1. Introduction
Radix bupleuri, a perennial herb, is also known as mushroom grass, firewood grass, and so on.
Radix bupleuri dried root, which originates from
Bupleurum Chinense DC. and
Bupleurum scorzonerifolium Willd. of the genus
Bupleurum (
Bupleurum L.) in the Umbelliferae family (Umbelliferae), is commonly used as a medicine and has a bitter and pungent taste. In China,
Radix bupleuri is extensively present in Shaanxi, Shanxi, Gansu, Hebei, Inner Mongolia and other regions [
1]. As a bulk medicinal material in China,
Radix bupleuri has the effects of harmonizing the exterior and interior, soothing the liver, relieving depression, and raising yang energy [
2]. It also plays a role in treating exogenous fever, stagnation of liver-qi, hypochondriac pain and chest distress [
3]. Saikosaponin is the main effective medicinal component of
Radix bupleuri, with anti-inflammatory [
4], anti-cancer [
5], anti-depressant [
6], antioxidant [
7] and anti-liver-fibrosis effectiveness [
8]. Recent medical research has exhibited that all 23 main active ingredients from
Radix bupleuri-Paeonia lactiflora compound medicine acted on potential depression target proteins and alleviated depressive symptoms [
9]. A compound of
Radix bupleuri and
Radix Scutellariae called ‘Chaiqin Kanggan Mixture’ had a substantial impact in preventing coronavirus disease 2019 (COVID-19) and influenza virus infection [
10]. The market demand for
Radix bupleuri is currently steadily rising as more and more pharmaceutical research findings are transitioning to industrialization. Expanding the breeding of high-quality
Radix bupleuri to increase yield for production will be the future trend.
However, high-quality
Radix bupleuri, or even the correct provenance selection of
Radix bupleuri, often causes confusion for growers. In addition to the above two kinds of
Radix bupleuri (
B. chinense DC. and
B. scorzonerifolium Willd.) stipulated in the Pharmacopoeia of the People’s Republic of China (2020 version) [
11], there are many species of
Radix bupleuri sold in the domestic medicinal material markets. As many as 44 species, 17 varieties and seven forms of
Radix bupleuri plants are used as medicinal herbs [
12]. They are used medicinally differently in various regions. In Guizhou Province, the medicinal standard contained
Bupleurum marginatum var.
stenophyllum (Wolff) Shan et Y. Li. and
Bupleurum scorzonerifolium Willd [
13].
Bupleurum smithii Wolff were officinal herbs in Gansu Province [
14]. In the early 1970s,
Bupleurum falcatum, a medicinal plant originally came from Japan, was introduced into China and cultivated in large quantities in Shandong, Hebei and other places, and mostly sold to Japan and Korea [
15]. Although the national and local standards positively regulated the medicinal species of
Radix bupleuri, counterfeits and substitutes are still available on the market. Many studies have demonstrated that the morphology, component composition and other characteristics of various
Radix bupleuri species vary greatly [
16]. However, growers who only rely on experience to judge the quality of varieties and their quality according to the appearance of plants often make mistakes, which further affects the efficacy and stability of downstream products. Therefore, it is necessary to explore a reliable and simple means of germplasm identification.
The species diversity of medicinal plants used as root medicines is mainly reflected in their morphological variations in the aboveground part, the content of active components in the underground part, and the molecular level. At present, the investigation of germplasm resources of
Radix bupleuri is from a single point of view, only considering the characteristics of one aspect. Zhang et al. compared several agronomic traits of various
Radix bupleuri species and found that the leaf characters of different types varied considerably and were significantly correlated with yield [
17]. Li et al. identified the main active ingredients of
Radix bupleuri from nine distinct sources and showed that the variation coefficient of the total content of saikosaponin a (SSa) and saikosaponin d (SSd) was 38.9%, and the difference between various
Radix bupleuri was significant [
18]. Wei et al. applied simple repeat sequence (SSR) molecular marker technology to identify six varieties of
Radix bupleuri and reported promising results for different species identification but unsuitable identification of similar species [
19]. Simple repeat amplification (ISSR), a molecular marker method based on microsatellite sequence, has established itself in recent years. Compared with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), ISSR has the advantages of strong polymorphism, high stability, reliable repeatability, cheap cost and straightforward operation [
20,
21]. A reliable ISSR-PCR reaction system for
Radix bupleuri has been developed [
22]. The genetic relationship of 11
Radix bupleuri was identified and analyzed, and 156 polymorphic bands were obtained, which accounted for 84.3% of all bands [
23]. Therefore, ISSR technology may have favorable effects in identifying
Radix bupleuri species, molecular marker-assisted selection breeding and genetic diversity research.
