*3.5. EST-SSR Amplification and Data Analysis*

The 81 pairs of randomly selected SSR primers were used for preliminary screening, and 10 individuals per population were used to test the primers amplified with clear bands in order to further determine the polymorphism of the SSR primers. PCR amplification was carried out in a 10 μL reaction mixture containing 10–30 ng of genomic DNA, 0.15 mmol/L of each dNTP, 1 μL of 10×Taq buffer, 0.6 mmol/L each of reverse and forward primer, and 1 unit of Taq DNA polymerase (TransGen, Beijing, China). The PCR amplification was carried out using a Bio-Rad C1000 Touch ™ Thermal Cycler (Bio-Rad Laboratories, CA, USA), as follows: 5 min of initial denaturation at 94 ◦C, followed by 30 cycles of denaturation for 30 s at 94 ◦C, a temperature gradient for annealing from 50 ◦C to 60 ◦C for 30 s, and extension at 72 ◦C for 1 min, with a final 10 min at 72 ◦C for final extension. The PCR products were resolved by electrophoresis in 1.5% agarose gels to determine whether amplification was successful. PCR fluorescent tagging and capillary electrophoresis were performed to further screen polymorphisms. The 5 end of each forward primer was labeled with FAM or HEX fluorescent dyes (Thermo Fisher Scientific, Wilmington, DE, USA). The PCR amplifications were performed using the PCR conditions mentioned above. The multiplex DNA products labeled with the above-mentioned fluorescent dyes were analyzed on an ABI 3730xl DNA Analyzer with a GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific, Wilmington, DE, USA), and allele sizes were assessed using GeneMapper 4.1 (Thermo Fisher Scientific, Wilmington, DE, USA). The parameters of genetic diversity including the number of alleles (Na), effective number of alleles (Ne), and Shannon's information index (I)—of these SSR loci were estimated using POPGENE 1.32 [55], and the

allelic polymorphism information content (PIC) for each SSR locus was calculated using CERVUS 3.0 (Field Genetics, London, UK) [56]. Cluster analysis of 6 populations on the basis of shared allele distance (DAS) was performed using POPULATIONS 1.2.30 [57], with the unweighted pair group method with arithmetic mean (UPGMA) method. The clustering tree was visualized and edited using Interactive Tree of Life (iTOL) version 3 [58]. The primers with good repeatability, clear bands, and high polymorphism were selected as candidate primers for molecular genetic diversity research on *L. yunnanensis*.
