Pipeline for developing polymorphic microsatellites in species without reference genomes

Microsatellites, also known as simple sequence repeats (SSRs), are the preferred type of marker for many genetic applications. In conjunction with the ongoing development of next-generation sequencing, several bioinformatic tools have been developed for identifying SSRs from genomic or transcriptomic sequences. Although these tools are handy for generating polymorphic SSRs, their application almost always depends on an existing reference genome or self-assembly of the reference genome. With this in mind, we propose a pipeline for developing polymorphic SSRs that may be applied to species without reference genomes. Using a species without a reference genome (black Amur bream; Megalobrama terminalis Richardson, 1846) as a model, our pipeline was able to effectively discover polymorphic SSRs. Under different R parameters of a reference-free single nucleotide polymorphisms (SNPs) caller (ebwt2InDel), a total of 258, 208, 102, and 11 polymorphic SSRs were mined. To quantify the accuracy of the polymorphic SSRs detected using our pipeline, we analyzed 25 SSRs with PCR experiments. All primers were successfully amplified, and most SSRs (23 SSRs, 92%) were polymorphic. From the 36 individual black Amur bream, we acquired an average of 3.36 alleles per locus, ranging from one to 11. This demonstrates the effectiveness of our pipeline in identifying polymorphic SSRs and designing primers for SSR genotyping. Ultimately, our pipeline can effectively mine polymorphic SSRs for species without reference genomes, complementing SSR mining approaches based on reference genomes and helping to resolve biological issues that accompany these methods.

Automated summary

Microsatellites, also known as SSRs, are widely used markers in genetic applications. This study presents a pipeline for discovering polymorphic SSRs in species without reference genomes, using black Amur bream as a model. The pipeline successfully identified polymorphic SSRs and designed primers for SSR genotyping, providing an alternative approach for mining SSRs in non-model organisms.