In this study, the species diversity of 13 different Radix bupleuri germplasms from the Baoji base in Shaanxi Province was investigated based on their phenotypic characteristics, saikosaponin content and molecular marker profiles during the growth periods. The germplasm difference of Radix bupleuri was explored from multiple angles, and a simple and convenient germplasm identification method was explored and aimed to offer a theoretical and practical foundation for breeding new cultivars of Radix bupleuri, germplasm innovation, and effective utilization of medicinal resources in China.
4. Discussion
Radix bupleuri is one of the major medicinal materials in China. With the research, development, and clinical application of Chinese patent medicines, such as granules, tablets and injections dominated by
Radix bupleuri, the annual demand for it has surged to 5000 tons, and the limited natural resources are far from meeting the market demand [
30]. Currently, the research on
Radix bupleuri germplasm resources is imperative for breeding improved strains and increasing the supply of high-quality germplasm resources. Due to its variety, extensive range, high phenotypic similarity and ambiguous categorization across species within the genus, it is not easy to identify and utilize the germplasm resources of
Radix bupleuri [
31]. For example,
B. falcatum is native and growing in Europe and Western Asia. Its main components and pharmacological effects are the same as those of domestic
Radix bupleuri [
15]. It is easy to be confused because of its similarity in shape to other
Radix bupleuri, especially
B. chinense DC.
Radix bupleuri is also mostly sold in the form of processed products (irregular thick slices, vinegar
B. chinense, turtle blood
B. chinense), in which the shape and color have changed. This makes it difficult for customers to determine authenticity. Therefore, it is crucial to recognize and safeguard
Radix bupleuri’s provenance. Numerous morphological, physiological and biochemical studies have already been conducted. However, the identification results of these methods among the more confusing species were far from satisfactory. Han et al. reported that the seeds of
Radix bupleuri from different habitats have little difference in morphology, which is easily affected by harvesting time and regional environment [
32]. Zhang et al. found that the seed phenotypes of
Radix bupleuri from different varieties showed great differences, as well as the seeds of
B. chinense DC. from different origins and the same variety, which were similar to the morphological observation results of mature plants [
33]. Wang et al. also proved that climatic and geographical factors affected plant growth and secondary metabolism, resulting in regional differences in the active components of
Radix bupleuri [
34]. These results indicated that it was not feasible to judge the variety of
Radix bupleuri solely by observing the plant appearance and the content of active components.
To eliminate the unpredictable difference between germplasm resources caused by environmental impact, we chose to plant the seeds of
Radix bupleuri with more varieties in the market in the same field. After studying the agronomic traits and active components, it was discovered that the aboveground part’s coefficient of variation for agronomic traits was 7.62–22.94%, a significant difference from the underground part’s coefficient of variation for agronomic traits, which was 19.01–41.54%. The underground part of
Radix bupleuri germplasm was where the diversity of the species was most pronounced. Moreover, the Chinese Pharmacopoeia listed SSa and SSd as indicator components for quality control of
Radix bupleuri, specifying the sum of the two contents ≥0.3% [
11]. The content of two saikosaponins in
Radix bupleuri ranges from 0.730 to 3.123%. All of the materials complied with the requirements, yet there was a substantial difference. Among them, the content of saikosaponin in Changzhi of Shanxi Province was much higher than that in other species of
Radix bupleuri, which was consistent with the results of cluster analysis based on content. Other different varieties of
Radix bupleuri fell indiscriminately into the other two categories. Additionally, the close correlation between morphological characteristics and compositional content of the underground parts indicated that environmental factors dominated over genetic impacts in the aboveground components. Morphological characteristics can also reflect a species’ genetic diversity under specific cultivation conditions to some extent. Therefore, plant characters with more notable variation, such as the root traits of
Radix bupleuri, should receive more focus. For example, the differentiation of commercial specifications and grades based on the size of harvested
Radix bupleuri is conducive to selecting high-quality medicinal materials. Based on the results of the above studies, it could be seen that even if the heterogeneous
Radix bupleuri was planted in the same background, differences in appearance and composition would still occur, but such differences could not distinguish the varieties in detail.
