毛世忠　李景剑　蒋小华　丁莉　赵博　邓涛　马虎生

摘要： 走马胎（Ardisia gigantifolia）是紫金牛科（Myrsinaceae）紫金牛属（Ardisia）多年生小灌木植物。走马胎作为我国传统中药材，已有多年的药用历史。目前，走马胎不再局限于临床药用，在食疗和保健方面的开发利用崭露头角，大大扩展了其应用范围。随着走马胎市场需求量的增大，野生走马胎植物被过度采挖，导致野生走马胎资源几乎枯竭。人工栽培走马胎逐渐成为供应药用市场的主力军，但是人工栽培走马胎种质、种子来源混杂，常会造成质量和疗效的不稳定，而利用分子标记技术可以从分子水平上对走马胎进行种质的区分和评价。该研究利用ISSR分子标记技术，对来自广西地区的36份走马胎种质资源进行了遗传多样性分析，采用POPGEN32软件进行数据分析，用UPGMA软件绘制聚类图。结果表明：14条ISSR引物共检测到136个清晰的扩增位点，多态性位点112个，多态位点百分率为82.35%；Neis基因多样性指数（H）为0.296 5，Shannon多样性指数（I）为0.441 7，基因分化系数（Gst）为0.855 8。个体间的遗传相似系数为0.667 8～0.838 2，平均为0.739 1。基于聚类分析可知，所有的个体被划分为5类，其中绝大多数来自相同或者邻近地区的个体严格按照地理位置聚为相同的一类或者亚类，只有少数个体在归类上与地理位置相悖。研究证明ISSR分子标记技术在评价走马胎种质资源亲缘关系和遺传变异等方面有很好的适用。该研究结果为该药用植物的种质资源评估和引种栽培提供了科学依据。

关键词： 走马胎， 分子标记， ISSR分子检测， 种质资源， 遗传多样性

Ardisia is the largest genus in the family of the Myrsinaceae， approximately consisting of 500 species of evergreen shrubs and trees throughout the subtropical and tropical regions of the world （Chen & Pipoly， 1996）. Ardisia species have been used as sources for both food and folk medicine. Ardisia gigantifolia， is mainly distributed in Guangxi Zhuang Autonanous Region， Guangdong， Fujian and Jiangxi provinces， China （Feng et al， 2011）. This plant has been used as an important traditional Chinese medicine to treat rheumatoid arthritis and inflammatory diseases in China for thousands of years as evident by ancient records （Mu et al， 2001）. A. gigantifolia is also used for treatment of bruises sprains， blood stasis， tumors and chronic ulcers diseases. Recently， diverse types of compounds have been isolated from this medicinal plant， such as saponins， coumarins， and quinones （Kobayashi & De Mejía， 2005）. In addition， very interesting bioactivities， such as antitumor， antiinflammation， antivirus， antiHIV， and antioxidation properties have been described for compounds isolated from this species （Kobayashi & De Mejía， 2005）. A literature survey showed that A. gigantifolia contained dimeric 1， 4benzoquinone derivatives that are rarely found in nature （Liu et al， 2009）. However， with the market demand increasing and destruction of wild resources intensifying， A. gigantifolia is being destroyed at an alarming rate， and has been listed as the rare medicinal plant （Mao et al， 2010）. Investigation of germplasm resources and evaluation of genetic diversity of A. gigantifolia will help ascertain the present condition of this medicinal plant and thus offer practical advices for its further breeding， utilization and conservation.

Genetic diversity analysis has proven to be a useful strategy for revealing genetic backgrounds and relationships of germplasm resources. In the past decades， research on A. gigantifolia has been carried out. However， most of these studies focused on morphology， cytology and biochemistry， very little is known about the genetic diversity and genetic variation in A. gigantifolia （Gao et al， 2015； Tuo & Wang， 2012； Shu et al， 2011）. Molecular markers based on polymerase chain reaction （PCR） are widely used since they require small amount of DNA and are effective and technically simple. The more popular markers are random amplified polymorphic DNA （RAPD） （Li & Nelson， 2002）， simple sequence repeat （SSR） （Qi et al， 2004）， and intersimple sequence repeat （ISSR） （Van der Nest et al， 2000）. The principle of ISSR is similar to RAPD in that randomly sequence primers used. However， the ISSR primer sequences consist of a dior trinucleotide simple sequence repeat amplifying the regions between two microsatellite repeats. As compared with RAPD， ISSR markers are more reproducible （Semagn et al， 2006） with polymorphic bands produced （Reddy et al，2002）. Since its establishment， ISSR has been widely used for analyses on genetic diversity， gene mapping and phylogenetic relationship in multiple species （Sun et al， 2014； Wei et al， 2014； Li et al， 2014； Li et al， 2015）.

