Abstract：Abstract Phoebe sheareri is one species of "Nanmu with golden tint", which is recognized as a vulnerable species in China. The existing natural populations of P. sheareri is only scattered in the southern region of the Yangtze River, and the wild resources are gradually reducing now. In order to further study genetic diversity and genetic structure of natural populations in P. sheareri, it was imminent to develop numerous EST-SSR markers. In the present study, transcriptome data of P.sheareri was obtained by high-throughput sequencing technology. The SSR loci were screened and the EST-SSR markers were developed, as well as the preliminary validation and the transferability within Phoebe species was conducted. The results showed that a total of 43 781 unigenes were obtained using de novo assembly, and 6 958 SSR loci were identified using MISA, at a frequency of 15.89%, with one SSR locus occurred in per 5.31 kb. The length and number of SSR repeats were largely different, the most abundant SSR length was 15~19 bp, accounting for 49.05% of the total repeat types，and the most abundant repeat number of SSR motif was 4~8 times, accounting for 60.25%. A total of 340 repeat types of SSRs were identified, dinucleotide and trinucleotide were the dominant repeat types, representing as AG/CT and AAG/CCT, accounting for 23.99% and 12.81%, respectively. Ninety-four primer pairs from typical types were synthesized, which were verified by PCR amplification, with genomic DNA from 10 P. sheareri samples. The transferability of SSR markers within Phoebe species was also conducted, using the genomic DNA from four Phoebe species. The results showed that 41 primer pairs could yield the expected PCR products, of which 32 primer pairs were polymorphic. The number of alleles was averagely 2.75 per locus. The average value of observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information content (PIC) were 0.369, 0.563 and 0.541, respectively. These markers also showed high transferability within Phoebe species. In conclusion, the unigenes generated from transcriptome data of P.sheareri can be used as an effective source to development EST-SSR markers. The EST-SSR markers could be used in genetic diversity analysis, genetic map construction and germplasm resources protection for P.sheareri and other Phoebe species.
