Epimedium koreanum is a perennial herb of the genus Epimedium in the family Berberaceae (Ma et al. 2011) and is found in China, Japan, Korea, and Russia (Ying et al. 2000;Stearn et al. 2002). The aboveground parts of the plant are very distinctive, with only three stems and three leaves on each stem (the whole plant has nine leaves), which is referred to as "San Zhi Jiu Ye Cao" in China (Administration Bureau in Traditional Chinese Medicines (China). 1998). The Epimedium genus is characterized by an evolutionarily conserved genome and high interspecific genetic similarity (Takahashi et al. 1989;Yan et al. 2016; Zhang et al. 2018 ), and plants of this genus are diploid (2n=2x=12), with karyotype characteristics according to Stebbins’ classification—all chromosomes having a pair of satellites. Because the seed cultivation technology is not developed, the propagation of E. koreanum relies mainly on rhizome division (Mihaljević et al. 2009), which has led to it being a vulnerable species.
Notably, E. koreanum is one of the herbal medicinal plants recorded in the Chinese Pharmacopoeia as Epimedium (National Commission of Chinese Pharmacopoeia. 2020). It grows mainly in the northeastern provinces of China. Epimedium is a commonly used Chinese herb that has been known for 2,000 years for its effectiveness in tonifying kidney yang, strengthening muscles and bones, and combating rheumatism (Li et al.1991). Previous experiments have proven that E. koreanum extract has a variety of biological activities, such as antibacterial, antioxidative, and anti-osteoporosis effects, among others (Meng et al. 2005). Even in modern Chinese medicine, it is utilized into tablets, capsules, and other drugs (Oh et al. 2015), such as Xianling Bone Capsules, which has been approved by the State Food and Drug Administration of China since 2002 and consists of six herbs, of which Epimedium accounts for 70% by weight (Cheng et al. 2013). Due to its medicinal properties, Epimedium is very popular in China and the herb is in high demand. Like most traditional Chinese herbs, medicinal plants of the genus Epimedium are not cultivated but collected from the wild (Guo et al. 2003), thus leading to a shortage in the number of wild E. koreanum plants in northeast China annually. Only a few conservation studies have been performed on this plant.
Many molecular marker methods are present for plant genetic diversity research, such as randomly amplified polymorphic DNA, simple sequence repeat, amplified fragment length polymorphism, and next-generation sequencing (Mondini et al. 2009). Inter simple sequence repeat markers (ISSR) combine the advantages of the aforementioned markers (Henareh et al. 2016), such as good stability, high reproducibility, rich polymorphism, simple operation, low cost, and good safety measures. They can also be used for organisms for which the genetic information available is insufficient to conduct a study (Mohammadi et al. 2020). ISSR is a PCR-based dominant marker, generally using primers of 16 to 25 bp in length, which can be dinucleotide, trinucleotide, tetranucleotide, or pentanucleotide (Zietkiewicz et al. 1994), and its usefulness as a molecular marker in plant genetic studies has been well established (Nidhal et al. 2016; Tiwari et al. 2017;Azizi et al. 2018 ).
Successful application of ISSR techniques has been reported for Epimedium brevicornum (Duan et al. 2021), and Epimedium elatum Morr & Decne (Lone et al. 2017), and this paper is the first report on E. koreanum. We selected ISSR markers to analyze the genetic diversity and genetic structure of 134 samples collected from 14 wild populations, and we believe that the results of our study provide valuable information for conservation strategies of E. koreanum.