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A rapid and cost-effective approach for the development of polymorphic microsatellites in non-model species using paired-end RAD sequencing

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Abstract

As one of the most informative and versatile DNA-based markers, microsatellites have been widely used in population and conservation genetic studies. However, the development of microsatellites has traditionally been laborious, time-consuming, and expensive. In the present study, a rapid and cost-effective “RAD-seq-Assembly-Microsatellite” approach was developed to identify abundant microsatellite markers in non-model species using the roughskin sculpin Trachidermus fasciatus as a representative. Overlapping paired-end Illumina reads generated by restriction-site-associated DNA sequencing (RAD-seq) were clustered based on the similarity of reads containing the restriction enzyme recognition site and then assembled into contigs, which were used for microsatellite discovery and primer design. A total of 121,750 RAD contigs were generated with a mean length of 522 bp, and 19,782 contigs contained microsatellite motifs. A total of 156,150 primer pairs were successfully designed based on 16,497 contigs containing priming sites. Experimental validation of 52 randomly selected microsatellite loci demonstrated that 45 (86.54%) loci were successfully amplified and polymorphic in two geographically isolated populations of T. fasciatus. Compared with traditional approaches based on DNA cloning and other approaches based on next-generation sequencing, our newly developed approach could yield thousands of microsatellite loci with much higher successful amplification rate and lower costs, especially for non-model species with shallow background of genomic information. The “RAD-seq-Assembly-Microsatellite” approach holds great promise for microsatellite development in future ecological and evolutionary studies of non-model species.

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Acknowledgements

We are thankful to the anonymous referee for critical and helpful comments on the manuscript.

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Author notes

  1. Dong-Xiu Xue and Yu-Long Li contributed equally to this work.

Authors and Affiliations

  1. CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071, Shandong, China

    Dong-Xiu Xue, Yu-Long Li & Jin-Xian Liu

  2. Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, Shandong, China

    Dong-Xiu Xue, Yu-Long Li & Jin-Xian Liu

Authors
  1. Dong-Xiu Xue
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  2. Yu-Long Li
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  3. Jin-Xian Liu
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Correspondence to Jin-Xian Liu.

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All authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Funding

This study was funded by the National Natural Science Foundation of China (NSFC) (Grant number 31502169), AoShan Talents Program supported by Qingdao National Laboratory of Marine Science and Technology (Grant Number 2015ASTP-ES05), the NSFC-Shandong Joint Fund for Marine Ecology and Environmental Sciences (Grant Number U1606404), and AoShan Science and Technology Innovation Plan (Grant Number 2015ASKJ02-05).

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Communicated by S. Hohmann.

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Xue, DX., Li, YL. & Liu, JX. A rapid and cost-effective approach for the development of polymorphic microsatellites in non-model species using paired-end RAD sequencing. Mol Genet Genomics 292, 1165–1174 (2017). https://doi.org/10.1007/s00438-017-1337-x

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  • DOI: https://doi.org/10.1007/s00438-017-1337-x

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