Mendel's Mutant Peas I-III

2009 Protocol

Mendel’s mutant peas I-III

 

Overview

 

During the next three weeks we will perform experiments to characterize an uncharacterized line of short mutant peas.  We will take two approaches, one that does not require any information about the gene (or genes) that are mutated and a second which does.  During the first two weeks we will perform a bioassay to determine which portion of the gibberellin signaling pathway may be affected in your peas.  During the second and third weeks we will use a PCR based genotyping assay to determine whether the peas are short because of the same mutation that made Mendel’s peas short.  These experiments will form the basis of a full lab report. 

 

Background

 

One of the traits that Gregor Mendel used to elucidate the fundamental concepts of genetics was stem length.  He referred to this as the Length (Le) trait.  Some of his pea plants had long stems and were tall (Le) while others had short stems and were short (le)[1].  Mendel figured out the genetics of these height differences but the tools and knowledge necessary to understand the molecular basis of these differences were not yet available.  Since then the Le gene has been cloned and shown to encode gibberellin 3b-hydroxylase, an[j2]  important enzyme in the gibberellin biosynthetic pathway (Lester et al., 1997). 

 

Gibberellin (also known as gibberellic acid or GA) is a plant hormone[2] with many effects on plant growth and development.  GA has been shown to play roles in the transition from vegetative to reproductive growth, in seed germination, and in cell expansion in stems.  GA was first identified by E. Kurosawa in the 1920’s from an extract of the fungus Gibberella fujikuroiG. fujikuroi causes a disease in rice called foolish seedling disease.  Infected plants grow very tall and have spindly stems which cannot support the plants in wind or once they set seed (if they survive that long).  Purified GA was shown to cause stem elongation not only in rice but in many other plants including dwarf varieties of many important agricultural crops (Hedden, 2003).

 

During the ‘green revolution’ in the 1960’s dwarf varieties of many cereal crops were developed.  These dwarves had higher yields than their tall counterparts for several reasons.  The plants put less energy into growing stems and more energy into their fruits (grains).  The shorter stems were sturdier and so were less susceptible to lodging (being blown over by the wind) and could also support more grain weight.  The breeding programs which produced these dwarf varieties, as well as increased use of fertilizers and pesticides, resulted in large yield increases in many important staple crops including wheat and rice.  Some of these dwarf plants responded to application of GA by growing to their normal heights, while others were GA insensitive (Taiz and Zeiger, 2006).

 

Because these crops are such important sources of food a lot of research has been performed in order to understand how GA is synthesized and how GA affects plant growth and development.  This research is ongoing; the GA receptor, which took decades to identify, was only cloned in 2005 (Ueguchi-Tanaka et al., 2005).  The results of many years of research can be simplified as a linear signal transduction pathway (Fig. 1).  Gibberellin precursors which are not biologically active are converted into a bioactive gibberellin (gibberellin A1 or GA1).  GA1 binds to the GA receptor resulting in the degradation of a protein that represses the transcription of GA responsive genes.  The expression of these genes results in GA responses at an organismal level. 

Figure 1.  A simplified GA signaling pathway.  Chemical precursors are converted into bioactive GA1 by GA biosynthetic enzymes (arrow A). GA1 binds to the GA receptor (B) which then activates a signal transduction pathway (C) which results in stem elongation and other GA responses.  Arrows (-->) represent activating interactions, bars (--|) represent repressive interactions.

As a result of this signal transduction pathway there are two categories of GA dwarves: 

-       GA responsive dwarves.   These dwarves have mutations in the biosynthetic portion of this pathway (ie. the conversion of GA precursors to GA1, Fig. 1A).  This results in plants with lower levels of bioactive GAs than wild type plants.  Since the receptor and signal transduction pathway in these mutants are functional, spraying plants with GA can ‘rescue’ the mutant phenotype.  Le encodes a GA biosynthetic enzyme and le mutants can be rescued with a GA spray.

-       GA insensitive mutants have mutations in the receptor or the pathway downstream from the receptor.  No matter how much GA is sprayed on the plant, there is no response because the GA signal is either not perceived (receptor mutation, Fig. 1B) or cannot be transmitted (signal transduction mutation, Fig. 1C).

