Molecular Diversity Analysis of Tetradium Ruticarpum (Wuzhuyu) In

Chinese Journal of

Natural Chinese Journal of Natural Medicines 2018, 16(1): 00010009 Medicines

doi: 10.3724/SP.J.1009.2018.00001

•Research Articles•

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers

XU Jing-Yuan, ZHU Yan, YI Ze, WU Gang, XIE Guo-Yong, QIN Min-Jian*

Department of Resources Science of Traditional Chinese Medicines, State Key Laboratory of Natural Medicines, China Pharma- ceutical University, Nanjing 210009, China Available online 20 Jan., 2018

[ABSTRACT] “Wu zhu yu”, which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

[KEY WORDS] Genetic diversity; Tetradium ruticarpum; iPBS; Retrotransposon; ISSR [CLC Number] R96 [Document code] A [Article ID] 2095-6975(2018)01-0001-09

 are known collectively as Tetradium ruticarpum (A. Jussieu) Introduction T. G. Hartley. “Wu zhu yu” has been used as a traditional Chinese T. ruticarpum is a shrub or tree widely distributed in the medicine for curing headaches, abdominal colic and hyper- southern Qinling Mountains. As a large number of the raw tension for thousands of years. According to “Chinese Phar- material demand for Chinese patent medicines, the wild re- macopoeia”, “Wu zhu yu” originates from the dried unripe sources of the plant are exhausted quickly. In some regions of fruits of Evodia rutaecarpa (Juss.) B enth., E. rutaecarpa var. China, the local farmers have begun to introduce and cultivate bodinieri (Dode) Huang, and E. rutaecarpa var. officinalis T. ruticarpum for meeting the market need. In previous stud- (Dode) Huang, which belongs to the genus Evodia of Ruta- ies, the morphology and chemical components of T. ruticar- ceae [1]. However, according to the latest classification system pum growing in different climatic and ecological environ- of the Rutaceae family in the “Flora of China” [2], the three ments showed significant variations [3-4], but the genetic basis varieties have been rearranged into the genus Tetradium, and of these variations has not been studied. Molecular markers are useful tools to evaluate the genetic variation of plants. [Received on]18-May-2017 Recently, Kalendar et al. [5] have developed an exceedingly [Research funding] This work was supported by the National Sci- efficient and universal molecular marker, the inter-primer ence and Technology Major Projects for “Major New Drugs Innova- binding site (iPBS), based on the conversed sequences of tion and Development” and the “Chinese Herbal Medicine Seeds and retrotransposons. As a class of repetitive and mobile se- Seedlings Planting (breeding) Standard Platform Topics” (No. quences, as well as ubiquitous and abundant components in 2012ZX09304006). [*Corresponding author] Tel: 86-25-86185130, Fax: 86-25-85301528, higher plants, retrotransposons have provided the potential for [6-7] E-mail: [email protected] the development of multiplex DNA-based marker systems . These authors have no conflict of interest to declare. The marker has been successfully employed in flax [8] and

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Saussurea [9] to evaluate genetic diversity. Based on size poly- collected samples were T. ruticarpum authenticated by Pro- morphisms of inter-microsatellite spacers, inter-simple sequence fessor Qin Min-Jian. The names, numbers, and geographic repeats (ISSR) has also been recognized as a useful molecular information are listed in Table 1. These accessions were marker for analyzing genetic diversity [10-11]. To evaluate genetic planted in the Medicinal Botanical Garden of the China variations of T. ruticarpum from different regions of China, we Pharmaceutical University, and their fresh leaves were ran- established iPBS and ISSR marker methods that would be ap- domly collected and stored with silica gel in zip-lock bags propriate for assessing genetic diversity of the species. It would until DNA extraction. provide basic genetic diversity information for the germplasm DNA extraction conservation and breeding of the species. Genomic DNA was extracted from the silica gel-dried leaves using a modified cetyltrimethyl ammonium bromide Materials and Methods method [12]. The quality of the DNA was determined by elec- Plant materials trophoresis in 1% agarose gels, and the concentration of the Several field investigation trips were conducted across DNA was determined using BioPhotometer plus (Eppendorf, the geographic range of T. ruticarpum in 2013, and 25 acces- Hamburg, Germany). DNA samples were diluted to 10 ng·µL−1 sions were sampled from 6 Chinese provinces (Fig. 1). All the and stored at –20 °C for PCR amplification.

