Analysis on Genetic Diversity of Radix Astragali by ISSR Markers

Radix Astragali has been an important traditional Chinese herbal medicine for over 2000 years. It is derived from two plant species, namely, Astragalus mongholicus [Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao] and Astragalus membranaceus [Astragalus membranaceus (Fisch.) Bge.] (Leguminosae), according to the Pharmacopoeia of the People’s Republic of China. In this study, the genetic diversity and genetic relationships of Radix Astragali in China were analyzed by Inter-Simple Sequence Repeat (ISSR) markers. A total of 25 highly polymorphic ISSR primers were selected to amplify 95 Radix Astragali samples. Among 273 DNA bands amplified, 213 are polymorphic (percentage of polymorphic bands: 78%). The average value of the amplified bands was 10.9 for each primer, and the number varied from 4 to 20. The genetic diversity of the 95 Radix Astragali samples was analyzed by using POPGENE 1.32 software. The Nei’s genetic diversity index (h) and Shannon’s information index (I) were 0.3590 and 0.5308, respectively, which indicated the abundant genetic diversity of Radix Astragali. The level of genetic diversity in A. membranaceus (h: 0.3109, I: 0.4657) was slightly lower than that in A. mongholicus (h: 0.3364, I: 0.4969). Considering the average genetic similarity coefficient by NTSYS analysis to cluster the A. membranaceus of nine habitats and A. mongholicus of five habitats, Radix Astragali samples were clustered into two groups according to place of origin. This clustering is different from traditional clustering, which divides groups according to species. Results obtained from this study will provide a theoretical basis for the molecular study on germplasm resources of Radix Astragali.


Introduction
Radix Astragali, which has a long history of medicinal use, is derived from Astragalus mongholicus and Astragalus membranaceus [1].Astragalus is mainly distributed in North, Northeast, Northwest, and Southwest China [2].However, the qualitative difference between wild and cultivated Astragalus owing to the determination of genuine raw materials results in significant difference in market price, which leads to excavation of wild herbs.Thus, the distribution and population of wild Astragalus have rapidly decreased.Astragalus has been included in the national list of protected rare and endangered plants [3].Effective conservation of germplasm resources of Radix Astragali is urgently needed.Although the artificial cultivation of Astragalus has achieved success, the qualitative and quantitative demands of raw material for medicine have greatly increased with the development of new drugs, medicine, and health products.Astragalus cultivation has produced hybrid varieties with unstable yield and low quality.This phenomenon has seriously affected the further development of Astragalus industry.Therefore, this preliminary study on Astragalus from different habitats was conducted to evaluate its genetic diversity by using the ISSR labeling technique.The findings will provide a theoretical basis for formulating Astragalus genetic diversity research, conservation strategies, and cross breeding.
ISSR was conducted by using a synthetic 16 -18 bp nucleotide repeat sequence as a primer.Genomic DNA was amplified by PCR, which employed a primer anchored at 3 or 5 end of oligonucleotides.The ISSR combines the advantages of RAPD and SSR markers at the same level.Thus, it can produce more polymorphism than RAPD, and the reaction system is more sensitive, more stable, and has good repeatability [4]- [7].This molecular marker has been widely used in studies on germplasm resource identification, phylogeny of species, plant taxonomy, evolution, and genetic diversity [8]- [10].Astragalus has several variations of morphological features in the actual production, such as stems with red or green color and stems with two colors.Variation and degradation of the Astragalus germplasm may occur in the introduction and domestication.Thus, studying the genetic diversity of Radix Astragali at the DNA level is necessary.This study provides molecular-level evidence for Astragalus germplasm resources by using the ISSR molecular marker.

