of chromosomes in single cells. The method hinges on the work of Takashi Nagano, a researcher at the Babraham Institute who figured out how to scale down the steps of the conventional Hi-C protocol.