Fig. 1 The collection sites of 25 accessions of Tetradium ruticarpum. The accession code at each point corresponds to those displayed in Table 1 iPBS-PCR amplification extension of 5 min at 72 °C. PCR products were separated on Ten iPBS primers that amplified strong and clear bands 4% non-denaturing polyacrylamide gels that were stained were selected (Table 2) for genetic diversity evaluation out of using a silver staining protocol for visual detection. the 30 designed by Kalendar et al. [5]. With slight modifica- ISSR-PCR amplification tions, the amplification reaction was performed as described A total of 13 primers that produced successful amplifica- by Kalendar et al. [5]. PCR reaction was set in volume of tion patterns were selected (Table 2) from the initial screening 20 µL containing 1 µL of the 10 × PCR buffer, 3 mmol·L−1 of of 35 ISSR primers. Primer sequences were obtained from the Mg2+, 0.4 mmol·L−1 of dNTPs, 1 µmol·L−1 of primer, 0.5 U UBC Primer Set #9 (Microsatellite) designed by University of Taq polymerase (Sango Co., Ltd., Shanghai, China), 0.5 U British Colombia (UBC) in Canada. PCR amplifications were Pfu polymerase (Sango Co., Ltd., Shanghai, China), and 40 carried out in a 20-µL volume solution containing 40 ng of ng of genomic DNA. The PCR program was run as follows: template DNA, 0.6 µmol·L−1 of primer, 2.25 mmol·L−1 of initial denaturation at 95 °C for 3 min, followed by 30 cycles Mg2+, 180 µmol·L−1 of dNTPs and 1.0 U of Taq polymerase of 15 s at 95 °C, 1 min at 55 °C, 1 min at 65 °C, and a final (Sango Co., Ltd., Shanghai, China). The protocol for PCR

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Table 1 T. ruticarpum accessions used for analysis in the present study Accession code Origins Latitude/° Longitude/° Altitude (m) ZWY Jiangning, Nanjing, Jiangsu 31.90 118.91 12 YY Qixia, Nanjing, Jiangsu 32.10 118.94 13 DG Yangzhou, Jiangsu 32.39 119.44 10 NC Nanchang, Jiangxi 28.67 115.75 40 JL Jinglou, Zhangshu, Jiangxi 28.06 115.41 1 011 DQ Daqiao, Zhangshu, Jiangxi 28.02 115.36 1 012 WC Wucheng, Zhangshu, Jiangxi 27.98 115.27 1 011 SL Xinwo, Jinhua, Zhejiang 28.95 120.38 339 DP Dapan, Jinhua, Zhejiang 29.00 120.55 488 JY Jinyun, Lishui, Zhejiang 28.82 120.40 237 JN Jinan, Shandong 36.56 116.80 65 HL Huanglei, Huaihua, Hunan 28.18 108.93 421 HHD Henghedi, Linxiang, Hunan 29.71 113.49 24 WL-A Wuli, Linxiang, Hunan 29.47 113.48 37 WL-B Wuli, Linxiang, Hunan 29.48 113.48 34 ND Nanda, Yuanjiang, Hunan 29.00 112.73 37 SHS Sihushan, Yuanjiang, Hunan 28.97 112.65 46 YS Yanshang, Tongren, Guizhou 27.72 109.02 408 SY Suyang, Zunyi, Guizhou 27.80 107.80 852 SQ Shiqian, Tongren, Guizhou 27.55 108.29 782 XS Xiaosai, Zunyi, Guizhou 27.13 107.47 782 BN Baini, Zunyi, Guizhou 27.21 107.90 703 ZZ Zhizhou, Zunyi, Guizhou 27.32 107.75 857 GZ Guanzhuang, Tongren, Guizhou 27.69 109.00 402 HJ Hongjun, Zunyi, Guizhou 27.18 107.43 863

