The study of genetic diversity is an important way to understand the genetic variation and relatedness of germplasm resources, which can promote the effective conservation, management, and utilization of medicinal plant species (Soltis and Soltis 1991). E. scandens is the only species belonging to the genus Erythropalum of the family Olacaceae. Field visits and research revealed that most of the E. scandens germplasms were extremely similar in morphology, making it difficult to distinguish between different germplasms. In this regard, DNA molecular markers, one of the most important techniques for examining genetic diversity, can rapidly identify and characterize germplasms that cannot be accurately distinguished phenotypically, regardless of the developmental stage and growing environment of the plant (Shahid et al. 2012). Each molecular marker has its advantages and disadvantages. However, ISSR markers combine the advantages of several molecular markers such as high polymorphism, reproducibility, low cost, ease of use, and no need for genome sequencing (Singh et al. 2021). Therefore, the analysis of the genetic diversity of 34 germplasms of E. scandens from different locations using ISSR markers provided the foundation for the identification of E. scandens germplasm resources for breeding.
Accurate and effective application of ISSR marker technology requires the establishment of a stable and reliable ISSR–PCR amplification (Deng et al. 2019). However, ISSR–PCR amplification is affected by several factors, and each factor exhibits different effects on the system based on the sample (Li et al. 2020; Huang et al. 2011; Mohamad et al. 2017; Jamil et al. 2022).
The widespread use of PCR MasterMix has resulted in a shift from the traditional five-component PCR reaction system (Mg2+, DNA template, primers, DNA polymerase, and dNTPs) to the three-component PCR system (MasterMix, DNA template, and primers), thereby reducing the additional steps and making PCR results highly stable and reliable with enhanced reproducibility (Khodaee et al. 2021; Akhtar et al. 2021; Sheikh et al. 2021). In this study, the optimization of the ISSR–PCR system was performed using a three-component, four-level orthogonal test. Finally, the optimal ISSR–PCR system (20 µL) was established as follows: 1 µL DNA template (60 ng·µL-1), 1.2 µL primer (10 µmol·µL-1), 10 µL MasterMix, and 7.8 µL H2O.
In total, 18 primer pairs were identified from 64 ISSR universal primers using the optimized ISSR–PCR system. The ISSR primer pairs with high polymorphism and adequate stability were used for analyzing the genetic diversity of E. scandens. Thus, in this study, PCR amplification was performed for 34 E. scandens germplasm with different geographical distributions using 18 pairs of ISSR primers, followed by data transformation for genetic diversity analysis. We amplified a total of 121 polymorphic markers (66.12%).
High genetic diversity indexes (H and I) indicated high genetic diversity and an increasingly complex genetic background (Zhang et al. 2011). In addition, the germplasms of E. scandens exhibited H and I values of 0.1946 and 0.2982, respectively, indicating some genetic variation among the germplasms from Guangxi, Guangdong, Hainan, Fujian, and Guizhou regions of China, with high genetic diversity. Furthermore, H and I were higher for Guangxi germplasms than for other regions, according to the statistical results of genetic diversity indexes based on different provinces.
According to the UPGMA clustering analysis of genetic similarity coefficients of the 34 germplasms of E. scandens, the germplasms of E. scandens for Guangxi were classified in the same cluster and could be distinguished from those in Guangdong, Hainan, Fujian, and Guizhou. Subsequently, PCoA was performed using the genetic distances among the 34 germplasms of E. scandens. The coordinates of the germplasm of E. scandens from each province could be roughly distributed together. The germplasms from Guangxi could be separated from those from other provinces. In contrast, the germplasms from Guangdong, Hainan, Fujian, and Guizhou were cross-distributed on the main coordinate map and did not exhibit an obvious distribution pattern, which was consistent with the results of UPGMA cluster analysis. The results of the Mantel correlation test revealed no significant correlation between the geographical and genetic distances among the germplasms. Moreover, the genetic variation among the 34 germplasms of E. scandens was not significantly related to the geographical distance, which was consistent with the results of the genetic diversity of the homolog of E. scandens—Malania oleifera Chun & S.K. Lee (Lai 2006).
The kinship (genetic) analysis can rapidly screen target germplasms with similar genetic backgrounds from numerous germplasm resources, which improves the screening efficiency of good germplasms (Sulima et al. 2017; Gupta et al. 2021) and facilitates the rapid advancement in breeding. In this study, 34 germplasms of E. scandens included two cultivated and 32 wild germplasms. The two cultivated germplasm (E. scandens No. 1 and 2) were obtained from the wild germplasm in Daxin County, Guangxi. E. scandens No. 1 exhibited outstanding growth potential and number of buds, whereas E. scandens No. 2 exhibited distinctive aroma and taste (Zhang et al. 2020). The results of cluster analysis suggested that E. scandens No. 1 could be distinguished from other germplasms from Guangxi, indicating that the genetic background of E. scandens No. 1 was different from those of other germplasms, which may be associated with its high-yield and high-quality germplasm characteristics. Nonetheless, the germplasm E. scandens No. 2 and those from Guangxi Lipu City and Guangxi Hepu County (No. 7 and 8) were clustered into a small category, indicating that the genetic backgrounds of these three germplasms were closely related and may exhibit similar phenotypic characteristics. Therefore, we must focus on the phenotypic traits related to aroma and taste in the two wild germplasms, E. scandens No. 7 and 8.
The medicinal components of E. scandens are primarily attributed to old stems and roots (Xu et al. 2019; Huang et al. 2021). Therefore, phenotypic characteristics, such as root biomass, stem thickness, and medicinal components, must be analyzed. In contrast, edible components are primarily distributed in newly sprouted shoots and branches (Long et al. 2017), which can be associated with phenotypic characteristics such as shoot number, taste, and aroma. The combination of phenotypes in future studies and the database of ISSR markers used in this study can further improve the screening efficiency of excellent germplasms.
The collection of E. scandens germplasm resources is difficult owing to their narrow distribution and severely damaged wild resources. Several locations from where the data were recorded previously are no longer available. Therefore, the ecological conservation of the germplasm resources of Erythronium is necessary. The number of germplasm samples and the level of genetic diversity among germplasms found in the wild in Guangxi were higher than those collected from Guangdong, Hainan, Fujian, and Guizhou. Nonetheless, the germplasms from each region can be propagated and transplanted to enhance the genetic exchange between regions and enrich genetic diversity. However, the samples collected in the present study were limited. Thus, future studies must focus on collecting and analyzing more germplasms to enrich the germplasm resource database. In addition, an extensive analysis of multiple molecular markers should be conducted to comprehensively and accurately evaluate the genetic diversity and relatedness of E. scandens, thereby facilitating the screening of excellent germplasms and meeting the increasing market demands.