Plant Materials and DNA Extraction
A total of 95 Radix Astragali individuals were sampled representing 10 natural populations, including Shanxi, Jilin, Inner Mongolia, Gansu, Ningxia, and Shaanxi Provinces of China (Table 1).Approximately 1 g of fresh leaf samples from each individual was collected, placed in a plastic bag with silica gel to dry the samples quickly, and brought back to the laboratory for DNA isolation.Total genomic DNA was extracted using a protocol described by Doyle and Doyle [11], and 0.6% agarose gel electrophoresis and a biospec-mini DNA/RNA/protein analyzer were used to detect the quality and yield of DNA.The DNA was later diluted to 50 ng•µL −1 working solution and placed in a refri

Primer Selection and ISSR-PCR Amplification
To determine the appropriate primers suitable for Astragali, we screened 100 ISSR primers purchased from the website of the University of British Columbia in Canada (https://www.ubc.ca/) and Synthesis Project.For each primer pair, two samples were amplified, and their amplification products were run on 1% agarose gels.PCR amplifications were performed in a 20 µL reaction that contained 2 µL of 10 × PCR buffer, 2.5 mM dNTPs, 10 µM primer, 5 unit of Taq DNA polymerase (Taka Ra Biotechnology Co., Ltd.), and 50 ng of template DNA.Double distilled water was added to make the volume 20 µL.Amplifications were performed as follows: 95˚C for 5 min, 35 cycles of denaturation at 95˚C for 30 s, annealing at 53˚C -57˚C for 40 s, extension at 72˚C for 40 s, and a final extension at 72˚C for 10 min.Afterward, the samples were held at 4˚C.
Highly polymorphic primers with clear bands and clean background were selected to amplify the genomic DNA of all Astragalus species.

Data Analysis
The amplified DNA polymorphic fragments (bands) were scored as present (1) or absent (0), and the data matrix of the ISSR phenotypes was assembled for further analysis.
The percentage of polymorphic bands (PPB), observed number of alleles (Na), effective number of alleles (Ne), Nei's genetic diversity index (h), and Shannon's information index (I) were calculated using POPGENE 1.32 software.NTSYSpc 2.10 software was used to calculate the genetic similarity (Gs) coefficient of the 183 Astragalus species.
The UPGMA tree [12] was constructed using NTSYS 1.8 software [13] to examine the relationship of the populations.

DNA Detection
The genomic DNA of the 95 A. mongholicus and A. membranaceus were extracted and detected by agarose gel electrophoresis.The results showed that the DNA bands were clear, and no diffusion phenomena were observed.The extracted genomic DNA detected by a nucleic acid protein detector revealed that the ratio of D260 nm/D280 nm was between 1.8 and 2.0, and the yield was between 100 and 1800 ng/µL.These findings indicate that the genomic DNA extracted from the Astragali leaves had high quality and high yield.

ISSR Polymorphism
A total of 35 primers were screened from 100 ISSR primers, which were published by the Columbia University, to study the genetic diversity of the two populations of Radix Astragali.Among these primers, 25 were polymorphic and 10 were monomorphic.The PPB was 71.4%.A total of 273 amplified bands were obtained from the 95 Radix Astragali samples using 25 polymorphic ISSR primers (Table 2).Among these bands, 213 were polymorphic, and the PPB was 78%.The average value of the amplified bands was 10.9 for each primer, and the number varied from 4 to 20 (Table 3).UBC835 and UBS855 presented the highest number of bands (20/19), whereas UBC811 and UBC808 had the lowest number (4/5).The results showed a significant genetic difference.

Genetic Diversity of Radix Astragali
The binary data matrix was scored as present (1) or absent (0) of homologous bands and loaded into POPGENE 1.32 software to analyze the genetic diversity of Radix Astragali.The mean Na was 1.9 in the two Astragalus species.For A. membranaceus, the Ne, h, and I were 1.5318, 0.3109, and 0.4657, respectively, which were slightly lower than those of A. mongholicus (Ne: 1.5861, h: 0.3364, and I: 0.4969).These results indicate that A. mongholicus have higher genetic diversity than A. mongholicus (Table 4).