Table 2 The iPBS primers and ISSR primers used in this the present study Marker Primer Sequence (5′-3′) 2 076 GCT CCG ATG CCA 2 237 CCC CTA CCT GGC GTG CCA 2 238 ACC TAG CTC ATG ATG CCA 2 079 AGG TGG GCG CCA 2 377 ACG AAG GGA CCA iPBS 2 270 ACC TGG CGTG CCA 2 271 GGC TCG GATG CCA 2 221 ACC TAG CTC ACG ATG CCA 2 230 TCT AGG CGT CTG ATA CCA 2 252 TCA TGG CTC ATG ATA CCA UBC836 AGA GAG AGA GAG AGA GYA UBC826 ACA CAC ACA CAC ACA CC UBC855 ACA CAC ACA CAC ACA CYT UBC890 VHV GTG TGT GTG TGT GT UBC808 AGA GAG AGA GAG AGA GC UBC809 AGA GAG AGA GAG AGA GG ISSR UBC810 GAG AGA GAG AGA GAG AT UBC812 GAG AGA GAG AGA GAG AA UBC834 AGA GAG AGA GAG AGA GYT UBC835 AGA GAG AGA GAG AGA GYC UBC816 AGA GAG AGA GAG AGA GYA UBC841 GAG AGA GAG AGA GAG AYC UBC857 ACA CAC ACA CAC ACA CYG

– 3 – XU Jing-Yuan, et al. / Chin J Nat Med, 2018, 16(1): 19 amplification was run as follows: an initial step of denatura- sions using the SHAN module of NTSYS-pc version 2.1 tion at 94 °C for 7 min, followed by 30 cycles of 94 °C for 30 s, (Exeter Software, Setauket, NY, USA). To construct a multi- 50–54 °C for 1 min, 72 °C for 2 min, and a final extension at ple dimensional array of eigenvectors, a principal coordinate 72 °C for 7 min. The amplified products were separated on a analysis (PCoA) was performed using the NTSYS program. 2% agarose gel containing 1.2 µL of GoldView in 1 × Tris- Results Acetate-EDTA buffer. The images were documented using the Gel Doc XR+ System (Bio-Rad, Hercules, California, USA) Polymorphism analysis under a UV light. Thirty iPBS primers were designed for the initial screen- Data analysis ing. 10 iPBS primers were selected for further analysis based Clear, reproducible and well-separated bands were se- on an evaluation of polymorphism performance, reproducibil- lected for scoring. Each iPBS or ISSR fragment was assigned ity, and readability (Fig. 2), and the amplified bands per as “1”, which indicated its presence, or “0”, which indicated primer varied from 24 (2 230) to 11 (2 076). The mean num- its absence. The polymorphic information content (PIC) was ber of bands per marker was 16.5. Of all of the amplified calculated to measure the effectiveness of the iPBS and ISSR bands, 152 were polymorphic, with an average of 15.2 poly- markers using the following formula [13]: PIC = 1 – ∑Pi2, morphic fragments per primer. The percentage of polymor- where Pi is the frequency of the genotype I. The resolving phic bands ranged from 78.9% (2 270) to 100% (2 076, 2 238, power (Rp) was calculated according to Gilbert et al. [14]: Rp = 2 230 and 2 252), with an average of 92.5%. The lowest

∑Ib, where Ib is the “band effectiveness”. Ib can be calculated (0.881) PIC was from iPBS primer 2 377 and the highest PIC by the formula: Ib = 1 – (2 × |0.5 – P|), where P is the fre- value (0.937) was from iPBS primer 2 230, with an average quency of varieties containing band I. of 0.914. The Rp values ranged from 11.20 (2 230) to 4.32 (2 The binary data matrix was analyzed using POPGENE 377) (Table 3). version 1.32 (Molecular Biology and Biotechnology Centre, 13 ISSR primers that showed potential polymorphisms University of Alberta, Edmonton, AB, Canada). The follow- were selected (Fig. 3). Total 151 bands were obtained, and ing parameters were obtained to estimate the genetic diversity 130 bands (86.1%) were polymorphic. The average number of at the species level: the percentage of polymorphic loci (PPL), bands and the polymorphic bands per primer generated were Nei’s gene diversity [15], Shannon’s information index (I) [16], 11.6 and 10, respectively. The percentage of polymorphic the total genetic diversity (Ht), genetic differentiation coeffi- markers produced by each primer ranged from 57.1% [17] cient (Gst), and gene flow (Nm) . Genetic distance and ge- (UBC836) to 100% (UBC809, UBC834, and UBC835). The netic identity were also generated by POPGENE to examine lowest PIC value (0.807) and the highest PIC value (0.926) the genetic relationships among these accessions. Based on were from ISSR primers UBC826 and UBC836, respectively. the unweighted pair group method with arithmetic averaging The Rp values ranged from 8.88 (UBC816) to 1.76 (UBC808) (UPGMA), a dendrogram was constructed for all 25 acces- (Table 3).

