Evaluating the genetic diversity of Erythropalum scandens based on using inter-simple sequence repeat markers

Erythropalum scandens Blume, an emerging medicinal plant with great potential for drug development, also possesses high edible value. In this study, we investigated the genetic diversity of the germplasm of E. scandens obtained from different geographical locations using inter simple sequence repeat (ISSR) markers. For this purpose, 18 ISSR primer pairs with a distinct background and adequate polymorphism were selected. We established an optimal ISSR–PCR reaction system (20 µL) with the following parameters: 1 µL DNA template (60 ng µL−1), 1.2 µL primers (10 µmol µL−1), 10 µL MasterMix, and 7.8 µL H2O. A total of 183 loci were amplified using the 18 primer pairs, of which 121 (66.12%) indicated polymorphism. Moreover, 34 germplasms of E. scandens exhibited genetic similarity coefficients ranging from 0.7104 to 0.9563, genetic distances ranging from 0.0447 to 0.3420, Nei’s genetic diversity index of 0.1946, and Shannon’s information index of 0.2982, suggesting high intraspecific genetic diversity. UPGMA cluster and PCoA analyses distinguished the germplasm of E. scandens obtained from Guangxi from those collected from Guangdong, Hainan, Fujian, and Guizhou. However, the Mantel correlation analysis revealed that the genetic variation among the 34 germplasms of E. scandens was not significantly related to geographical distance. The analysis of the genetic background of wild and cultivated germplasms of E. scandens can help guide variety selection and breeding. Furthermore, the present study revealed the genetic background and affinities among 34 germplasms of E. scandens. Overall, our findings lay the foundation for the conservation and utilization of germplasm resources, identification and classification of varieties, and variety selection and improvement of E. scandens at the molecular level.


