COMPLETE LISTING OF METHODS
1. 48 Canadian wheat cultivars were obtained from Plant Gene Resources of Canada (PGRC). These cultivars represent the approximate 160 years of wheat cultivar development in Canada.
2. Phenotypic experiments were conducted in a controlled-environment greenhouse. Phenotypic information was collected from 48 cultivars, six plants per cultivar. The 288 plants were examined almost daily for heading and maturity. Upon maturity, plant height was measured and the heads were cut and placed in envelopes allowing further study. I obtained the following phenotypic data by recording:
a. days from planting until the first heading (when 50% of the head is exerted from the boot);
b. days from planting to maturity (all heads on plant);
c. maximum height of the plant from ground to the top of the head (excluding awns);
d. maximum length of each head;
e. number of kernels per head;
f. weight of kernels per head;
g. weight of kernels per plant;
h. number of heads per plant;
i. 100 kernel weight, and
j. whether the heads are awned or awnless.
3. The phenotypic data was entered into an Excel file. SAS (Statistical Analysis System) and statistical packages within Excel were used to analyze the data for correlation coefficients, regression and significance levels.
4. The genotypic information was collected from 288 individual plant samples. DNA was extracted from the leaf tissue of six plants of each cultivar and later grouped.
5. DNA samples were prepared for the Polymerase Chain Reaction (PCR) by diluting the samples to a standard concentration and placing them in a thermocycler.
6. 24 primers were tested on four samples of wheat DNA. The 12 most precise primers (UBC 111, UBC 230, UBC 250, UBC 276, UBC 296, UBC 336, UBC 367, UBC 389, UBC 411, UBC 417, UBC 611 and UBC 626) were chosen to be used in the PCR reaction to determine the polymorphic differences.
7. The RAPD (Randomly Amplified Polymorphic DNA) technique, which separates the DNA strands by heat and recopies the nucleotides thousands of times, was used to amplify the DNA in the thermocycler.
8. Samples were placed in pre-prepared agarose gels in a gel tray and an electric current was used to move the DNA through the gels. DNA has a slight negative charge, so was repelled by the negative electrodes and attracted to the positive electrodes.
9. The gels were placed in ethidium bromide, and photographed in a UV box with a digital camera.
10. Banding patterns were analyzed for polymorphisms by scoring the amount of base pairs in each sample compared to the standard amount.
11. Data was entered in an Excel file and analyzed using correlation coefficient and linear regression and by the UPGMA (unweighted pair-group methods using arithmetic averages) algorithm.