Genetic Diversity of Two Radix Astragali from Different Regions
A. membranaceus was collected from nine provinces including Inner Mongolia, Ningxia, Shandong, Shaanxi, Gansu, Hebei, Heilongjiang, Jilin, and Liaoning (Table 5).The highest number of polymorphic bands (Np) of 190 was observed in Inner Mongolia.
The PPB, h, and I were 89.20%, 0.2538, and 0.3714, respectively.The lowest Np was found in Shaanxi with only 83.The PPB, h, and I were 38.97%, 0.0861, and 0.1260, respectively.These results indicate the different origins and order of A. membranaceus genetic diversity from highest to lowest: Inner Mongolia, Heilongjiang, Ningxia, Shandong, Heibei, Gansu, Jilin, Liaoning, and Shaanxi.Meanwhile, A. mongholicus was collected from five provinces including Shanxi, Inner Mongolia, Shaanxi, Gansu, and Hebei (Table 5).The highest Np of 203 was observed in Shanxi.The PPB, h, and I were 95.3%, 0.4877, and 0.5212, respectively.The lowest Np was observed in Shaanxi with

Clustering Analysis of Radix Astragali
The Gs coefficient of nine origins of A. membranaceus and five origins of A. mongholicus was calculated using NTSYSpc 2.10 software, and UPGMA was used to generate a dendrogram (Figure 1).The 14 different origins of two Astragali could be completely

Genetic Diversity
Genetic diversity is the product of the long-term evolution of a species or a population.It is the basis of existence, development, and evolution of species.Therefore, the study on the genetic diversity of species is fundamental to understand the taxonomy, origin, and evolution of species.Furthermore, such study will provide a theoretical basis for the germplasm resource conservation, development, utilization, and breeding [14].In the present study, the genetic diversity of 95 Astragalus materials was analyzed by using 25 highly repeatable and polymorphic ISSR primers.A. mongholicus had higher genetic diversity (h: 0.3364, I: 0.4969) than A. membranaceus (h: 0.3109, I: 0.4657) at the species level (Table 4).The results of this study were consistent with the findings (A. mongholicus, h: 0.822; A. membranaceus, h: 0.689) of the research on genetic diversity of the two species by SSR molecular marker [15].Compared with a relevant research [16], we speculated that A. mongholicus had a higher level of genetic diversity.Astragalus mainly depends on insect for pollination and on humans for seed dispersal; it also possesses distant hybridization properties [17]- [19].These life history traits are dominated by outcrossing breeding system and considered as an important factor to maintain higher genetic diversity of species [20]- [22].The wild Astragalus resource gradually decreased mainly because of the recent over-excavation and habitat deterioration caused by human activity [23] [24].However, the interference is likely only the effect of the number and genetic diversity of parts of a local area of Astragalus, in addition to the fact that the species are perennial herbaceous plants with high medicinal value.Thus, the large range of distribution, long life cycle, and germplasm (seed) exchanges in the development and propagation can be conducive to the genetic diversity at the species level [21] [25].Therefore, the genetic diversity of the two species of Astragalus is unaffected by severe loss of habitat and human destruction.

Analysis and Diversity of Two Species of Astragalus in Different Habitats
The genetic diversity of different habitats of the two species of medicinal Astragalus was analyzed.

Analysis on the Genetic Relationships between Two Species of Astragalus in Different Habitats
Results showed that the two medicinal Astragalus species from 10 different habitats could be clustered into two groups (Figure 1).In these groups, major clustering was based on the geographical habitats (Shanxi and Inner Mongolia) of A. membranaceus and A. mongholicus.The experimental results also presented that these species were divided according to origin and not from species distinction.This clustering also showed that the genetic relationship was close and may be related to the dominant characteristics of ISSR molecular markers.