 

The experiment

The late Dr. G.A. Marx was a pea geneticist at Cornell University who amassed a large collection of pea varieties (~80,000!).   Several hundred of these are labeled as lel; , however, there are no records detailing how he determined that these varieties are true le mutants as opposed to being short for another reason.  Because of this uncertainty, we will refer to these mutants as le* mutants.  In this lab we will investigate why uncharacterized le* mutants are short.  We will use known Le and le plants  as controls for our experiments.

 

Based on our knowledge that Le encodes a GA biosynthetic gene we can generate several testable hypotheses about the behavior of le mutants and we will perform experiments to test two of these hypotheses: 

 

1) le mutants should be GA responsive, and

2) le mutants will have a change in the DNA sequence at the Le locus.

 

[Can you think of any other testable hypotheses about the basis of the stature of these plants?]

 

Before you begin collecting data you should have a clear understanding of each hypothesis you plan to test and generate an appropriate experimental design to test each. 

 

In the first two weeks of this lab we will treat pea seedlings with GA3 to test if the le* lines are GA responsive.  GA3 is a commercially available gibberellin which is quickly converted into active GA1 by plants.  If the mutation in the le*  lines is in fact due to a lesion in the Le gene then a GA3 spray should result in a rescue of the short stem phenotype.   In the second and third weeks of the lab we will isolate DNA and use it to genotype the plants for Mendel’s le allele.

 

Week

GA response

Genotyping

1

Discuss experimental design

Measure plant height before spray

Spray plants with GA3

Discuss experimental design

2

Measure plant height after spray

Pool data

Make DNA

Set up PCR

Set up restriction digest

3

Discuss results

Pour and run gel

Document and discuss results

Materials

The following materials will be available in lab: 

GA response:

2 pots of Le peas planted 6-7 days ago (6 pea plants/pot)

2 pots of le peas planted 6-7 days ago (6 pea plants/pot)

2 pots of le*  peas planted 6-7 days ago (6 pea plants/pot)

Stakes, tape, and Sharpies to label the pots

Rulers, scissors, and gloves

10-4M GA3 and a surfactant in a spray bottle

Water and the surfactant in a spray bottle

 

Genotyping:

DNA extraction buffer

Eppendorf tubes

Blue pestles for grinding tissue

Isopropanol

1x TE

PCR master mix

PCR strips

Pipettors and pipette tips

Centrifuges

Agarose gels, gel rigs, buffers, and power supplies

 

Read through this lab handout before the lab starts.  You will discuss how you will design your experiments as a lab group.  Sketch your design ideas in your lab notebook and check your design with an instructor before you begin.

 

Although we have made the GA3 solution for you, go through the exercise of calculating how much GA3 (in grams) you would need to make 100ml of a 10-4M GA3 solution (molecular weight 346.38.)  Write down your calculations in your lab notebook.  Are you surprised by the amount of hormone needed to make this solution?

 

GA response week 1

 

1. Label your pots with tape on their sides as well as on a stake (in case the stakes get knocked out of the pots in the greenhouse).  On the labels, indicate your group, the lab section you are in, and the genotype of the peas in the pot.  We will treat one pot of each genotype with a GA spray; the other pot of each genotype will be used as a control.

 

2. Measure and record the height of each seedling in your pots, measuring from the soil to the top of the seedling.  Keep track of which measurement is from which pea plant.  You will need this data to determine how much your peas grow over the next week.

 

3. Count the number of leaves on each seedling.  Peas have compound leaves so make sure that you know what a single pea leaf looks like.  Keep track of which measurements are from which plants.  You will need this data to determine how many leaves your peas made over the next week.

 

4. Take your pots of peas for each spray treatment to the appropriate spraying area.  The GA3 and water control sprays will be along the back bench.  Wear gloves and be careful not to spray yourself with the spray.  Spray the plants thoroughly making sure to wet the stems and the leaves completely. 

 

5. Return the pots to the trays they came in.  They will be returned to the greenhouse where they will receive care and attention until next week.  Next week you will be measuring how much the plants grew with various treatments.  Feel free to visit your peas in the greenhouse during the week.  Visiting hours are while Bill Pinder, the greenhouse manager is in (generally from noon until 4pm every day).

