PBG431 – ‘Recitation’ Thursday January 29th 2015
The spreadsheet ‘DxB Genotypes.xlsx’ contains marker data for 141 barley DH lines produced from the
F1 of a cross between Derkado and B-83-12/21/5 together with the parents. All the individuals were
scored for two naked eye polymorphisms and two SNP markers. Derkado carries one naked eye
polymorphism (the semi-dwarfing gene sdw1) and B83-12/21/5 carries the other naked eye
polymorphism (the semi-dwarfing gene ari-e.GP). All the data are scored with reference to the parental
alleles that they possess so that Derkado alleles are coded ‘a’ and B83-12/21/5 alleles are coded ‘b’.
The object of the exercise is to test the null hypotheses that all 4 loci are segregating in a Mendelian
fashion and that they are all truly independent via a series of chi-square tests performed in Excel.
Task 1. Produce Graphical Genotypes for the 4 markers
1. Use Conditional Formatting to highlight cells with Derkado alleles in red
2. Use conditional formatting to highlight cells with B83-12/21/5 alleles in green
QUESTION 1. Note down any loci that appear to be associated from the red and green patterns
produced?
Task 2. Produce pairwise scores for each possible combination of markers – already done for you
1. Use a formula of the form ‘=$b3&c3’ and fill down for the first three combinations with SNP
11_21508
2. Repeat for the two combinations with ari-e.GP by starting with the formula ‘=$c3&d3’
3. Repeat for the combination of sdw1 with 11_20392
QUESTION 2. What is the formula to express the number of possible pairwise combinations of n
markers?
Task 3. Produce Punnett Squares for each pairwise marker combination
1. Produce Punnett Squares by writing the following formulae into the pre-existing grids on the
spreadsheet with the top LH corner in e.g. cell F150:
=G2&" a"
=G2&" b"
11_21508 a =COUNTIF(G5:G145,"aa") =COUNTIF(G5:G145,"ab")
11_21508 b =COUNTIF(G5:G145,"ba") =COUNTIF(G5:G145,"bb")
Total
=SUM(G150:G151)
=SUM(H150:H151)
2. Edit these formulae into the appropriate places for the other 5 pairwise combinations
QUESTION 3. Does a visual inspection of the squares lead you to conclude that the two dwarfing genes
are linked?
QUESTION 4. Does a visual inspection lead you to conclude that any of the markers are linked to either
of the dwarfing genes and, if so, which ones?
Task 4. Test the null hypothesis that the segregation of all 4 classes in each Punnett Square is random
1. Use the 4 cells in the Punnett square to produce a chi-square test for independent segregation
by writing the following formulae into a grid with the top LH corner in e.g. cell :
11_21508 a
11_21508 b
Total
(O-E)2/E
ari-eGP a
=(G150-K150)^2/K150
=(G151-K151)^2/K151
(O-E)2/E
ari-eGP b
=(H150-L150)^2/L150
=(H151-L151)^2/L151
2. Sum up the values in the four cells by writing a formula of the form =SUM(O150:p151).
3. Find the probability that the null hypothesis of independent segregation is accepted by writing a
formula of the form: =CHIDIST(Q152,3), where Q152 would be the cell holding your suum from
Task 4.2 and 3 is the degrees of freedom
4. Use Conditional Formatting to highlight if the probability is less than 0.05
5. Repeat for all the remaining Punnett squares.
Task 5. Test the significance of the assortment of parental and recombinant (non-parental)
combinations.
1. Use the values in the Punnett Square to test whether the totals of recombinant allele
combinations are the same as the parentals using the formula below
Obs
Expected
(O-E)2/E
Parentals
Recombinants
=G150+H151
=G151+H150
=I152/2
=I152/2
Chi-Square
=(U150-U151)^2/U151=(V150-V151)^2/V151=SUM(U152:V152)
Probability
=CHIDIST(W152,3)
2. Compare the value of the chi-square from Task 5 with that found for Task 4 to at least 3 decimal
places.
QUESTION 5. Is there a difference between the two chi-square values? If so, what do you think it
represents?
Task 6. Calculate the recombination frequencies for each pair of loci and order if more than two are
linked together.
1. Use a formula in excel to divide the recombinants by the total to estimate the recombination
fraction (r).
2. Produce a triangular matrix of the values for all 6 recombination fractions
3. Deduce the likely order of any groups of more than two loci.
QUESTION 6. Is there a group of more than two loci and, if so, what is it’s likely order (either
orientation)