Genetic patterns observed among populations by neutral molecular markers is a major method used to reconstruct the invasion history of invasive species. The Shimofuri goby (Tridentiger bifasciatus) has invaded several lakes along the East Route of the South‐to‐North Water Transfer Project, China (ENST). However, neutral molecular markers for this goby are not currently available. In this study, fourteen novel polymorphic microsatellite loci were developed through next generation sequencing technology. These markers were also tested in an invasive population (Nansi Lake, a storage lake of ENST) consisting of 54 individuals. The average number of alleles per locus was 7.6 and ranged from 4 to 13. The average Ho was 0.587 and ranged from 0.404 to 0.852, and the average He was 0.733 and ranged from 0.564 to 0.851. The average PIC per locus was 0.689 and ranged from 0.521 to 0.824. Four markers (TB4, TB65, TB66 and TB26) were found to deviate from HWE after correction following the False Discovery Rate method. Null alleles were found in four loci (TB11, TB65, TB66 and TB26). These highly polymorphic microsatellite markers are useful for further study to reconstruct invasion history of Shimofuri goby.
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