For studies with many accessions (more than 50), we usually run a dry gel apparatus based on the design that was developed in Jeff Palmer's lab. For smaller studies, we use submerged gels. Results from both types of gel apparatus are similar. For each gel we use three 1 kb DNA size ladders (Gibco BRL) -- one on each side and one in the middle. We also use marker taxa from the other gel sets to make our comparisons between gels more robust.
At least two replicate experiments are conducted for each primer/taxon pair. The results from replicate gels are compared. If a band is present on one gel (for a particular accession), but not the other, that band is eliminated from the data set. We haven't had to discard many data points in that the replicability of these experiments is high.
We do not run ethidium bromide in the agarose, but stain and destain gels after the bromophenol blue marker has migrated 10 cm. We capture the gel images with a digital camera, saving the file in a TIFF format. We then use the BioMax 1D software package for scoring and sizing band fragments.
The data matrix is compiled from spreadsheets imported from each BioMax 1D analysis (one per gel). Adjustment of loci is necessary to compile the data matrix. For example, BioMax 1D doesn't line up the loci from different lanes according to size. If one lane has three bands and the next lane has four bands, manual adjustment is necessary to collate the loci from the two lanes into order by fragment size.
Analysis of ISSR data is dependent on the type(s) of question(s) addressed in a particular study. My lab has been involved with studies of hybridization, conservation biology, and systematics. Most studies have common themes in data analysis, but there are specific types of analyses unique to some studies. The following examples are not all-inclusive, but should serve as a starting point for similar studies.
|1.||Import data into a spreadsheet with rows = loci and columns = taxa. Arrange fragment sizes by locus. Convert the actual fragment sizes to 1's and the blanks to 0's. Reduce the size of the cells and page layout to get as much readable information as possible on one page to reduce the number of printed pages that need to be taped together.|
|2.||Examine data for species-specific and population-specific bands (especially for pure populations outside the hybrid zone if conducting a study on hybridization) by highlighting bands present for a locus with a specific color. We usually rotate six colors down the spread sheet. Representing the data in this fashion allows for a quick assessment of # bands/primer, # bands/taxon, bands shared, bands present in all accessions, % polymorphic bands, etc.|
|3.||If the number of accessions and/or populations is large, condense the information from each accession into a summary spreadsheet for each population and/or each taxon. For example, the rows are the same as in the previous spreadsheet, but the information in the column cells contains the number of accessions in the population with a particular band. The highlighting color scheme is the same. This approach makes assessing species specific and population-specific patterns much easier than examining the raw data.|
|4.||Format data for computer programs that estimate distance and/or similarity. This usually means transposing the rows and columns. In Excel, this is done by the "Paste Special" command. Run data through algorithm to generate a triangular matrix of distances for use in PHYLIP or some other program that can generate trees based on phenetic measures (e.g., UPGMA, neighbor-joining). Software information is listed under the "Resources" section of this manual.|
|5.||Print tree and examine for anomalous placement of taxa -- accessions with a lot of missing data will fall out basal in the diagram if the clustering algorithm was UPGMA. Go back and examine the raw data to decide whether the accession should be kept, discarded, or if more data gathering should be attempted.|
|1.||Calculate: # species-specific bands (potential marker bands); # population-specific bands; # marker bands shared by taxa in pairwise comparison -- may need to consider patterns of sympatry if hybridization is known to occur (i.e., if there are F1's present). See Wolfe, Xiang and Kephart (1998a, b) for examples.|
|1.||Determine number of genotypes present. If information on the accession transects was collected, examine correlation between genotypes and the physical arrangement of individuals in the study population(s).|
|2.||Calculate measures of genetic diversity (e.g., % polymorphic loci, average similarity, Fst and Nm).|
|1.||Components of the previous two types of studies are useful in studies of systematics.|
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