 

Before next week, start thinking about how you will decide if the GA treatments have any effect.  To get you started:

 

-       Calculate average heights and leaf numbers for the plants you treated today. 

-       Graph these averages in a bar chart.  Are the tall plants actually taller than the short plants?  How do you know this? 

 

We will use a statistical test called the t-test to determine if the heights in our treated and untreated plants are statistically different.   The t-test will be discussed more while your gels are running in week 3.

 

GA response week 2

 

This week in lab we will measure the growth of the pea seedlings that were treated with GA3 last week.   Your peas have been growing since last week in the greenhouse attached to Martin Hall.  They will be returned to you in lab so that you can observe the effects that the GA treatment and control spray had on the growth of the peas.  You will measure the height and the number of leaves on each plant as you did last week.

 

1. Retrieve your peas from the cart.

 

2. Measure and record the height of each seedling in your pots, measuring from the soil to the top of the seedling.   Keep track of which measurements are from which plants.

 

3. Count the number of leaves on each plant.  Remember that peas have compound leaves so make sure that you know what a single pea leaf looks like.   Keep track of which measurements are from which plants.

 

 

GA response week 3

 

Once you have measured the pea plants, we will test some of hypotheses we generated about the data last week using the t-test and present these data in a table.  This will introduce you to the methods you will use in your writing assignment.  The data will be used to test hypotheses about the effects of these compounds using a statistical test called the t-test, which[j3]  is used to determine whether the means (averages) of two samples are different. 

 

The t-test is a statistical test that is used to determine the probability that the average values of two samples are the same.  See the Statistics Appendix for a brief overview of t-tests and hypothesis generation and testing[3].  We will be calculating the t statistic based on the instructions provided in the Statistics Appendix.  The t-test was designed to be used with small sample sizes, and so is well suited for the analysis of our pea plant growth data[4].  Based on our calculated t statistic and degrees of freedom of our test (related to sample size) we will use Student's t distribution to determine the P-value, which represents the probability that two sets of data compared in our test are the same. 

 

In class last week you were asked to define your experimental questions.  For example

one question might be ‘are dwarf peas treated with GA taller than those treated with water? ‘  The t test cannot "prove" that the two samples of peas (treated with GA or water) have different mean stem lengths.  Instead the t test provides the probability that the two samples have the same mean stem lengths.  This theoretical possibility is called a "Null Hypothesis.".

 

To determine the probability that this null hypothesis is correct, you will enter your data in the online t-test calculator (which can be found on the Bio 1 Blackboard site in the Lab Resources section under Mendels Peas).  Enter your data, click the ‘calculate now’ button, and record the P value, mean heights, and standard deviations (SD) for each t-test you perform.  You will need these data for your lab write up.

 

The P-value is the probability that the average values of the two samples of data that you entered are actually the same.  The significance is an arbitrary measure, with a commonly accepted value of 0.05 being considered ‘significant..  Remember that a P-value of 0.05 means that you would find similar differences between samples once every twenty experiments (5%) you analyze.  Another way of saying this is that a small P-value (5% or less) means that there is only a small chance that the two samples are actually from the same population,; thus we can be confident that a real difference exists. Also keep in mind that while a ‘significant’ difference may be statistically supported, it may have very little biological relevance.

 

The null hypothesis will be rejected if the P-value is less than 0.05.  For instance, if the P-value for the heights of the control and GA-treated dwarf plants is <0.01 then there is only a one in a hundred chance that the stem lengths are actually the same but we happened to measure the tallest plants in the GA-treated pots and the shortest plants in the control pots.

 

How might we express this in a written report?  One example could be, "The mean stem length of the GA-sprayed dwarf pea plants was significantly greater than the mean stem length of plants sprayed with water (Table 1) after one week of treatment."

 

In our example sentence above we referred the reader to a table.  Figures or tables provide the reader with a clear presentation of your results.  Figures and tables must contain a table heading or a figure caption that is descriptive enough that the reader can understand your results without ever reading the rest of your paper. 