The identification and estimation of genetic diversity and relationships of elite varieties or promising varieties of A. gigantifolia is important because it provides the basic information for breeding programs and conservation and can ultimately have a direct effect on the production and quality of this medicinal plant. In this work， our aims were to construct the fingerprinting and assess the genetic diversity of A. gigantifolia germplasm resources from different places of Guangxi in China using ISSR markers. Our study will uncover valuable raw materials that provide crucial genetic diversity information for conservation and breeding of A. gigantifolia.

1Materials and Methods

Though wild A. gigantifolia is distributed in Guangxi Zhuang Autonornous Region， Guangdong， Jiangxi and Fujian provinces， etc. in China， among which Guangxi is the main distribution region. Unfortunately， the wild A. gigantifolia is dispersedly distributed with very small number of individuals so that it is very difficult to find and collect. To evaluate the sampling strategies， we surveyed， collected， selected， conserved and documented 52 germplasm resources of A. gigantifolia mainly by phenotypic traits over a period of five years. According to geographic origins， all accessions of germplasm resources have been planted and conserved in the germplasm repository in Guangxi Institute of Botany， Guilin， China. For molecular assays， a total of 36 samples were originally and randomly collected from these plants. Fresh leaves in squaring period were randomly collected and desiccated in silica gel for molecular analyses. The details of the studied samples are summarized in Table 1.

Extraction of DNA was performed with 0.1 g starting material of leaves by using the Plant Genomic DNA Extraction kit （Tiangen Biotech Co.， China）， according to the manufacturers instructions. The DNA concentration was estimated by standard spectrophotometric method at 260 and 280 nm UV lengths by Thermo Scientific Nanodrop 2 000 and the integrity by gel electrophoresis in a 0.8% agarose gel. DNA samples were then diluted to 30 ng·μL1 work concentration.

A total of 100 ISSR primers were synthesized by Sangon Biotech （Shanghai） Co.， Ltd. （China）， according to the public biotechnology website of University of British Columbia. These primers were then screened using a few DNA samples and the primers which yielded polymorphism bands were used initially for amplification to optimize the PCR conditions. Finally， fourteen of them yielded clearly， reproducible and relatively high polymorphism bands were used for ISSR analysis （Table 2）. The PCR reaction was carried out in 25 μL of mixture containing 30 ng of genomic DNA， 2.0 mmol·L1 MgCl2， 0.2 mmol·L1 dNTP， 10 × PCR buffer， 1 mmol·L1 primer and 1 U Taq DNA polymerase （Sangon Biotech. Co.， Ltd.， Shanghai， China）. The PCR cycling conditions were as follows： 94 ℃ for 3 min （initial denaturation）， then followed by 39 cycles at 94 ℃ for 30 s （denaturation）， annealing at optimal temperature for 45 s， 72 ℃ for 2 min （extension）， with a final 7 min extension at 72 ℃ and then a cool down to 4 ℃. The amplified ISSR fragments along with DL2000 DNA marker （TaKaRa Co.， Ltd.， Dalian， China） were resolved in 1.5% agarose gels in TAE buffer at 110 V for 50 min. Gels with amplification fragments were visualized and photographed under UV light.

The amplified DNA fragments were identified as present （1） or absent （0）. The dataset was converted into a mathematical matrix used by the POPGENE32

software to perform statistical analyses， and calculate the index of genetic diversity， the observed total number of alleles （Na）， effective allele number （Ne）， percentage of polymorphic bands （PPB）， Nei gene diversity （He， Nei， 1973）， and Shannons information index （I， Shannon & Weaver， 1949）. A cluster dendrogram （UPGMA） was constructed to evaluate the genetic relationship for these accessions based on the average genetic distances using the NTSYS software （Yeh et al， 1997； Rohlf， 2000）.

2Results and Analysis

The extent of genetic diversity among the thirtysix accessions of A. gigantifolia， a total of 100 ISSR primers were screened and fourteen primers yielded clearly， reproducible and relatively high polymorphism bands were selected for further ISSR analysis （Fig. 1）. ISSR primers produced different numbers of DNA fragments， depending upon their simple sequence repeat motifs. The fourteen selected primers generated altogether 136 unambiguous and reproducible bands， of which 112 （82.35%） were polymorphic， the sizes ranging from 250 to 2 000 bp， the numbers of bands varied from eight to twelve， with an average of 9.7 bands per primer （Table 2）.