Protection Strategy of Astragalus Germplasm Resources
In summary, two Astragalus species currently maintained a middle genetic diversity level.Genetic diversity is the basis of species to adapt to the environment and evolution to avoid extinction and survive.Genetic diversity also provides theoretical basis for further development and utilization of germplasm resources.The current anthropogenic deforesting and picking activities as well as habitat deterioration do not seriously affect the Astragalus genetic diversity.However, if the deterioration of the environment and damage induced by human activities are ineffectively prevented, the fragmentation of Astragalus habitat and population scale is bound to accelerate.This phenomenon can cause a genetic drift in its population and lead to a substantial loss of the current abundant genetic diversity.Such loss will affect the survival of the species and the availability of excellent germplasm resources.The main reasons of wild Astragalus resource loss are deliberate cutting and picking, as well as habitat destruction.We suggest that Astragalus protection area should be established as soon as possible in wild Astragalus habitat to prohibit unauthorized and wasteful mining.Detailed plan about devel- opment and utilization should also be designed.Considering that the current biological research on Astragalus is few, we suggest further research on the reproductive biology, conservation genetics, and physiological and ecological aspects to provide basis for the conservation and sustainable utilization of these species.
separated into two parts based on 0.80 coefficient using the 25 ISSR primers.One group cluster consisted of the northern regions including Inner Mongolia, Shanxi, Shandong, Heilongjiang, Hebei, Jilin, and Liaoning, and the other group cluster consisted of Ningxia, Gansu, and Shaanxi.This first group was clustered according to place of origin and conformed to the geographical characteristic of the traditional Chinese medicinal material geoherbalism.The large group was also divided into two groups according to production.One group includes Shanxi and Inner Mongolia.Cluster was initially observed with A. mongolian of Shanxi and Inner Mongolia at 0.93 and then with A. membranaceus of Inner Mongolia at 0.91.The other group includes five districts, namely, Shandong, Heilongjiang, Hebei, Jilin, and Liaoning, which were mostly from A. membranaceus.The major second group mainly includes three origins, namely, Ningxia, Gansu, and Shaanxi.The membrane pod of A. membranaceus and A. mongholicus of Gansu Province was clustered as a group at 0.88.After Shaanxi A. mongolian clustering, A. membranaceus of Ningxia formed the second group.

Figure 1 .
Figure 1.Dendrogram of Gs among the radix astragali from different habitats.

Table 1 .
Resources of the test samples.

Table 2 .
Primer name and sequences used in the ISSR analysis.

Table 3 .
Statistical results of polymorphic bands of ISSR primers.
Note: Nb (Number of bands); Np (Number of polymorphic bands); PPB (Percentage of polymorphic bands).

Table 4 .
Genetic diversity of two species of radix astragali.

Table 5 .
Genetic diversity of radix astragali of different origins.87.The PPB, h, and I were 40.85%, 0.0869, and 0.126, respectively.These results indicate the different origins and order of A. mongholicus genetic diversity from highest to lowest: Shanxi, Heilongjiang, Hebei, Gansu, and Shaanxi.At a general level, the average value (h: 0.1516, I: 0.2245, PPB: 60.51%) of the genetic diversity of A. membranaceus from the nine provinces was slightly lower than the value (h: 0.2324, I: 0.3044, PPB: 68.17%) of A. mongholicus from the five provinces.
[2] morphological characteristics and classification status of the two species showed a very close genetic relationship[2].In addition, Shanxi and Inner Mongolia are areas where authentic Astragalus herbal medicine is real estate areas.Thus, the first clustering was according to place of origin.However, Hebei A. mongholicus merged into different geographical positions in the clustering, which may be due to its mixed medicinal material source.Hebei is the largest business center; thus, seed and germplasm mix into the genetic information of the two species.Another group of three habitats of A. membranaceus and A. mongholicus, including Ningxia, Gansu, and Shaanxi, was clustered into one category from Gansu.The recorded change for the famous region of Astragalus in history is from Sichuan to Gansu, Shaanxi, Ningxia, Shanxi, Inner Mongolia, and gradual migration to Northeast Heilongjiang.Main cultivated varieties of A. membranaceus also replaced A. Mongholicus.Based on the classification of A. mon-gholicus from A. membranaceus variants, the genetic relationship is very close.