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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.

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Availability of data and materials

The genome sequence data supporting this study's findings are available in GenBank of NCBI at [] under the accession number OM982470 to OM982494. The associated BioProject number is PRJNA813998. Other sequence read archives were downloaded under the BioProject numbers PRJNA756243, PRJNA640946, and PRJNA688781. The reference genome of Acanthopagrus latus was downloaded under the Assembly number GCF_904848185.

Code availability

All codes related to the current pipeline are included as supplementary material.


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This work was supported by China Agriculture Research System under Grant CARS-45-39; Science and Technology Innovation Program of Hangzhou Academy of Agricultural Sciences under Grant 2019HNCT-01.

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KL and NX conducted the experiments; KL analyzed the data and wrote the manuscript; All authors read and approved the final manuscript.

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Correspondence to Kai Liu.

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The authors declare that they have no conflict of interest in the publication.

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The approval from the Science and Technology Bureau of China and the Department of Wildlife Administration is not required for the experiments conducted in this paper when the fish in question are neither rare nor near extinction (first- or second-class state protection level). According to Measures of Zhejiang Province on Administration of Laboratory Animals, ethical approval was not required, because the approval is only necessary when researchers will use mammals.

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Liu, K., Xie, N. Pipeline for developing polymorphic microsatellites in species without reference genomes. 3 Biotech 12, 248 (2022).

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  • Polymorphic
  • Microsatellites
  • Simple sequence repeats
  • Nucleotide insertions and deletions
  • Reference genome