*2.3. SSR Primer Screening and Verification*

A total of 9195 primer pairs were designed using Primer 3.0, and 81 primer pairs were randomly selected to amplify across the genomic DNA template of *L. yunnanensis*. Of the 81 primer pairs, 44 of them successfully amplified fragments from *L. yunnanensis* genomic DNA, with a success rate of 54.32%. Therefore, the primers developed and designed in this study have a certain applicability to the population of *L. yunnanensis*. According to the preliminary screening results, 10 individuals were randomly selected from each population (a total of 60 individuals from 6 populations) to further verify the polymorphism of the 44 primer pairs that could amplify bands. Among them, 17 primer pairs were found to be polymorphic in *L. yunnanensis*, producing clear amplicons of the expected size, which were suitable for the detection of SSR loci by capillary electrophoresis. The specific information of 17 EST-SSR primers is shown in Table 5. A dendrogram clustered the 6 populations of *L. yunnanensis* into 2 clusters (Figure 5). One cluster (I) included 2 populations (GDX and GCG) of Gongshan County and one population (FMM) of Fugong County. The other cluster (II) included 2 populations (LCM and LLO) of Lushui County and one population (FPK) of Fugong County. Geographically, FMM is close to Gongshan County and FPK is close to Lushui County, which is consistent with the results of cluster analysis.


**Table 5.** The information of 17 EST-SSR primers of *L. yunnanensis*.

Na = number of different alleles; Ne = number of effective alleles; *I* = Shannon's information index; PIC = polymorphism information content; *Ta* = annealing temperature.

The SSR marker diversity index was estimated using POPGENE 1.32; the effective number of alleles (Ne) and Shannon's information index (I) per locus varied from 1.298 to 2.690 (average of 1.925), and from 0.476 to 1.239 (average of 0.837), respectively. Using CERVUS 3.0 (Field Genetics, London, UK), we calculated that the allelic polymorphism information content (PIC) for each SSR locus ranged from 0.223 to 0.580, with an average of 0.403. Botstein [45] put forward a criterion that primers are highly polymorphic when PIC > 0.5, moderately polymorphic when 0.5 > PIC > 0.25, and low-grade polymorphic when PIC < 0.25. Most of the 17 EST-SSR primer pairs were moderately or highly polymorphic, indicating that these primers could be used for genetic diversity analysis and fingerprint construction of *L. yunnanensis*.

Today, plant diversity is gradually decreasing on a global scale, leading to a related decline in ecosystem service function [46]. Many rare species, such as *L. yunnanensis*, have seldom been studied, and lack protection due to a lack of social awareness. Thus, the numbers and distribution ranges of the species are shrinking. Using transcriptome sequencing technology to study the genetic diversity of *L. yunnanensis* can quickly and efficiently develop mass EST-SSR markers. The application of these markers is conducive to quickly supplementing the molecular biological information of this species, and provides a theoretical basis and scientific evidence for research on the genetic resources, conservation, and utilization of *L. yunnanensis*. In addition, due to the high ornamental value of *L. yunnanensis*, we can refer to Ding's [47] study on *Chrysanthemum*, in which the development of SSR markers for genes related to the regulation of flower shape and color were studied, and these SSR markers were applied to the classification of more than 100 *Chrysanthemum* germplasm resources with different flower colors and shapes. In the same way, we could also screen the SSR loci of important ornamental traits such as the flower shape and color of *L. yunnanensis*, which could lay the foundation for breeding new varieties of *L. yunnanensis*.

**Figure 5.** Cluster analysis of 6 *L. yunnanensis* populations based on 17 EST-SSR markers.
