Calculating Genetic Distances from RAPD Data

The image above shows DNA profiles of the population of var. arizonicus plants from the type locality (plants 4A-6D) for Primer G11. You may also choose to look at the image "Primer G11 4A-14B" to get a better look.

1) Score the DNA profiles for the presence or absence of the band. Only those bands which can be scored as clearly present or absent in each plant should be scored. For example, the 1.74 kb band is only present in plants 4D, 5B, and 5C. Score a "1" for presence, a "0" for absence. Do this for all scorable bands, as below.

 Band (kb) 4-A 4-B 4-C 4-D 5-A 5-B 5-C 5-D 6-A 6-B 6-C 6-D 1.74 0 0 0 1 0 1 1 0 0 0 0 0 1.56 1 1 1 1 1 1 1 1 1 1 1 1 1.21 1 1 1 1 1 1 1 1 1 1 1 1 0.79 0 0 1 1 0 1 0 1 1 1 1 1 0.59 0 0 0 0 0 0 1 1 0 0 0 0 0.53 0 0 0 0 0 0 1 0 0 0 0 0 0.51 0 1 1 1 1 0 0 0 1 1 0 0

NOTE: Band 0.59 may look as though it is faintly present in plants 4A - 5B and 6A - 6D. However, these bands are slightly higher and probably derived from a different locus on the genome.

2) When you have scored all of your profiles for all plants, you can then begin your genetic analysis. Start by doing a pair-wise comparison, i.e. compare each plant to all others. For example, to start, compare plant #4A to plant #4B to determine the number of bands shared. For this primer, plants 4A and 4B share two bands out of a total of seven bands scored. Only count the shared presence of a band, and not the shared absence of a band. Next, compare 4A to 4C, and so on, until you have compared each plant to all of the others. Making a table with all of the plants across the top and the plants down the side will help you keep track of which plants you have compared.

Pair-wise Comparisons Table: Number of Bands Shared Out of 7 Bands

 Band 4-A 4-B 4-C 4-D 5-A 5-B 5-C 5-D 6-A 6-B 6-C 6-D 4-A ----- 2 2 2 ____ ____ ____ ____ ____ ____ ____ ____ 4-B ----- ----- ____ ____ ____ ____ ____ ____ ____ ____ ____ ____ 4-C ----- ----- ----- ____ ____ ____ ____ ____ ____ ____ ____ ____ 4-D ----- ----- ----- ----- ____ ____ ____ ____ ____ ____ ____ ____ 5-A ----- ----- ----- ----- ----- ____ ____ ____ ____ ____ ____ ____ 5-B ----- ----- ----- ----- ----- ----- ____ ____ ____ ____ ____ ____ 5-C ----- ----- ----- ----- ----- ----- ----- ____ ____ ____ ____ ____ 5-D ----- ----- ----- ----- ----- ----- ----- ----- ____ ____ ____ ____ 6-A ----- ----- ----- ----- ----- ----- ----- ----- ----- ____ ____ ____ 6-B ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ____ ____ 6-C ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ____ 6-D ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- -----

3. To calculate the genetic distance of each band to all others you can use the simple equation:

For our example above, the genetic distance between plant #4A ("x") and plant #4B ("y") is:

For this very small number of bands scored, the calculation of genetic distance says that these plants are 33% different or 67% the same genetically. Obviously, the more bands you score and the more plants you have will increase the statistical significance of your calculations. You might use these calculations as a way to introduce the idea of statistical significance. For my research purposes, I will score about 100 bands to obtain statistically significant results.

4) Calculate the average genetic distance between plants within the same population. Use the chart below to determine to which population each plant belongs. Each number represents a different location, while each letter represents a different plant collected from that location. For example, four arizonicus plants (A, B, C, D) were collected from Location #4; they are named 4A, 4B, 4C and 4D.

 Population Plant Numbers arizonicus 4, 5, 6 neomexicanus 13, 14, 15 triglochidiatis 7, 9, 10

Compare the average genetic distance within a population to the average genetic distance between populations. Is there more variation within the population or between different populations?