Introduction
Erythropalum scandens Blume, a perennial vine of the genus Erythropalum belonging to the family Olacaceae, is native to the tropical and subtropical regions of the world.In China, E. scandens is primarily distributed in Guangxi, Guangdong, Yunnan, and Hainan (Zhang et al. 2020).According to the Flora of China (Chinese Flora Committee 1988), E. scandens is a medicinal and edible plant, its stem possesses Abstract Erythropalum scandens Blume, an emerging medicinal plant with great potential for drug development, also possesses high edible value.In this study, we investigated the genetic diversity of the germplasm of E. scandens obtained from different geographical locations using inter simple sequence repeat (ISSR) markers.For this purpose, 18 ISSR primer pairs with a distinct background and adequate polymorphism were selected.We established an optimal ISSR-PCR reaction system (20 µL) with the following parameters: 1 µL DNA template (60 ng µL −1 ), 1.2 µL primers (10 µmol µL −1 ), 10 µL MasterMix, and 7.8 µL H 2 O.A total of 183 loci were amplified using the 18 primer pairs, of which 121 (66.12%) indicated polymorphism.Moreover, 34 germplasms of E. scandens exhibited genetic similarity coefficients ranging from 0.7104 to 0.9563, genetic distances ranging from 0.0447 to 0.3420, Nei's genetic diversity index of 0.1946, and Shannon's information index of 0.2982, suggesting high intraspecific genetic diversity.UPGMA cluster and PCoA analyses Vol:.( 1234567890) diuretic properties and can be used to treat jaundice, rheumatism, and ostealgia, whereas its roots dispel edema and bruises.The antigout effects of the stem and leaf extracts of E. scandens have been associated with the promotion of uric acid metabolism, antiinflammation, and protection or improvement of renal function (Xu et al. 2019).Furthermore, coumarins, flavonoids, phenols, triterpenoids, polysaccharides, and other phytochemicals contained in the alcoholic extracts of E. scandens can significantly reduce serum uric acid and creatinine levels, thereby enhancing renal function, promoting uric acid excretion, and reducing uric acid levels in the body.The alcoholic extracts of E. scandens also exhibit low acute toxicity and exert significant inhibitory and protective effects in both rat and mouse models of hyperuricemia.Therefore, this species is an excellent potential alternative for the treatment and prevention of hyperuricemia (Pan et al. 2020;Huang et al. 2017).
In addition to its therapeutic applications, the young leaves of E. scandens are used as a vegetable with a pleasant aroma and unique flavor and can be consumed as fresh, fried, stuffed, boiled in soup, congee, and pickled.Moreover, the nutritional value of its edible shoots is high, with protein, fat, fiber, vitamin B1, vitamin B2, and vitamin C contents comparable to those of common nutritive vegetables such as lettuce, choy sum, mustard, water spinach, sweet potato leaves, pumpkin seedlings, boxthorn leaves, and cauliflower.E. scandens also exhibits high levels of minerals, such as copper, zinc, iron, calcium, and phosphorus, and its calcium content is as high as 1040 mg kg −1 , making it a calcium-rich vegetable (700-2300 mg kg −1 ) (Huang et al. 2021).
In recent years, the demand for E. scandens has also been increasing owing to its widely recognized edible and medicinal values.However, the supply of seedlings of E. scandens is insufficient to meet the present market demands, and the breeding technology is still evolving.As a result, growers primarily rely on cutting wild branches and or whole wild-type plant for propagation, which severely damages the wild resources and environment of E. scandens (Huang et al. 2021).Furthermore, varieties suitable for edible and medicinal purposes have not yet been identified and developed.In addition, few reports are available on the germplasm resources of E. scandens, resulting in inefficient germplasm collection, conservation, and seedling breeding.Therefore, genetic-diversity studies on the germplasm of E. scandens obtained from different geographical locations are necessary to improve the collection and conservation of germplasm resources, evaluation, and efficient utilization, as well as the selection and breeding of superior varieties to establish the theoretical basis for the breeding and improvement research programs on E. scandens.
Molecular markers-one of the most important tools used for analyzing genetic diversity-have been widely used in several plant species.The most commonly used molecular markers include simple sequence repeat (SSR), inter SSR (ISSR), start codon-targeted polymorphism (SCoT), and random amplified polymorphic DNA (RAPD) markers (Bernard et al. 2020;Sirangi et al. 2020).Of these, ISSR markers, which are used for amplifying simple, repetitive sequences, exhibit several advantages, such as high polymorphism, adequate stability, low cost, and simple operation (Shi et al. 2010;Li et al. 2020).They have been successfully applied for analyzing the genetic diversity of plants such as Cassia tora (Vikas and Krishna 2018), sand ginger (Subositi et al. 2020), and Dendrobium huoshanense (Tikendra et al. 2019).

Sampling
The samples were obtained from 34 germplasm resources (Table 1).The geographical distribution of the germplasms is shown in Fig. 1.The phenotypes of two cultivated germplasm (E.scandens No. 1 and 2) are shown in Fig. 2.
In this study, we used the ISSR primers UBC800-864 out of the 100 primers published by the University of British Columbia, Canada (http:// www.biote ch.ubc.ca/ servi ces/ naps/ prime rs.html).

Genomic DNA extraction
Genomic DNA was extracted using a modified CTAB method (Safeena et al. 2021) that had revised 'chloroform: IAA (24:1)' to 'phenol: chloroform: IAA (25:24:1)', and its quality was examined using 0.8% agarose gel electrophoresis.The concentration and purity of DNA were determined using an ultra-micro UV-Vis spectrophotometer (Thermo NanoDrop One, America).Subsequently, high-quality DNA was diluted to appropriate concentrations for PCR amplification, and the remaining DNA was stored at − 20 °C.

