Molecular Diversity Analysis of Tetradium Ruticarpum (Wuzhuyu) In
Total Page：16
File Type：pdf, Size：1020Kb
Chinese Journal of Natural Chinese Journal of Natural Medicines 2018, 16(1): 00010009 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 am- plified 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 – 1 – XU Jing-Yuan, et al. / Chin J Nat Med, 2018, 16(1): 19 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 dis- played 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 – 2 – XU Jing-Yuan, et al. / Chin J Nat Med, 2018, 16(1): 19 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.