The number of alleles is one of the most important genetic components for genetic diversification in populations. The percentage of polymorphicbands （PPB） for this species was 82.35% （Table 2）. The mean observed number of alleles （Na） was 1.823 5， while the mean effective number of alleles （Ne） was 1.510 1. The Neis gene diversity （H） was 0.296 5， and Shannons indices （I） was 0.441 7. These results demonstrate that A. gigantifolia has a relatively high level of genetic diversity.

The total gene diversity （Ht） and the gene diversity within populations （Hs） were 0.274 1 and 0.039 5 respectively. According to Gst = Ht-Hs/Ht， all the samples of this species from different locations had a coefficient of genetic diversification （Gst） of 0.855 8.

Based on similarity coefficient among 36 samples， cluster analysis was carried out using UPGMA method and resulted in a phylogenetic dendrogram shown in Fig. 2. All the samples that could be classified into two

Fig. 3Threedimensional plot of the principal coordinate analysis （PCoA） of distance among 36 A. gigantifolia accessions

big groups and then further divided into five subgroups with a similarity coefficient value of 0.70. The clustering result of A. gigantifolia samples corresponded generally with the geographical distribution of their collections. However， there were still a few accessions could not be distinguished clearly. Generally， the result of PCoA was in accordance with that of genetic clustering analysis （Fig. 3）. These findings clearly indicate a distinct differentiation between A. gigantifolia germplasms from various geographic origins.

3Discussion and Conclusion

In recent years， a number of quantitative measures of genetic diversity have been proposed in the context of species conservation （Krajewski， 1994）. The studies of the genetic diversity do not only help us to understand the present condition and appreciate genetic structure of our resources， but also help conserve biodiversity， as well as development and utilization of germplasms. In China， there are countless medicinal plants that have been widely used for treatment of many kinds of diseases. Nevertheless， with the increasingly serious environmental damage and excessive collection， many wild germplasm resources are facing destruction and are in danger of extinction. It would be an unprecedented disaster if medical cures were lost due to the extinction of these plants. Although A. gigantifolia had a long cultivating history as medicinal plant， there is little research on evaluating its germplasm diversity and how to construct a core collection. We investigated and evaluated the wild and cultivated A. gigantifolia in the past few years. Our results showed that the wild resources had been excessively excavated in the past decade and the wild resources reserves were not sufficient to meet the growing demands of medical utilization and chemical extraction industry. The cultivating area of A. gigantifolia has been increasing in recent years but still inadequate to meet the supply and demand in China. Some old problems are still unresolved and new problems are arising， such as decline in genetic diversity， yield and quality， etc. All these problems need to be resolved in the future. The development of a core collection will help cultivators create lowcost strategies to evaluate this medicinal plant germplasm.

There are different sampling strategies to construct core collection for germplasm resources. Cluster and principal coordinate analysis have been widely used as an important tool to group samples for constructing core collections （Zhang et al， 2004； Hintum， 1995） and grouping data based on molecular markers （Chabane & Valkoun，2004）. In the present study， we tested the usability of ISSR primers to investigate the level and distribution of genetic diversity in wild and cultivated populations of A. gigantifolia from Guangxi. We used fourteen out of the 100 ISSR primers tested， and 112 polymorphic bands in a total of 136 bands （PPB=82.35%） were identified in the 36 accessions. The analyzed results of genetic diversity parameters showed that the initial collection of A. gigantifolia germplasm resource had large genetic diversity at a molecular level （Na=1.823 5，Ne=1.510 1，He=0.296 5，I=0.441 7）. The cluster dendrogram （UPGMA） and principal coordinate analysis （PCoA） clearly showed the genetic relationship among all germplasms. All the germplasms and genetic information not only provided valuable raw materials to construct core collection of A. gigantifolia， but also provided the information for selecting and perserving the quality of this medicinal plant.

In conclusion， the results in this study indicated a great level of genetic variation among Guangxi wild A. gigantifolia germplasm. The majority of these natural accessions from similar or adjacent regions were clustered into one group with a few exceptions. Those accessions growing in similar environment generally were also clustered together. This result indicated that differentiation of A. gigantifolia gene pools from different regions might have resulted from reproductive isolation and divergent natural selection arising from wide geographic separation. However， there were still a few accessions showed a great difference from the majority of accessions， which might be due to the diffusion of introduced species or the introgression. We assume that human activities maybe one possible reason for the anomaly. These accessions were collected from their original populations and may have been cultivated in different places. It is important to collect and protect natural populations of A. gigantifolia in Guangxi， and contamination or difusion of introduced species must be avoided. In addition， in order to broaden genetic bases and improve genetic depression of the cultivated accessions， it is necessary to select wild accessions with greater genetic differences as parent accessions for crossbreeding， and these combinations might have potential for improving the quality and yield of the cultivars.

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