Optimization of the ISSR-PCR amplification
The three factors affecting the ISSR-PCR amplification, including the amount of template, primers, and 2× ES Taq MasterMix (CW0690M; Kangwei Century Biotechnology Co. Ltd.China), were optimized using the L9 (34) orthogonal test table (Table 2).The PCR (20 µL) conditions were as follows: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s (35 cycles), and extension at 72 °C for 5 min.The results of the orthogonal test were analyzed using intuitive analysis (He et al. 1998).Finally, the number and definition (clarity) of amplified bands were scored to identify the most suitable reaction conditions.

Primer screening
We performed the screening of 64 ISSR primers for clear and high polymorphism bands using temperature gradient PCR machine programmed with annealing temperatures of 49 ℃, 51 ℃, 53 ℃, 55 ℃, 57 ℃, and 59 °C.

PCR amplification
The reaction conditions for ISSR-PCR amplification was as follows: initial denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing for 30 s (annealing temperature of primers), extension at 72 °C for 30 s (ramp temperature of annealing to extension is 5 ℃/s), final extension at 72 °C for 5 min, and held at 4 °C.Finally, the results were analyzed using agarose gel electrophoresis.

Data processing
The number and position of DNA bands were converted into binary data (0 and 1), where the presence or absence of bands at the same locus with the same mobility was recorded as "1" and "0," respectively, to form a (0, 1) matrix.Thereafter, the genetic diversity indexes were calculated and clustering analysis was performed for 34 germplasms of E. scandens using PopGene 32, GenAlEx 6.51b2 and STRU CTU RE software.Finally, the correlation between geographical location and germplasm was determined using the RStudio software.

Results
Quality and quantity of genomic DNA extracted from E. scandens germplasms The optical density (A260/A280) and concentration of genomic DNA isolated from 34 germplasms of E. scandens were in the range of 1.81-1.97and 298.1-1693.9ng µL −1 , respectively.Agarose gel electrophoresis depicted single bands with no smears for all samples, indicating that the extracted DNA was of good quality and could be used for ISSR analysis (Fig. 3).34,34 germplasms of E. scandens.

Optimization of the ISSR-PCR system
The PCR amplification results suggested that the different orthogonal combinations amplified bands with large differences in the number and the degree of clarity (Fig. 4).Among them, treatment 16 exhibited the best amplification, with the highest score of 16 in the visual analysis method (Table 3).In general, a good amplification profile resulted in high scores.
The mean Ki of each factor at different levels and the extreme difference of the mean between different levels of the same factor was derived from the scoring result R. The degree of influence of the three factors on the ISSR-PCR amplification of E. scandens germplasms was ranked using the R-value: DNA template amount > primer amount > Mix mixture amount.Finally, the following optimal reaction composition (20 µL) was determined using the Ki value: 1 µL DNA template (60 ng µL −1 ), 1.2 µL primer (10 µmoL µL −1 ), 10 µL MasterMix, and 7.8 µL H 2 O.

Polymorphic amplification and genetic diversity analyses using ISSR markers
The initial screening of primers UBC800-864 and the re-screening of a few primers (using gradient PCR) indicated the primer pairs and annealing temperatures suitable for ISSR-PCR amplification of E. scandens germplasms (Table 4).A total of 18 primer pairs resulting in bands with distinct backgrounds, adequate stability, and high polymorphism were identified.In total, 183 markers were amplified using the 18 ISSR primers, including 121 polymorphic markers (66.12%), with an average of 10.2 markers and 6.7 polymorphic markers per primer pair, an average allele number (Na) of 1.66, and an average effective allele number (Ne) of 1.32.The lowest percentage of polymorphism was 25% from primer UBC808, The genetic diversity indexes of Guangxi, Guangdong, Hainan, Fujian, and Guizhou regions are shown in Table 5.We observed that the number of samples, Nei's genetic diversity index (H), and Shannon's information index (I) were higher in germplasms collected from Guangxi than those collected from other regions.

