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

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

Chinese Journal of Natural Medicines

•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 Pharmaceutical 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

Introduction

are known collectively as Tetradium ruticarpum (A. Jussieu) T. G. Hartley. T. ruticarpum is a shrub or tree widely distributed in the southern Qinling Mountains. As a large number of the raw material demand for Chinese patent medicines, the wild resources of the plant are exhausted quickly. In some regions of China, the local farmers have begun to introduce and cultivate T. ruticarpum for meeting the market need. In previous studies, the morphology and chemical components of T. ruticarpum growing in different climatic and ecological environments showed significant variations [3-4], but the genetic basis of these variations has not been studied. Molecular markers are useful tools to evaluate the genetic variation of plants. Recently, Kalendar et al. [5] have developed an exceedingly efficient and universal molecular marker, the inter-primer binding site (iPBS), based on the conversed sequences of retrotransposons. As a class of repetitive and mobile sequences, as well as ubiquitous and abundant components in higher plants, retrotransposons have provided the potential for the development of multiplex DNA-based marker systems [6-7]. The marker has been successfully employed in flax [8] and

“Wu zhu yu” has been used as a traditional Chinese medicine for curing headaches, abdominal colic and hypertension for thousands of years. According to “Chinese Pharmacopoeia”, “Wu zhu yu” originates from the dried unripe fruits of Evodia rutaecarpa (Juss.) B enth., E. rutaecarpa var. bodinieri (Dode) Huang, and E. rutaecarpa var. officinalis (Dode) Huang, which belongs to the genus Evodia of Rutaceae [1]. However, according to the latest classification system of the Rutaceae family in the “Flora of China” [2], the three varieties have been rearranged into the genus Tetradium, and [Received on]18-May-2017 [Research funding] This work was supported by the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” and the “Chinese Herbal Medicine Seeds and Seedlings Planting (breeding) Standard Platform Topics” (No. 2012ZX09304006). [*Corresponding author] Tel: 86-25-86185130, Fax: 86-25-85301528, E-mail: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

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Saussurea [9] to evaluate genetic diversity. Based on size polymorphisms of inter-microsatellite spacers, inter-simple sequence repeats (ISSR) has also been recognized as a useful molecular marker for analyzing genetic diversity [10-11]. To evaluate genetic variations of T. ruticarpum from different regions of China, we established iPBS and ISSR marker methods that would be appropriate for assessing genetic diversity of the species. It would provide basic genetic diversity information for the germplasm conservation and breeding of the species.

collected samples were T. ruticarpum authenticated by Professor Qin Min-Jian. The names, numbers, and geographic information are listed in Table 1. These accessions were planted in the Medicinal Botanical Garden of the China Pharmaceutical University, and their fresh leaves were randomly collected and stored with silica gel in zip-lock bags until DNA extraction. DNA extraction Genomic DNA was extracted from the silica gel-dried leaves using a modified cetyltrimethyl ammonium bromide method [12]. The quality of the DNA was determined by electrophoresis in 1% agarose gels, and the concentration of the DNA was determined using BioPhotometer plus (Eppendorf, Hamburg, Germany). DNA samples were diluted to 10 ng·µL−1 and stored at –20 °C for PCR amplification.

Materials and Methods Plant materials Several field investigation trips were conducted across the geographic range of T. ruticarpum in 2013, and 25 accessions were sampled from 6 Chinese provinces (Fig. 1). All the

Fig. 1 The collection sites of 25 accessions of Tetradium ruticarpum. The accession code at each point corresponds to those displayed in Table 1

extension of 5 min at 72 °C. PCR products were separated on 4% non-denaturing polyacrylamide gels that were stained using a silver staining protocol for visual detection. ISSR-PCR amplification A total of 13 primers that produced successful amplification patterns were selected (Table 2) from the initial screening of 35 ISSR primers. Primer sequences were obtained from the UBC Primer Set #9 (Microsatellite) designed by University of British Colombia (UBC) in Canada. PCR amplifications were carried out in a 20-µL volume solution containing 40 ng of template DNA, 0.6 µmol·L−1 of primer, 2.25 mmol·L−1 of Mg2+, 180 µmol·L−1 of dNTPs and 1.0 U of Taq polymerase (Sango Co., Ltd., Shanghai, China). The protocol for PCR

