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SSR POPGEN TOOLKIT

A Comprehensive Workflow for SSR Genotyping, UPGMA, PCoA & STRUCTURE

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ssr-popgen-toolkit

ssr-popgen-toolkit is a modular, user-friendly R-based workflow for population genetics using microsatellite (SSR) datasets.
It provides fully automated preprocessing, clustering, ordination, STRUCTURE-format conversion, and publication-ready visualizations.
Works for any diploid species (plants, animals, fungi, insects, etc.).

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FEATURES

• Import SSR matrices from GeneMapper, GenAlEx, or custom formats
• Automated cleaning:
  • averaged duplicated loci
  • missing data handling (–9 → NA → column mean)
  • removal of non-variable markers
• UPGMA clustering (average linkage)
• PCoA (2D, Scree plot, optional 3D)
• STRUCTURE-format conversion
• STRUCTURE barplots (K = 2–8)
• Combined multi-K visualization
• High-resolution export suitable for publication

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DIRECTORY STRUCTURE

ssr-popgen-toolkit/
│
├── scripts/
│   ├── 01_upgma_pcoa.R
│   ├── 02_convert_genealex_to_structure.R
│   ├── 03_structure_plots.R
│
├── ssr_popgen_toolkit.pbs
├── LICENSE
├── README.md
└── example_data/

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INSTALLATION

Using Conda (recommended)

conda create -n ssr_popgen r-base r-dplyr r-tidyr r-stringr \
    r-ggplot2 r-ggrepel r-patchwork r-plotly -c conda-forge -c bioconda
conda activate ssr_popgen

Install ape:

install.packages("ape")

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SCRIPT 1 — UPGMA + PCoA

File: scripts/01_upgma_pcoa.R

Performs:

• Load SSR data
• Average duplicate loci
• Replace –9 with imputed means
• Z-score scaling
• Euclidean distance
• UPGMA dendrogram (PNG)
• PCoA 2D plot
• Scree plot
• Optional 3D PCoA

Usage

Rscript scripts/01_upgma_pcoa.R input.txt prefix

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SCRIPT 2 — GenAlEx → STRUCTURE

File: scripts/02_convert_genealex_to_structure.R

Converts standard GenAlEx SSR tables into STRUCTURE-compatible format.

• Converts diploid alleles
• Handles missing data (NA → –9)
• Outputs clean STRUCTURE file

Usage

Rscript scripts/02_convert_genealex_to_structure.R genalex_input.txt structure_input.txt

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SCRIPT 3 — STRUCTURE BARPLOTS (K = 2–8)

File: scripts/03_structure_plots.R

Produces:

• Sorted STRUCTURE barplots
• Multi-K combined figure (K = 5 → 2)
• Publication-quality PNGs

Usage

Rscript scripts/03_structure_plots.R Qmatrix.txt output_prefix

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PBS SCRIPT FOR HPC

File: ssr_popgen_toolkit.pbs

Example qsub command

qsub -v INPUT=ssr_data.txt,PREFIX=myrun,CONDA_ENV=/path/to/env ssr_popgen_toolkit.pbs

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COMPLETE WORKFLOW OVERVIEW

1️⃣ Export SSR peak data in GeneMapper.
2️⃣ Import to GenAlEx → generate allele matrix.
3️⃣ Run 01_upgma_pcoa.R for clustering + PCoA.
4️⃣ Convert GenAlEx file → STRUCTURE with 02_convert_genealex_to_structure.R.
5️⃣ Run STRUCTURE (K = 1–8).
6️⃣ Upload results.zip to StructureSelector or CLUMPAK.
7️⃣ Identify optimal K using Evanno's method.
8️⃣ Visualize Q matrices using 03_structure_plots.R.

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CITATION — SSR POPGEN TOOLKIT

If you use this toolkit, please cite:

Boutsika, A. (2025). ssr-popgen-toolkit (v1.0.0). Zenodo.
https://doi.org/10.5281/zenodo.17856732

BibTeX

@software{boutsika_2025_ssr_popgen_toolkit,
  author       = {Boutsika, Anastasia},
  title        = {ssr-popgen-toolkit: A Comprehensive Workflow for SSR Genotyping, UPGMA Clustering, PCoA, and STRUCTURE Analysis},
  year         = {2025},
  publisher    = {Zenodo},
  version      = {v1.0.0},
  doi          = {10.5281/zenodo.17856732},
  url          = {https://doi.org/10.5281/zenodo.17856732}
}

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REQUIRED METHOD CITATIONS

STRUCTURE

Pritchard, J.K., Stephens, M., & Donnelly, P. (2000). Genetics 155:945–959.

Evanno ΔK

Evanno et al. (2005). Mol Ecol 14:2611–2620.

CLUMPAK

Kopelman et al. (2015). Mol Ecol Resour 15:1179–1191.

pophelper

Francis (2017). Mol Ecol Resour 17:27–32.

ape (UPGMA tree)

Paradis et al. (2004). Bioinformatics 20:289–290.

ggplot2 / ggrepel

Wickham (2016), Slowikowski (2023).

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LICENSE

This project is released under the MIT License.
See LICENSE for details.