Fig. 2 Amplification profile of 25 accessions with iPBS primer 2270 . Lanes: 1) ZWY, 2) YY, 3) DG, 4) NC, 5) JL, 6) DQ, 7) WC, 8) SL, 9) DP, 10) JY, 11) JN, 12) HL, 13) HHD, 14) WL-A, 15) WL-B, 16) ND, 17) SHS, 18) YS, 19) SY, 20) SQ, 21) XS, 22) BN, 23) ZZ, 24) GZ, 25) HJ; M: DNA Marker-D (Sangon Biotech)

Genetic diversity and distance est genetic identity value were found between accessions YS In the evaluation of genetic diversity and differentiation, and GZ. the data generated by iPBS primers were useful. The value of For the ISSR marker, Ht = 0.2624, Gst = 0.5694, and Nm =

Ht = 0.282 0, Gst = 0.477 2, Nm = 0.547 8, h = 0.285 3, and I = 0.3782 at the species level (Table 4). The other two genetic 0.431 6 at the species level (Table 4). The genetic distances diversity indices, I and h, had similar values to those of the were calculated to estimate the extent of their divergence. The iPBS markers. The genetic distance and genetic identity genetic distances (Online Resource 1) ranged from 0.082 1 to (Online Resource 2) ranged from 0.040 5 to 0.562 9 and from 0.886 4 and genetic identities ranged from 0.412 1 to 0.921 2, 0.569 5 to 0.960 3, respectively. The greatest genetic distance among which, the largest genetic distance value and the and the smallest identity were both found between the acces- smallest genetic identity value were found between accessions sions JL and GZ. The shortest genetic distance and the largest XS and JL. The smallest genetic distance value and the great- genetic identity values were found between ZWY and DG.

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The accessions XS and SQ had the same genetic distances divided into three sub-clusters. The accessions of sub-clusters and genetic identity values as those of ZWY and DG. d and e, except for GZ and YS, were collected from Hunan Cluster analysis Province. Accessions in sub-cluster f were all from Zunyi Based on the iPBS data, a dendrogram (Fig. 4) was con- City in Guizhou Province. structed using the UPGMA analysis. The 25 T. ruticarpum Using the ISSR data, a dendrogram (Fig. 5) grouped the accessions were divided into two major clusters (A1 and B1) 25 accessions into three main clusters (A2, B2, and C2) with a with a genetic similarity of 0.67. Cluster-A1 was further sub- genetic similarity of 0.682. Cluster A2 mainly included 18 divided into three sub-clusters. Sub-cluster a, which included accessions from Zhejiang, Guizhou and Hunan Provinces. It eight accessions from Jiangsu, Shandong and Jiangxi Prov- could be further divided into three sub-clusters, a (three ac- inces, could be further grouped into two clusters (I and II). cessions from Jiangsu), b (eight accessions from Guizhou, Sub-cluster b contained only one accession “HHD”. except JN from Shandong) and c (seven accessions from Hu-

Sub-cluster-c included all the three accessions collected from nan, except YS from Guizhou). Cluster B2 included three

Zhejiang Province. Cluster-B1 included 13 accessions mainly accessions that were collected from Zhejiang. The accessions from Hunan and Guizhou Provinces, and they also could be from Jiangxi were all placed in Cluster C2.