iPBS-PCR amplification Ten iPBS primers that amplified strong and clear bands were selected (Table 2) for genetic diversity evaluation out of the 30 designed by Kalendar et al. [5]. With slight modifications, the amplification reaction was performed as described by Kalendar et al. [5]. PCR reaction was set in volume of 20 µL containing 1 µL of the 10 × PCR buffer, 3 mmol·L−1 of Mg2+, 0.4 mmol·L−1 of dNTPs, 1 µmol·L−1 of primer, 0.5 U Taq polymerase (Sango Co., Ltd., Shanghai, China), 0.5 U Pfu polymerase (Sango Co., Ltd., Shanghai, China), and 40 ng of genomic DNA. The PCR program was run as follows: initial denaturation at 95 °C for 3 min, followed by 30 cycles of 15 s at 95 °C, 1 min at 55 °C, 1 min at 65 °C, and a final

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Table 1 T. ruticarpum accessions used for analysis in the present study Accession code

Origins

Latitude/°

Longitude/°

ZWY

Jiangning, Nanjing, Jiangsu

31.90

118.91

Altitude (m) 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

iPBS

ISSR

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

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

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

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amplification was run as follows: an initial step of denaturation at 94 °C for 7 min, followed by 30 cycles of 94 °C for 30 s, 50–54 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 7 min. The amplified products were separated on a 2% agarose gel containing 1.2 µL of GoldView in 1 × TrisAcetate-EDTA buffer. The images were documented using the Gel Doc XR+ System (Bio-Rad, Hercules, California, USA) under a UV light. Data analysis Clear, reproducible and well-separated bands were selected for scoring. Each iPBS or ISSR fragment was assigned as “1”, which indicated its presence, or “0”, which indicated its absence. The polymorphic information content (PIC) was calculated to measure the effectiveness of the iPBS and ISSR markers using the following formula [13]: PIC = 1 – ∑Pi2, where Pi is the frequency of the genotype I. The resolving power (Rp) was calculated according to Gilbert et al. [14]: Rp = ∑Ib, where Ib is the “band effectiveness”. Ib can be calculated by the formula: Ib = 1 – (2 × |0.5 – P|), where P is the frequency of varieties containing band I. The binary data matrix was analyzed using POPGENE version 1.32 (Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, AB, Canada). The following parameters were obtained to estimate the genetic diversity at the species level: the percentage of polymorphic loci (PPL), Nei’s gene diversity [15], Shannon’s information index (I) [16], the total genetic diversity (Ht), genetic differentiation coefficient (Gst), and gene flow (Nm) [17]. Genetic distance and genetic identity were also generated by POPGENE to examine the genetic relationships among these accessions. Based on the unweighted pair group method with arithmetic averaging (UPGMA), a dendrogram was constructed for all 25 acces-

sions using the SHAN module of NTSYS-pc version 2.1 (Exeter Software, Setauket, NY, USA). To construct a multiple dimensional array of eigenvectors, a principal coordinate analysis (PCoA) was performed using the NTSYS program.

Results Polymorphism analysis Thirty iPBS primers were designed for the initial screening. 10 iPBS primers were selected for further analysis based on an evaluation of polymorphism performance, reproducibility, and readability (Fig. 2), and the amplified bands per primer varied from 24 (2 230) to 11 (2 076). The mean number of bands per marker was 16.5. Of all of the amplified bands, 152 were polymorphic, with an average of 15.2 polymorphic fragments per primer. The percentage of polymorphic bands ranged from 78.9% (2 270) to 100% (2 076, 2 238, 2 230 and 2 252), with an average of 92.5%. The lowest (0.881) PIC was from iPBS primer 2 377 and the highest PIC value (0.937) was from iPBS primer 2 230, with an average of 0.914. The Rp values ranged from 11.20 (2 230) to 4.32 (2 377) (Table 3). 13 ISSR primers that showed potential polymorphisms were selected (Fig. 3). Total 151 bands were obtained, and 130 bands (86.1%) were polymorphic. The average number of bands and the polymorphic bands per primer generated were 11.6 and 10, respectively. The percentage of polymorphic markers produced by each primer ranged from 57.1% (UBC816) to 100% (UBC809, UBC834, and UBC835). The lowest PIC value (0.807) and the highest PIC value (0.926) were from ISSR primers UBC826 and UBC836, respectively. The Rp values ranged from 8.88 (UBC836) to 1.76 (UBC808) (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)

