Pipeline for developing polymorphic microsatellites in species without reference genomes.

Area of Science:

• Genetics
• Bioinformatics
• Genomics

Background:

• Microsatellites, or simple sequence repeats (SSRs), are crucial genetic markers.
• Existing SSR identification tools often require a reference genome, limiting their application.
• Developing SSRs for species without reference genomes presents a significant challenge.

Purpose of the Study:

• To propose and validate a novel bioinformatics pipeline for identifying polymorphic SSRs in species lacking a reference genome.
• To demonstrate the pipeline's effectiveness using the black Amur bream (Megalobrama terminalis) as a model organism.

Main Methods:

• A reference-free pipeline was developed for SSR discovery.
• The pipeline utilized a reference-free single nucleotide polymorphisms (SNPs) caller (ebwt2InDel).
• Polymorphic SSRs were identified and validated using PCR experiments and genotyping.

Main Results:

• The pipeline successfully identified polymorphic SSRs in black Amur bream.
• PCR validation confirmed high accuracy, with 92% of tested SSRs being polymorphic.
• Genotyping revealed an average of 3.36 alleles per locus, indicating marker utility.

Conclusions:

• The developed pipeline is effective for mining polymorphic SSRs in species without reference genomes.
• This approach complements existing reference-genome-based methods.
• The pipeline facilitates genetic marker development and aids in resolving biological questions for non-model organisms.