Table 3 The observed genetic diversity based on iPBS markers and ISSR markers Marker Primer TBa PBb PPLc(%) PICd Rpe 2 076 11 11 100.0 0.904 7.28 2 237 17 16 94.1 0.905 6.24 2 238 15 15 100.0 0.924 9.92 2 079 19 16 84.2 0.931 5.60 2 377 12 11 91.7 0.881 4.32 iPBS 2 270 19 15 78.9 0.927 7.44 2 271 15 14 93.3 0.906 5.20 2 221 17 14 82.4 0.915 5.20 2 230 24 24 100.0 0.937 11.20 2 252 16 16 100.0 0.913 8.80 Average 16.5 15.2 92.5 0.914 7.12 UBC836 17 14 82.4 0.926 8.88 UBC826 7 5 71.4 0.807 3.36 UBC855 15 14 93.3 0.900 6.48 UBC890 11 10 90.9 0.866 5.12 UBC808 11 7 72.7 0.859 1.76 UBC809 11 11 100.0 0.871 3.76 UBC810 10 9 90.0 0.845 3.52 ISSR UBC812 8 5 71.4 0.828 2.88 UBC834 16 16 100.0 0.905 6.48 UBC835 15 15 100.0 0.886 6.64 UBC816 7 4 57.1 0.847 3.12 UBC841 9 8 88.9 0.854 3.12 UBC857 14 12 85.7 0.903 4.20 Average 11.6 10 84.9 0.869 4.56

aTB: The number of total bands; bPB: The number of polymorphic bands; cPPL: The percentage of polymorphic loci; dPIC: Polymorphic information content; eRp: Resolving power

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Fig. 3 Amplification profile of 25 accessions with ISSR primer, ISSR profiles of 25 accessions using primer UBC857; 1–25 Lanes represent the same accessions as listed in Fig. 2. M: DNA Marker-D (Sangon Biotech)

Table 4 Nei’s analysis genetic diversity of T. ruticarpum accessions based on iPBS and ISSR data Marker Hta Gstb Nmc hd Ie iPBS 0.282 0 0.477 2 0.547 8 0.285 3 0.431 6 ISSR 0.262 4 0.569 4 0.378 2 0.259 6 0.395 7 Note: aHt = The total genetic diversity; bGst = genetic differentiation coefficient; cNm = gene flow; dh = Nei’s (1973) gene diversity; eI = Shannon's Information index

Fig. 4 Dendrogram of 25 T. ruticarpum accessions obtained using UPGMA cluster analysis of iPBS data (the accession code corresponds to those displayed in Table 1)

Fig. 5 Dendrogram of 25 T. ruticarpum accessions obtained using UPGMA cluster analysis of ISSR data (the accession code corresponds to those displayed in Table 1)

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The UPGMA cluster (Fig. 6), which was constructed of Rutaceae in the “Flora of China” [2]. Based on the data in using a combination of data from iPBS and ISSR markers, the present study, we no longer consider varieties during the revealed more genetic relations than the individual markers, sampling of the accessions, all of the collected accessions and it separated the 25 accessions into 5 groups mainly were treated as equivalent, which was different from the based on the geographic locations. The dendrogram was methods used in the assessment of the genetic diversity of similar to that constructed using iPBS or ISSR data. Asso- Evodia rutaecarpa (synonym of T. ruticarpum) reported by ciations among the 25 accessions were also resolved by the Huang et al. [19] and Wei et al. [20]. The samples of the present PCoA (Fig. 7). Five main groups were shown in the diagram study were collected from 6 Chinese provinces, representing 4 generated by the PCoA, and they revealed a similar cluster different geographical regions of China, according to differ- result as that shown in Fig. 4. The two principal axes in the ences in climate and terrain, and the sampling sites covered PCoA plot accounted for 15.13% and 13.34% of the total the major distribution areas of T. ruticarpum in China. variation, respectively. As wide geographical distribution and long-time natural Discussion selection, the complicated climate and ecological environ- ments may cause some variations in morphological characters T. ruticarpum is one of the most widely distributed spe- and interior chemical composition of the plant. Genetic vari- cies in the genus Tetradium. According to old classification of ability is the basis for plants adapting to different environment Rutaceae, the species, named as Evodia rutaecarpa (Juss.) and a species without enough genetic diversity is thought to be Benth., includes three varieties [18]. As the boundary and dis- difficult to cope with the changing environment [21]. Genetic tinction among those varieties is not obvious, the three varie- variations of plants could be evaluated by loci polymorphism, ties have been abolished from the latest classification system and high, medium and low loci polymorphisms are in accorded