 

For your report we expect you to conduct unpaired t tests for each experimental question related to stem length (height) using compiled data for each pea.  For leaf count data we expect that you will present descriptive statistics only (mean, SD, n).  Why do you think we are not recommending that you conduct t tests for leaf count data? We expect that all data will be presented in tables in your write up.

 

An explanation of dCAPS genotyping

Cleaved Amplified Polymorphic Sequence (CAPS) genotyping is a technique for differentiating between two alleles (ie wild type and mutant) containing a single nucleotide polymorphism (SNP) using PCR and restriction enzyme digests.  If the SNP results in the presence of a restriction enzyme site in one sequence and not the second, by amplifying the region of DNA around the SNP and subsequently digesting it with the appropriate restriction enzyme, the amplified and digested DNA from the allele containing the site will be cleaved while the DNA from the allele missing the site will not.  The cleaved product will consist of two DNA fragments that are smaller than the uncleaved PCR product.  This difference in size can be detected by separating the cleaved PCR products by electrophoresis on an agarose gel (Figure 2).  If the SNP does not create a restriction site difference then a difference can be introduced by one of the PCR primers, a technique called derived CAPS or dCAPS (Figure 3).  The PCR primer is designed to include a mismatch so that the PCR products from one allele will contain a restriction site while the products from the other allele do not contain the site.  A program that will design a mismatched primer based on two polymorphic sequences can be found at http://helix.wustl.edu/dcaps/dcaps.html (Neff et al., 2002).  

Figure 2.  dCAPS genotyping results for BOB1, an Arabidopsis gene.  The primers used in this genotyping assay introduce an EcoRI site into wild type but not mutant PCR products.

Lane 1 – 100bp ladder

Lane 2 – bob1/bob1 mutant individual (no EcoRI site)

Lane 3 – BOB1/bob1heterozygous individual

Lane 4 – BOB1/BOB1 wild type individual (PCR product is cut by EcoRI)

Note – in a dCAPS reaction, the smaller cleaved band is too small to be visualized (it is the same size as the primer used, normally 20-25bp).

Figure 3.  An example  of a dCAPS assay.  CRY1 refers to the wild type allele of CRYPTOCHROME1, an Arabidopsis blue light receptor.  cry1-102 is a mutant allele of CRY1, 102 is the allele designation.  From (Neff et al., 2002).


dCAPS genotyping protocol week 2

 

1. Label an eppendorf tube with the genotype of each of your pea lines.  Use the lid of the tube as a punch to collect a leaf disk from each line.  Make sure that you do not cross contaminate your samples.

 

2. Add 500ml of DNA extraction buffer and grind each sample with a separate blue pestle until the extraction buffer turns green.

 

3. Spin at maximum speed for 1 minute in the centrifuge.  Label a new set of tubes and transfer 250ml of the supernatant (making sure not to disturb the pellet) to the new tubes.  Remember to change the pipette tip between each sample to avoid DNA cross contamination.  PCR = amplification of tiny amounts of DNA!

 

4. Add 250ml of isopropanol to each tube.  Close each tube, invert gently five times, and let the tubes sit on your bench for 2 minutes.  Genomic DNA should become visible as white strands in the solution.

 

5. Centrifuge your tubes at maximum speed for 5 minutes.  A pellet of DNA should be visible in the bottom of each tube.

 

6. Carefully remove as much of the solution as you can from each tube without disturbing the DNA pellet.  Invert the tubes over a paper towel and air dry for ten minutes.

 

7. Resuspend each pellet in 100ml of TE and store your DNA on ice.

 

From this point on keep all of your DNA samples and other reagents on ice at all times.

 

8. Label an empty eppendorf tube with ‘Master Mix’.  Each 20ml PCR reaction will contain 1ml of pea DNA and 19ml  of master mix.  Calculate how much master mix you will need for all of your PCR reactions (remember to set up a negative control with no DNA).  Add an additional 10ml to this volume to ensure that you don’t run out in the event of minor pipetting errors and using a filter pipette tip get this amount of master mix from a lab instructor.  The master mix contains water, PCR buffer, dNTPs (nucleotides), primers, and Taq polymerase. 

