Lab 13 – Population Genetics Analysis I

The first part of this lab was performing a gel electrophoresis of our ISSR samples. After obtaining the ISSR strips (17898 and Omar ISSRs were used) assembled last week, three samples from each strip were selected to perform the run. The following samples were selected from strip 1 (17898): PRK01, PRK02, PRK03. From strip 2 (Omar), samples PRK13, PRK14, PRK15 were selected. 1 µl of loading dye was transferred to each of the six samples using a p10.

The samples were loaded into the appropriate ISSR gel tray. The gels ran at 60 volts for 1.5 hours.

Next, an ITS alignment was assembled in Geneious utilizing forward and reverse reads of the ITS marker for 41 individuals of Lupinus arboreus collected from 13 geographic regions.

An assembly was constructed using the forward and reverse sequences for each individual. Once the assembly was complete, the sequences were edited. Unsuccessful regions at the beginning and at the end were trimmed and incorrect base calls and ambiguous bases were amended.   Then, edited consensus sequences were extracted. These steps were repeated for all 41 individuals.

An alignment was assembled using the 41 consensus sequences extracted in the previous step. The alignment was edited by trimming the ends so that sequence lengths were in agreement. The edited ITS alignment was used to construct a phylogenetic tree using the MrBayes tool in Geneious. The MrBayes analysis was run using the following parameters: Chain length was set as 1,500,000; Burn-in Length was set at 100,000; HKY85 was selected as the substitution model; and 500 was set for the Subsampling Frequency.

The following phylogenetic tree with support values was generated.

Below are the posterior distribution and the trace from the MrBayes run.

The tree included one clade with a support value of 0.9895. The individuals of this clade were PRD05, PSF03, GWC01, and GWC02. PRD05 was located at Drakes Beach in Point Reyes, PSF03 was obtained from Presidio, GWC01 and GWC02 were obtained from Grey Whale Cove State Beach.  PRD05 was collected from a mixed lupine plant. PSF03, GWC01, and GWC02 were sampled from a yellow flowered lupine plant.

Based on the individuals represented within each clade of the phylogenetic tree, it can be concluded that the individuals are found in geographically close populations, rather than in the same population. Some individuals with purple flowers were found in the larger clade, where most yellow flowered individuals were grouped. In addition, not all purple flowered individuals were grouped in the same clade. Individuals from purple flowered samples (PHO, PHT, and PRD) were present in the largest clade and in a clade with a support value of 0.9805 or 0.7472.

As previously mentioned, the internal transcribed spacer (ITS) is found in fungi and was utilized as a primer to perform PCR reactions with DNA extracted from the leaflets collected from lupine plants. In assessing the phylogenetic relationships of the Lupinus populations, it appears that ITS does not provide enough resolution to distinguish populations phylogenetically. Some samples of either the purple or yellow flowered individuals were not grouped together in the same clade. If these samples were present together, then ITS could have been successful at the population level.  Finally, the presence of the polytomy indicates phylogenetic uncertainty, in that we cannot determine how the individuals are related.

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