est genetic identity value were found between accessions YS and GZ. For the ISSR marker, Ht = 0.2624, Gst = 0.5694, and Nm = 0.3782 at the species level (Table 4). The other two genetic diversity indices, I and h, had similar values to those of the iPBS markers. The genetic distance and genetic identity (Online Resource 2) ranged from 0.040 5 to 0.562 9 and from 0.569 5 to 0.960 3, respectively. The greatest genetic distance and the smallest identity were both found between the accessions JL and GZ. The shortest genetic distance and the largest genetic identity values were found between ZWY and DG.

Genetic diversity and distance In the evaluation of genetic diversity and differentiation, the data generated by iPBS primers were useful. The value of Ht = 0.282 0, Gst = 0.477 2, Nm = 0.547 8, h = 0.285 3, and I = 0.431 6 at the species level (Table 4). The genetic distances were calculated to estimate the extent of their divergence. The genetic distances (Online Resource 1) ranged from 0.082 1 to 0.886 4 and genetic identities ranged from 0.412 1 to 0.921 2, among which, the largest genetic distance value and the smallest genetic identity value were found between accessions XS and JL. The smallest genetic distance value and the great-

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divided into three sub-clusters. The accessions of sub-clusters d and e, except for GZ and YS, were collected from Hunan Province. Accessions in sub-cluster f were all from Zunyi City in Guizhou Province. Using the ISSR data, a dendrogram (Fig. 5) grouped the 25 accessions into three main clusters (A2, B2, and C2) with a genetic similarity of 0.682. Cluster A2 mainly included 18 accessions from Zhejiang, Guizhou and Hunan Provinces. It could be further divided into three sub-clusters, a (three accessions from Jiangsu), b (eight accessions from Guizhou, except JN from Shandong) and c (seven accessions from Hunan, except YS from Guizhou). Cluster B2 included three accessions that were collected from Zhejiang. The accessions from Jiangxi were all placed in Cluster C2.

The accessions XS and SQ had the same genetic distances and genetic identity values as those of ZWY and DG. Cluster analysis Based on the iPBS data, a dendrogram (Fig. 4) was constructed using the UPGMA analysis. The 25 T. ruticarpum accessions were divided into two major clusters (A1 and B1) with a genetic similarity of 0.67. Cluster-A1 was further subdivided into three sub-clusters. Sub-cluster a, which included eight accessions from Jiangsu, Shandong and Jiangxi Provinces, could be further grouped into two clusters (I and II). Sub-cluster b contained only one accession “HHD”. Sub-cluster-c included all the three accessions collected from Zhejiang Province. Cluster-B1 included 13 accessions mainly from Hunan and Guizhou Provinces, and they also could be

Table 3 The observed genetic diversity based on iPBS markers and ISSR markers Marker

iPBS

ISSR

a

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

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

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

b

c

d

TB: The number of total bands; PB: The number of polymorphic bands; PPL: The percentage of polymorphic loci; PIC: 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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of Rutaceae in the “Flora of China” [2]. Based on the data in the present study, we no longer consider varieties during the sampling of the accessions, all of the collected accessions were treated as equivalent, which was different from the methods used in the assessment of the genetic diversity of Evodia rutaecarpa (synonym of T. ruticarpum) reported by Huang et al. [19] and Wei et al. [20]. The samples of the present study were collected from 6 Chinese provinces, representing 4 different geographical regions of China, according to differences in climate and terrain, and the sampling sites covered the major distribution areas of T. ruticarpum in China. As wide geographical distribution and long-time natural selection, the complicated climate and ecological environments may cause some variations in morphological characters and interior chemical composition of the plant. Genetic variability is the basis for plants adapting to different environment and a species without enough genetic diversity is thought to be difficult to cope with the changing environment [21]. Genetic variations of plants could be evaluated by loci polymorphism, and high, medium and low loci polymorphisms are in accorded

The UPGMA cluster (Fig. 6), which was constructed using a combination of data from iPBS and ISSR markers, revealed more genetic relations than the individual markers, and it separated the 25 accessions into 5 groups mainly based on the geographic locations. The dendrogram was similar to that constructed using iPBS or ISSR data. Associations among the 25 accessions were also resolved by the PCoA (Fig. 7). Five main groups were shown in the diagram generated by the PCoA, and they revealed a similar cluster result as that shown in Fig. 4. The two principal axes in the PCoA plot accounted for 15.13% and 13.34% of the total variation, respectively.

