Download Laboratory #4: Segregation of Traits According to Mendel

Laboratory #4: Segregation of Traits According to Mendel
Background
Gregor Mendel was a monk who studied genetic inheritance using pea plants. Peas are self-pollinating
plants, so after multiple generations (without interference) of self-fertilization they become
homozygous for all their important qualitative genes and are known as a pure line. Mendel crossfertilized different homozygous pea lines to see what would happen.
As a result of his work with peas, Mendel concluded that physical traits are passed from one generation
to the next as discrete units, which we now call genes. Genes are a section of DNA that codes for a
protein. These proteins create particular traits, often through complicated interactions and pathways.
Alleles are possible forms a gene can take. In peas as in humans and other diploid species, there are
two alleles for each gene. These alleles may be the same (homozygous) or they may be different
(heterozygous).
Alleles can be either dominant or recessive. An allele that is dominant will be expressed any time it is
present in either the homozygous dominant or heterozygous state. A recessive allele will only be
expressed if no dominant alleles are present and the recessive allele is in the homozygous recessive
state.
The physical appearance of an organism is its phenotype (wrinkled, smooth, yellow, green). This
appearance is coded for by the alleles present for the gene. The actual alleles and genes that determine
the phenotype are the genotype of a trait, and are illustrated using capital and lower case letters.
Let us consider Mendel’s round pea x wrinkled pea example. In the first generation (F1), all the peas
resulting from a cross between wrinkled and round, were round. Thus the round pea allele (gene) is
dominant to the wrinkled pea allele. Taking a look at the F2 generation of progeny in the smooth
versus wrinkled pea example, we can see that 75% of the progeny are smooth, which also demonstrates
that the round characteristic is dominant over the wrinkled. We can illustrate this by assigning the
capital letter ‘S’ to the dominant allele controlling the round pea and the small letter ‘s’ to the recessive
allele that results in the wrinkled trait. Mendel’s experiment with smooth and wrinkled peas would
look like:
Crossing the parents to create the F1 generation.
Homozygous smooth ‘S’ peas crossed to homozygous wrinkled ‘s’ peas
SS x ss
s
S Ss
S Ss
s
Ss
Ss
100% of the F1 generation have the heterozygous genotype ‘Ss’ and will have the smooth
phenotype as a single copy of the ‘S’ allele is sufficient to attain round peas.
Self-pollinating the F1 generation plants to create the F2 generation.
Heterozygous smooth ‘Ss’ by heterozygous smooth ‘Ss’
Ss x Ss
S
S SS
s
Ss
S
Ss
Ss
From this we have that 25% of the progeny have the ‘SS’ genotype and are hence round (homozygous),
50% have the ‘Ss’ genotype and are also round (heterozygous), and 25% of the progeny have the ‘ss’
genotype and are hence wrinkled. Pulling like phenotypes together we have that 75% (25% + 50%) of
the F2 generation will have the smooth phenotype (genotype ‘Ss’ and ‘SS’) and 25% of the F2
generation will have the wrinkled phenotype (homozygous recessive genotype ‘ss’).
A number of agronomically important characteristics (i.e. yield, protein content, drought resistance,
etc) are not controlled by a single gene but are controlled by many, sometimes thousands. Rather than
produce discrete classes (i.e. round v wrinkled) variation within progeny for quantitatively (many
genes) inherited characteristics show continuous variation. To relate this concept to Mendelian
genetics, if the smooth versus wrinkled characteristic of Mendel’s peas was continuous and therefore
controlled by a number of genes, Mendel would have observed phenotypes intermediate to either of the
parents (i.e. a little bit wrinkled, or almost round) when he crossed smooth and wrinkled pure lines.
This experiment will demonstrate Mendelian segregation using an easy to observe trait (Roundup herbicide resistance). The species that will be used is canola (Brassica napus L.). One of the parents
used in this crossing study had been transformed to have a gene that confers resistance to glyphosate,
the active ingredient in Roundup herbicide. This cultivar (genotype) was crossed to a second genotype
which did not have resistance to Roundup herbicide. The crossing and F1 part of the study was
completed last year.
To complete this genetic study you will be provided with F2 seed from the cross between a glyphosate
(Roundup) resistant parent and glyphosate susceptible parent. You will use the progeny from these
seeds to determine whether the inheritance of Roundup resistance is controlled by a single gene
according to Mendel’s Laws of Segregation. Herbicide susceptibility will be tested by spraying the
plants with glyphosate (Roundup).
Objectives
Observe segregation in Roundup resistant canola.
Determine whether glyphosate resistance is controlled by a single gene.
Materials and Methods
Each group will plant 80 F2 seeds of unknown genotype along with 10 seeds from the susceptible
parent and 10 seeds from the resistant parent in a seedling flat. Three weeks after planting, when plants
are at approximately at the three to four leaf stage, each group will spray their plants with glyphosate
solution. In the following weeks the resistant and susceptible plants will be noted. In two weeks, when
herbicide damage symptoms are clearly visible, results will be compiled as a class.
Calulating Chi-square
It is often necessary to test whether the distribution of progeny we observe from a cross does indeed
follow the frequency expected given a simple Mendelian inheritance. This can be achieved by doing an
appropriate Chi-square calculation. Chi-square calculations compare the expected phenotypic or
genotypic ratio to the observed results of the experiment to mathematically determine the distance
between an acceptable fit for the ratio to the results and the actual fit of the ratio.
The formula to calculate Chi-Square is:
X2obs=Σ[((obss-exps) 2/exps)+((obsw-expw) 2/expw)]
Where obss = Observed number of smooth peas,
exps = Expected number of smooth peas,
obsw = Observed number of wrinkled peas,
expw = Expected number of wrinkled peas.
Expected values are calculated by multiplying the total number of peas by the expected percentage of
the peas that should have the trait (i.e. 75% should be smooth).
X2table is then looked up on a Chi-Square chart. These can be found in any statistics book, and will be
provided for you in a lab handout. When looking up a chi-square value, you must know the amount of
certainty you want and the degrees of freedom. Scientific experiments are typically judged at the 95%
certainty level. The degrees of freedom is the number of independent categories (usually the number of
categories minus one). In Mendel’s pea experiment there are two categories (smooth and wrinkled) so
the degrees of freedom would be one, making the X2 value 3.84.
If the ratio fits the data (in this experiment, if there is a 3:1 ratio of smooth versus wrinkled peas) then,
X2obs < X2table, and the ratio is said to have a good fit. This means the results are as expected based on
the laws of Mendelian genetics.
If the ratio is not a good fit for the data then, X2obs > X2table, the data is said to not fit the 3:1 ratio. This
means that the results of the experiment are not as expected based on the laws of Mendelian genetics.
Assignment
1. What is the difference between a homozygous genotype and a heterozygous genotype?
2. Why would you want homozygous alleles in crops?
3. What was your group's ratio of resistant:susceptible?
4. What was the class' ratio of resistant:susceptible?
5. What are the results of a chi-square test of the resulting ratio?
6. Based on your results is glyphosate resistance a single-gene trait? Why?
7. What did you learn in this laboratory?
8.
How would you change this laboratory?