As an effective tool for germplasm resource identification and genetic diversity analysis, molecular marker technology has been widely used in genetic background analysis, re-collection of germplasm resources and selection of breeding parents [
35]. Many molecular markers have been used to estimate the genetic diversity of
Radix bupleuri in several investigations. Zhao et al. used the dry roots of cultivated
Radix bupleuri as the materials and adopted the two analytical methods of RAPD and AFLP to analyze the genetic diversity of
Radix bupleuri, and they found that the results of the two molecular marker clustering were not completely consistent and could not distinguish
B. falcatum from
B. chinense DC [
36]. Kang et al. used ITS2 sequences to determine the DNA sequence of the sample seeds. The results of the systematic clustering tree showed that some varieties, such as
B. yinchowense, were not strictly clustered together. In addition, the clustering of a variety of
Radix bupleuri plants from different regions further indicated that the ecological environment also had inevitable effects on the genetic variation of
Radix bupleuri [
37]. Song et al. conducted genetic diversity analysis of cultivated and wild
Radix bupleuri from different regions based on SSR molecular marker technology and found that SSR markers could distinguish the wild population from the cultivated population [
38]. However, SSR markers require prior knowledge of the genome, clone or primer design, and the experimental process and data analysis are relatively complex [
39].
ISSR is a molecular marker technology with simple operation and high stability. The markers amplify the inter-repeat region. When the plant genome has a high proportion of repeat sequences, it will produce a high polymorphism rate, which can well reveal the polymorphism of species [
40]. ISSR has been widely utilized in plant genetic diversity studies. Araújo et al. provided a new approach to determining molecular differences between two cotton species (
Gossypium hirsutum L. r. marie-galante and
Gossypium barbadense L.) using the ISSR marker system [
41]. Mesfer ALshamrani et al. explored the genetic diversity of six sesame landraces by ISSR maker. They amplified 233 alleles, while the average polymorphism percentage (P%) of all the genotypes studied was 65.32% and the ISSR markers utilized were highly reproducible [
42]. Gholami et al. also confirmed that ISSR markers could be useful for distinguishing the relationships among species of Iranian terrestrial orchids [
43]. Li et al. analyzed the genetic diversity of 11 samples from 6 populations of
B. chinense DC. using the ISSR technique and showed that the intraspecific genetic differences of
B. chinense DC. were related to the environmental differences [
44]. Ma and Ke conducted ISSR markers on different varieties of
Radix bupleuri from different origins but failed to completely distinguish
B. chinense DC.,
B. smithii Wolff and
B. scorzonerifolium Willd. It was possible that
Radix bupleuri was a cross-pollinated plant, and many varieties grew mixed during the cultivation, resulting in germplasm confusion [
23]. In addition, the diversity of
Radix bupleuri based on ISSR molecular marker was rarely reported. In our research, the nine primers reassessed could amplify polymorphic bands, according to our prior laboratory experience, and the UBC 810 primer performed the best. The germplasm materials of No. 4 (
B. falcatum), No. 5 (
B. smithii var.
parvifolium Shan et Y. Li), and
B. chinense DC. were clustered in different groups, indicating that ISSR markers could accurately and reliably identify
B. chinense DC. and its adulterants. This approach may prevent errors in judgment caused by the appearance and composition of Chinese herbal medicines. Overall, accurate identification of
Radix bupleuri germplasm resources are of great significance for the utilization, breeding and production of germplasm and pharmaceutical production with different active ingredients.