Discussion T. ruticarpum is one of the most widely distributed species in the genus Tetradium. According to old classification of Rutaceae, the species, named as Evodia rutaecarpa (Juss.) Benth., includes three varieties [18]. As the boundary and distinction among those varieties is not obvious, the three varieties have been abolished from the latest classification system

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

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to PIC > 0.5, 0.5 > PIC > 0.25 and PIC < 0.25, respectively, according to Vaiman et al. [22] and Xie et al. [23]. The average PIC values in the present study were higher than 0.5, indicating that T. ruticarpum possessed high loci polymorphism. Polymorphism detected in a species can be given in terms of estimate of gene flow (Nm) and coefficient of population differentiation (GST) [24]. As an indicator of gene movements, gene flow is negatively correlated with gene differentiation [25], and is very important for population transfers and evolution. The gene flow (Nm) for T. ruticarpum (Table 4) was lower than the limit value set by Wright [26], where values below 1 indicate genetic isolation. This result clearly indicated that the gene migration was limited. Hamrick [27] has reported that 16 species of cross-pollinating plants have a higher gene flow (Nm), with an average of 1.15. In the present study, as related to the iPBS and ISSR markers, the Nm for T. ruticarpum was 0.547 8 and 0.378 2, respectively, which was significantly lower than the average of the cross-pollinated plants. The possible reasons for lower Nm may be that most of seeds of T. ruticarpum are infertility and difficult to germinate and that the T. ruticarpum usually reproduces asexually. According to Wright [28], the values of Gst higher than 0.25 indicate a very great gene differentiation between the accessions being compared and it may be explained by geographic isolation of populations [29]. In our study, the Gst for T. ruticarpum was higher than 0.25 (Table 4). It seems that gene differentiation which might be caused by geographical obstacles had occurred in the T. ruticarpum distributed in different regions. As the differentiation of gene pools from different regions has arising by reproductive isolation and divergent natural selection, different populations can be geographically clustered [30]. The UPGMA dendrogram and the PCoA clustering data of the present study separated the 25 samples from 6 Chinese provinces into five major distinct groups (Figs. 6 and 7), reflecting the geographic distribution patterns of the plant. ISSR and iPBS are quite efficient tools in exploring genetic variations and assessing diversity. To best of our knowledge, this was the first study to investigate the genetic diversity of T. ruticarpum by combining iPBS and ISSR data. The increased capacity of ISSR and iPBS markers are likely to provide more specific genetic information compared to SRAP and AFLP [19-20] because of the high number of PPL that can be obtained (Table 3). The parameters of PIC and Rp were calculated to further evaluate the performance of the iPBS markers. As a quite efficient tool for exploring genetic variations and assessing diversity, ISSR has been widely used to identify the germplasm in many plant species [31-33]. Thus, the iPBS results were compared with those of the ISSR markers. The average PIC value of iPBS primers was 0.914, greater than that of the ISSR markers (0.869) in the present study. It showed that the iPBS markers can detect more abundant loci polymorphism of T. ruticarpum. When describing the discriminating ability of primers in a genetic diversity study, Rp was found to be more suitable [34]. The average Rp

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values of iPBS for T. ruticarpum (Table 3) were higher than those of the ISSR and SSR markers for Triticum dicoccon Schrank from India reported by Salunkhe et al. [35], indicating that the iPBS markers were suitable for the rapid determination of T. ruticarpum genetic diversity. In conclusion, the present study represented a first effort to investigate the genetic diversity of T. ruticarpum by combining iPBS and ISSR data. Compared to the classical molecular genetic markers, the iPBS marker was an effective new approach to evaluating the genetic diversity of plants. The results showed that the accessions of T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The accessions of T. ruticarpum in the present study could be clustered into several groups which approximately matched the geographical distribution of the species. The present study also found that the gene flow of the T. ruticarpum was significantly lower than the average of the cross-pollinated plants, which might mainly be due to the seed infertility and lower seed germination ratio, asexual reproduction, and geographical obstacles. These findings from the present study would contribute to the germplasm conservation and further breeding of T. ruticarpum.

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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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