Correlation between genetic similarity coefficients and genetic distances of E. scandens germplasms
The ISSR data of 34 germplasms of E. scandens were analyzed using PopGen32 (Fig. 5).The genetic similarity coefficients and genetic distances among the germplasms of E. scandens based on ISSR markers were 0.7104-0.9563(mean: 0.7995) and 0.0447-0.3420(mean: 0.2247), respectively.The lowest genetic similarity coefficient was observed for the germplasms obtained from both Cengxi City, Guangxi (No. 4) and Shangsi County, Fangchenggang city, Guangxi (No. 6) (0.7104) with Ledong County, Hainan (No. 24), and the germplasm obtained from them showed the maximum genetic distance (0.3420), indicating a high genetic variation and distance between them.The genetic similarity coefficient between the two germplasms collected from Qiongzhong County, Hainan (No. 26 and 27), was the highest (0.9563), and their genetic distance was the lowest (0.0447), indicating less genetic variation and relatedness between them.Vol.: (0123456789)

ISSR marker-based clustering analysis
The genetic similarity coefficients of the 34 germplasms of E. scandens were analyzed using UPGMA clustering and mapped using the software PopGen32 and MEGA7.The results of cluster analysis (Fig. 6) revealed that the 34 germplasms of E. scandens were classified into three clusters.Cluster 1 contained 93.75% of Guangxi and 33.3% of Fujian germplasms, cluster 3 contained 50% of Guangdong and 20% of Hainan germplasms, and cluster 2 included germplasms collected from Guangxi (6.25%), Guangdong (50%), Hainan (80%), Fujian (66.7%), and Guizhou (100%) regions.The genetic similarity coefficients among the germplasms of E. scandens in Guangxi were high, with a few differences in genetic backgrounds among the germplasms of E. scandens from Guangdong, Hainan, Fujian, and Guizhou.The ISSR data of the 34 germplasms of E. scandens were transformed into genetic distances for PCoA analysis using the GenAlEx software (Fig. 7).The two-dimensional PCoA plot of the 34 germplasm resources of E. scandens was constructed using the first and second principal components as the horizontal and vertical coordinates, respectively.The two principal components explained 10.45% and 8.87% of the variations, respectively.PCoA result could not distinguish the five groups of germplasm distributed by geographical sources.However, the germplasms in Guangxi were gathered together and could distinguish with germplasms derived from other regions in the coordinate map.The germplasms of E. scandens from Guangxi could be distinguished from those collected from Guangdong, Hainan, Fujian, and Guizhou, whereas those from Guangdong, Hainan, Fujian, and Guizhou were not strictly divided by geographical location.

Structure analysis
The Bayesian clustering model was performed to evaluate the population structure of 34 E. scandens samples.The optimal cluster value (K) was two (Fig. 8a), with the highest values of both LnP(K) (log probability of data, −1921.17)and delta K (5.27) obtained from the STRU CTU RE.The results showed that it was more appropriate to divide 34 germplasms

Correlation analysis of genetic and geographical distances
The geographical distances between sampling sites were calculated using RStudio based on the latitude and longitude information of each sampling site.Subsequently, the correlation between geographic and genetic distances among different germplasms was analyzed using Mantel's correlation test [Mantel's r = 0.1176; P = 0.084) (Fig. 9)], and no significant correlation between geographic and genetic distances was observed among germplasms of E. scandens obtained from different locations.

