| Astragalus mongholicus and Astragalus membranceus are members of the genus Astragalus (Fabaceae) and mainly distributed in northern China and Mongolia. According to Pharmacopoeia of the People’s Republic of China (2010), they are widely used medicinal plants in Traditional Chinese Medicine (TCM) known as Radix Astragali (or Huangqi in Chinese), and a well-known ethnomedicine in many ethnic groups. The roots have been used for centuries in China as a therapeutic agent for Yang weakness and Qi deficiency. Modern studies have shown that the main active ingredients in Huangqi were the triterpenoid saponin, flavonoids, polysaccharides, amino butyric acid and various trace elements. It also reported that Huangqi have hepatoprotective, antidiabetic, diuretic, sedative, immunomodulatory, anti-cancer, anti-inflammatory, anti-viral, anti-aging and antioxidant effects, and could also protect the neuron cells. This medicine has attracted global attention, and the demand for it increased rapidly these years. With the decrease of wild Huangqi resources, there is an increasing amount of cultivated resources. In this study, we carried out surveys on Huangqi resources, developed ten polymorphic microsatellite markers for A. mongholicus, evaluated the genetic diversity of Huangqi resources using these primers, and authenticated the Huangqi purchased from the market. The main conclusions are as follows:(1) We conducted the field survey in Inner Mongolia Autonomous Region, Gansu, Shaanxi, Shanxi, Heilongjiang and Jilin Province. With the pursuit of healthy life and Radix Astragali has the role of improving immunity, the demand for this medicine increased rapidly which had led to the diminishing of wild resource. Nowadays, there are only a small amount of wild plants in remote mountainous areas, such as the Greater Khingan Mountains and Changbai Mountain area, and the wild resources can not meet the demand of the people for Radix Astragali, In our field work, we collected7wild populations of A. membranaceus in Inner Mongolia Autonomous Region, Heilongjiang and Jilin Province. We also collected7populations of cultivated A. mongholicus in Inner Mongolia Autonomous Region, Gansu and Shanxi Province, and1population of wild A. mongholicus in Inner Mongolia. During our survey, we also found some problems in cultivation of these plants, and the most serious problem is the disease, which has negative effects on Huangqi quality and quantity. On the other hand, some businessmen use sulfur and other chemical substances to fumigate Huangqi in order to prevent it from bugs, seriously affecting the quality of the medicine.(2) Modern studies on Huangqi mainly focused on separation of active ingredients and pharmacological analysis, and only a few studies on germplasm resources. Microsatellite markers are powerful tools for evaluating genetic diversity and have the advantage of codominant and high polymorphism. But so far, there is no report on microsatellite primers development for A. mongholicus or A. membranaceus. In this study, for the first time, ten novel microsatellite loci were developed for A. mongholicus. Through the construction of microsatellite DNA library, screening positive clones, sequencing, primers design and polymorphism screening,10pairs of primers with high polymorphism were developed.30individuals were used to evaluate these primers and123alleles were detected. The number of alleles ranged from4to19, with an average number of alleles12.3. The observed and expected heterozygosities ranged from0.367to1.000and from0.395to0.912, respectively. These markers will be useful for population genetics and molecular ecology studies of this plant.(3) We evaluated the genetic diversity of eight A. mongholicus populations and seven A. membranaceus populations using the ten polymorphic microsatellite primers. The results showed that A. mongholicus populations had high level of genetic diversity. The average number of alleles for each population was from6.2to13.8(with average of11.2), of which WC population collected from Inner Mongolia had the highest average number of alleles (13.8); followed by GY population (13.0), and the lowest was HS population (6.2). The average gene diversity was0.822, and the highest was0.863(WC population), followed by0.852(LX population). Average observed heterozygosity was0.789and average expected heterozygosity was0.797. The study also found cultivated A. mongholicus populations had higher level of genetic diversity than that of wild population, which might because we only have one wild A. mongholicus population and it can not reflect the total genetic diversity of wild A. mongholicus. Genetic diversity of the wild A. membranaceus populations was also evaluated. The results showed that the average number of alleles for each population was from5.0to8.8(with average of7.2), and the highest number of alleles was8.8(ED population), followed by8.6(CH population), and the lowest was5.0(XL population). The average gene diversity was0.689, with the highest in CH population (0.807) and the lowest in DYS population (0.608). The average observed heterozygosity and expected heterozygosity were0.738and0.667, respectively. Molecular variance (AMOVA) analysis showed that genetic variation of A. mongholicus was mainly within populations (94%), and only6%of the genetic variation was among populations. Therefore, there was only a little degree of differentiation between the populations. For wild A. membranaceus,72%of genetic variation was within populations and28%among populations, therefore there was a certain degree of differentiation among populations. The number of gene flow also showed similar results. Based on SSR analysis, genetic differentiation coefficient (FST) of A. mongholicus populations was0.0658, and gene flow Nm=3.549. For wild A. membranaceus, genetic differentiation coefficient was0.2262and gene flow Nm=0.855. Furthermore, we used principle coordinates analysis to investigate relationship between A. membranaceus and A. mongholicus, and found that the two species are different morphologically.(4) In this study, we also authenticated the Huangqi purchased from the market. The experiments showed that the candidate DNA barcoding markers ITS and trnH-psbA had the ability to distinguish Huangqi from its adulterants. Compared with ITS, the trnH-psbA fragment had more variation sites, providing more ability to distinguish Huangqi from its adulterants. |
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