 

9.  Label a PCR strip with your group and record which sample will go in which tube.  Since it is easier to load your gel in the order that you load your samples into the strip, think about the order you want your samples to be on the gel.  Using a filter tip transfer 19ml of master mix to each tube you are going to use.  Using fresh filter tips add 1ml of the appropriate DNA sample or water to each reaction.  Make sure that the DNA or water is added directly to the master mix.  Tightly seal the strip with the strip cap and give your labeled strip to a lab instructor.

 

10.  The lab instructors will start your PCR reactions in class[5].  When the reactions are finished they will add 10ml of HhaI[6] reaction mix (containing water, buffer, and HhaI) to each tube and the tubes will be transferred to a 37oC incubator overnight.  After the digest is complete your samples will be transferred to the -20oC freezer until week 3.

  

dCAPS genotyping protocol week 3

 

We will use ethidium bromide (EtBr) to visualize the DNA in the gels.  EtBr binds to DNA (including yours) and is thus considered to be a potential carcinogen and should be treated with great care.  Wear gloves whenever you touch gels, gel rigs, or the transilluminator and discard your gloves in a biohazard bin after contact with materials that are potentially contaminated with EtBr.

 

Agarose gel electrophoresis involves high voltage electrical currents. 

Never put anything in a gel rig or remove a gel from a gel rig unless the power supply has been turned off.

 

1. Pour a 3.5% Metaphor agarose TBE gel.  Instructions will be provided in lab.

 

2. Add 6ml of 6x gel loading buffer to each reaction tube.

 

3. When the gel has set up transfer it to the gel rig and pour in enough TBE buffer to completely cover the gel.  Load 10ml of 100bp ladder in the first lane and 20ml of each sample in separate lanes.  Make sure that you write down which sample was loaded in each lane!

 

4. Run the gels at 100v until the dye front has migrated ~2/3 of the way to the bottom of the gel. 

 

5. Remove the gel from the gel rig and take a picture of your DNA using the transilluminator.  Print out a copy of your picture using the printer and save the image so that you have an electronic copy for inclusion in your lab report.

 

6. Record the genotype of all of the samples in your lab notebook.

References:

 

Hedden, P. (2003). The genes of the Green Revolution. Trends Genet 19, 5-9.

Lester, D.R., Ross, J.J., Davies, P.J., and Reid, J.B. (1997). Mendel's stem length gene (Le) encodes a gibberellin 3 beta-hydroxylase. The Plant cell 9, 1435-1443.

Neff, M.M., Turk, E., and Kalishman, M. (2002). Web-based primer design for single nucleotide polymorphism analysis. Trends in Genetics 18, 613-615.

Taiz, L., and Zeiger, E. (2006). Plant Physiology. (Sinauer Associates).

Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M., Itoh, H., Katoh, E., Kobayashi, M., Chow, T.-y., Hsing, Y.-i.C., Kitano, H., Yamaguchi, I., and Matsuoka, M. (2005). GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437, 693-698.

 

 

 




[1] In most genetic nomenclatures capitalized gene names represent dominant alleles and lower case names represent recessive alleles.  For this lab we will follow this convention and designate the dominant (and tall) allele as Le versus  le for the recessive (and short) allele.

[2] Hormone – hormones are chemicals that are produced in one part of an organism and have biological effects in other parts of the organism.  Hormones produce their effects at very low concentrations.

[3] You can also find a good science specific t-test tutorial as part of the Really Easy Statistics Site (http://tinyurl.com/3xqycc) which also has a good overview of other statistical tests.

[4] The t-test assumes a Gaussian (normal) distribution of data, which we will assume we have for this experiment

[5] PCR conditions are: 40 cycles, 60oC  annealing temperature, 20 second extensions.

PCR primers sequences are:                le-HhaI-R 5’-TGTCGTGCAATATGATGAAACCATGA -3’

                                                                        le-HhaI-F 5’-TGGTTAAAAATGTTGAGTCTGTGTGCGGCG-3’

 

[6] The primers we use introduce a HhaI site into PCR products amplified from wild type but not from le mutant alleles of gibberellin 3b-hydroxylase.


 [j1]Which and that appear throughout this part of your manuscript, also.

 [j2]You may have to check with Dr. Jim Clack, the editor, to determine how to retain formatting in your manuscript.

 [j3]Be consistent in your use of t-test or t test.