Discussion
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 (Mg 2+ , DNA template, primers, DNA polymerase, and dNTPs) to the threecomponent 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 H 2 O.This is different from the ISSR reaction system of Diospyros (Mansoory et al. 2022), Vigna subterranea (Khan et al. 2021), and Prunus salicina (Li et al. 2022), which indicates that the ISSR optimal reaction system of each plant might not be universal.Various factors in the ISSR reaction system need to be optimized for screening the ISSR markers of a new plant.
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%).These results were consistent with the study of Dalbergia nigra by Silva Júnior et al. (2020), which demonstrated the percentages of polymorphic loci detected by 8 ISSR primers in 24 germplasms were 68.04%.Moreover, the percentage of polymorphic sites detected by ISSR markers is more than 90% in Kaempferia galanga (Subosti et al. 2020), Aniba rosaeodora (Guizado et al. 2020) and Diospyros (Mansoory et al. 2022), while the percentage of polymorphic sites detected in exotic ginger is only 35.14% (Jithin et al. 2008).Thus, the detection efficiency of ISSR markers in E. scandens of this study is above the average overall.
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 germplasms from Guangxi could be separated from those from other provinces.In contrast, the germplasms from Guangdong, Hainan, Fujian, and Guizhou were crossdistributed on the main coordinate map and did not exhibit an obvious distribution pattern, which was consistent with the results of UPGMA cluster analysis.The cluster pattern of germplasms from Guangdong, Hainan, Fujian and Guizhou has no obvious relationship with geographical distribution.This indicates that samples from different geographical locations may have the same origin and genetic structure.Similar results were also obtained in the study of Diospyros (Mansoory et al. 2022) and Andrographis paniculata (Tiwari et al. 2016).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 Fig. 9 Mantel test to determine the correlation between geographical and genetic distances 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.About wild germplasm, the buds of E. scandens No.3 were red, with special aroma and vigorous growth.The leaves of E. scandens No.7 and No.22 were larger and the number of buds was more, which may have advantages in yield.Cross breeding between germplasms with far genetic distance is more conducive to obtain new varieties with excellent traits of both parents.Combined with the genetic distance and the characteristics of some germplasms, the recommended breeding combinations are shown in Table 6.
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.

Conclusion
In the present study, an optimal ISSR-PCR system was established for analyzing the genetic diversity of E. scandens.Eighteen ISSR primer pairs that could amplify genetic markers with distinct backgrounds and adequate polymorphism in 34 germplasms of E. scandens were selected.The germplasm resources from Guangxi, Guangdong, Hainan, Fujian, and Guizhou in China exhibited a relatively high level of genetic diversity.However, a few genetic differences were observed between the germplasms of Guangxi and those of Guangdong, Hainan, Fujian, and Guizhou, but the genetic variation among the 34 germplasms was not significantly correlated with geographical distance.Thus, the 34 E. scandens germplasms could be propagated and transplanted onto each other to enhance genetic exchange between regions and improve genetic diversity in each region, thereby elevating the overall genetic diversity of E. scandens.The kinship analysis of wild and cultivated germplasms of E. scandens indicated that wild germplasms exhibited genetic backgrounds similar to those of cultivated varieties, which could be used for variety selection and breeding.Thus, in this study, we used ISSR markers to study the genetic diversity of E. scandens and revealed the genetic background and affinities of 34 E. scandens germplasms.The findings of this study lay the foundation for further research focusing on the conservation and utilization of E. scandens germplasm resources, identification and classification of varieties, and variety selection and improvement at the molecular level.

Fig. 5
Fig. 5 Genetic distance and similarity coefficient matrix of 34 germplasms of E. scandens based on ISSR markers.Genetic similarity coefficient is indicated above the diagonal line and genetic distance below it; 1-34, 34 germplasms of E. scandens

Fig. 8 a
Fig. 8 a Estimated K values for the structure analysis of E. scandens germplasm.b Two run results ( K = 2 and K = 3) by STRU CTU RE. 1-34, 34 germplasms of E. scandens

Table 2
Orthogonal test factors and levels Fig.3Agarose gel electrophoresis of 34 germplasms of E. scandens.M, DL15000 DNA Marker; 1-whereas the highest percentage was 92.31% from primer UBC827.

Table 3
Combinations of orthogonal tests and derived scores K1, K2, K3, and K4 are the mean values of scores at different levels for each factor, and R is the extreme difference in the mean values of scores between different levels for the same factor

Table 4
Statistics of ISSR primer pairs and their polymorphism and genetic diversity indexes