Fig. 6 Dendrogram of 25 T. ruticarpum accessions obtained using UPGMA cluster analysis of iPBS and ISSR data (the accession code corresponds to those displayed in Table 1)

Fig. 7 PCoA plot of the first two principal component of principal coordinate analysis based on iPBS and ISSR data. The accession code at each point corresponds to those displayed in Table 1

– 7 – XU Jing-Yuan, et al. / Chin J Nat Med, 2018, 16(1): 19 to PIC > 0.5, 0.5 > PIC > 0.25 and PIC < 0.25, respectively, values of iPBS for T. ruticarpum (Table 3) were higher than according to Vaiman et al. [22] and Xie et al. [23]. The average PIC those of the ISSR and SSR markers for Triticum dicoccon values in the present study were higher than 0.5, indicating Schrank from India reported by Salunkhe et al. [35], indicating that T. ruticarpum possessed high loci polymorphism. Poly- that the iPBS markers were suitable for the rapid determina- morphism detected in a species can be given in terms of esti- tion of T. ruticarpum genetic diversity. mate of gene flow (Nm) and coefficient of population differ- In conclusion, the present study represented a first effort [24] entiation (GST) . As an indicator of gene movements, gene to investigate the genetic diversity of T. ruticarpum by com- flow is negatively correlated with gene differentiation [25], and bining iPBS and ISSR data. Compared to the classical mo- is very important for population transfers and evolution. The lecular genetic markers, the iPBS marker was an effective gene flow (Nm) for T. ruticarpum (Table 4) was lower than the new approach to evaluating the genetic diversity of plants. limit value set by Wright [26], where values below 1 indicate The results showed that the accessions of T. ruticarpum pos- genetic isolation. This result clearly indicated that the gene sessed high loci polymorphism and genetic differentiation migration was limited. Hamrick [27] has reported that 16 spe- occurred in this plant. The accessions of T. ruticarpum in the cies of cross-pollinating plants have a higher gene flow (Nm), present study could be clustered into several groups which ap- with an average of 1.15. In the present study, as related to the proximately matched the geographical distribution of the spe- iPBS and ISSR markers, the Nm for T. ruticarpum was 0.547 8 cies. The present study also found that the gene flow of the T. and 0.378 2, respectively, which was significantly lower than ruticarpum was significantly lower than the average of the the average of the cross-pollinated plants. The possible rea- cross-pollinated plants, which might mainly be due to the sons for lower Nm may be that most of seeds of T. ruticarpum seed infertility and lower seed germination ratio, asexual re- are infertility and difficult to germinate and that the T. ruti- production, and geographical obstacles. These findings from carpum usually reproduces asexually. According to Wright [28], the present study would contribute to the germplasm conser- the values of Gst higher than 0.25 indicate a very great gene vation and further breeding of T. ruticarpum. differentiation between the accessions being compared and it may be explained by geographic isolation of populations [29]. 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An initial investigation of the taxonomic greater than that of the ISSR markers (0.869) in the present status of Saussurea esthonica, Baer ex Rupr. utilising DNA study. It showed that the iPBS markers can detect more markers and sequencing [J]. Plant Syst Evol, 2012, 298(5): abundant loci polymorphism of T. ruticarpum. When describ- 913-919. ing the discriminating ability of primers in a genetic diversity [10] Ajal EA, Jbir R, Melgarejo P, et al. Efficiency of Inter Simple study, Rp was found to be more suitable [34]. The average Rp Sequence Repeat (ISSR) markers for the assessment of genetic

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Cite this article as: XU Jing-Yuan, ZHU Yan, YI Ze, WU Gang, XIE Guo-Yong, QIN Min-Jian. Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers [J]. Chin J Nat Med, 2018, 16(1): 1-9.

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