Sassafras tzumu (Hemsl.) Hemsl., as an Arctic Tertiary relict woody species, is an ecologically and economically important deciduous tree endemic to southern China. Nonetheless, the genetic resources and backgrounds of S. tzumu are still lacking and remain largely unclear. Here, we predicted 16,215 candidate polymorphic nuclear microsatellite (nSSR) loci from the assembled nucleus databases of six geographic-distant individuals of S. tzumu via CandiSSR. Among these nSSRs, the di- (75.53%) and tri-nucleotide (19.75%) repeats were the most abundant, and 27 new polymorphic SSRs were developed and characterized in 136 individuals from six natural populations of S. tzumu. The majority of the above 27 SSRs (24 loci, 88.89%) presented moderate polymorphism (mean PIC = 0.356), and the transferability of these markers in other Sassafras species was high (85.19%). A moderately low level of genetic diversity and a high variation (F_ST = 0.286) of six wild populations of S. tzumu were illuminated by 16 selected polymorphic nSSRs, with the average expected heterozygosity (H_E) of 0.430 at the species level and H_E ranging from 0.195 to 0.387 at the population level. Meanwhile, a bottleneck effect was shown in two populations. Consistent with the results of the principal coordinate analysis (PCoA) and phylogenetic trees, structure analysis optimally divided these six S. tzumu populations into two clusters, and the further strong population subdivision appeared from K = 2 to K = 5, which corresponded to two evolutionarily significant units (ESUs). Moreover, the significant correlation between genetic and geographic distance was tested by the Mantel test (r = 0.742, p = 0.006), clarifying the effect about isolation by distance (IBD), which could be possibly explained by the low gene flow (N_m = 0.625), a relatively high degree of inbreeding (F_IS = 0.166), a relatively large distribution, and mountainous barriers. Above all, our research not only enlarged the useful genetic resources for future studies of population genetics, molecular breeding, and germplasm management of S. tzumu and its siblings but also contributed to proposing scientific conservation strategies and schemes for the better preservation of S. tzumu and other Sassafras (Lauraceae) species.

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