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Revised 11/11

RESEARCH

Analysis of the Genetic Diversity of Natural Populations of Alpinia oxyphylla Miquel Using Inter-Simple Sequence Repeat Markers Haiyan Wang,* Xiaojing Liu, Mingfu Wen, Kun Pan, Meiling Zou, Cheng Lu, Shisheng Liu, and Wenquan Wang*

ABSTRACT Alpinia oxyphylla Miquel is an endangered but economically valuable species. Given that it is one of the four most important medicinal plants in southern China, surprisingly few studies have characterized the species at the molecular level. In this study, 163 accessions were collected from seven natural populations of A. oxyphylla, along with one outgroup species, to investigate the genetic diversity of the species using intersimple sequence repeat (ISSR) analysis. A total of 365 polymorphic markers were detected from the general population when it was analyzed using 20 ISSR primers. Nei’s genetic diversity and Shannon’s information index were 0.2129 and 0.3373, respectively, indicating considerable genetic diversity at the species level. Less genetic differentiation took place among populations, with evidence of coefficient of gene differentiation 0.338 and gene flow 0.907, suggesting that there was more genetic variation within populations than between populations. An unweighted pair group method with arithmetic averaging dendrogram divided all accessions into eight groups that coincided almost completely with their geographical origin, with the exception of just a few crosses. This suggests that the geographic distribution of accessions coincided closely with the distribution of genetic diversity within A. oxyphylla, indicating weak gene flow. These results may provide valuable guidance regarding the conservation and genetic improvement of the species.

H. Wang, M. Wen, K. Pan, M. Zou, C. Lu, and W. Wang, Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou, Hainan 571101, People’s Republic of China; X. Liu, Guangxi Sugarcane Research Insititute, Nanjing, Guangxi 530001, People’s Republic of China; S. Liu, College of Food Science of Technology, Hainan Univ., Haikou, Hainan 570228, People’s Republic of China. Received 16 June 2011. X. Liu and M. Wen are joint first authors. *Corresponding authors ([email protected]; [email protected]). Abbreviations: AMOVA, analysis of molecular variance; ISSR, intersimple sequence repeat; ITS, internal transcribed spacer; A.oxyphylla, Alpinia oxyphylla Miquel; PCA, principal component analysis; PCR, polymerase chain reaction; PPL, percentage of polymorphic loci; RAPD, random amplified polymorphic DNA.

A

Miquel (A. oxyphylla) is a perennial herb within the genus Alpinia (Zingiberaceae). Given the Chinese name Yizhi, it is one of the four main medicinal herbs in southern China. It originates from Hainan Island (Wu, 1996), is mainly distributed throughout the Hainan and Guangdong provinces, and is scattered across Fujian and Guangxi provinces (Wu, 1964). Since ancient times, the fruits of A. oxyphylla have been widely used in Chinese traditional medicine. They are rich in phenolic compounds, such as yakuchinone A and nootkatone, which are useful for strengthening the heart, protecting kidney function, treating nausea and stomach aches, and countering the accumulation of reactive oxygen species implicated in the onset of many human diseases. The fruits of A. oxyphylla also contain amino acids and minerals such as manganese, calcium, and phosphorus, which are often present in health foods (Shoji et al., 1984; Itokawa et al., 1979; Luo et al., 2001). lpinia oxyphylla

Published in Crop Sci. 52:1–9 (2012). doi: 10.2135/cropsci2011.07.0392 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

crop science, vol. 52, july– august 2012

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Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

Recently, with the increased growth of China’s pharmaceutical industry, there has been an expansion of the production of medicinal crops. However, there has been a decline in the quality of cultivated A. oxyphylla compared with wild germplasm resources (Fu et al., 2005; He et al., 1992). Unfortunately, the wild germplasm of A. oxyphylla has undergone significant damage over the last 20 yr (Yang et al., 2010). This is regrettable, as wild populations can be valuable resources for plant breeding as well as for the preservation of genetic diversity. Genetic diversity is the basis of evolution by natural selection. Yet this is gravely threatened for the progenitors of many cultivated plants. Thus, the evaluation and conservation of genetic diversity, both in situ and ex situ, is imperative for guaranteeing sustainable development (Nevo, 1998). Inter-simple sequence repeat (ISSR) markers are being used increasingly for the analysis of genetic diversity because of their dominant inheritance, transferability, ease of use, reproducibility, and independence from prior knowledge of the target sequences flanking the repeat regions (Fang and Roose, 1997; Godwin et al., 1997). Compared with other methods involving polymerase chain reaction (PCR)–based markers, ISSR analysis is more specific than random amplified polymorphic DNA (RAPD) analysis, because the longer ISSR primers enable more stringent and reproducible amplifications than RAPD primers (Qian et al., 2001; Tsumura et al., 1996). To date, ISSR markers have been widely used to identify genetic variations in many medicinal herbs, such as Siraitia grosvenorii (Peng et al., 2005), Ginkgo biloba (Ge et al., 2003), Piper spp. (2011), Iris lactea var. chinensis (2009), Gastrodia elata (Chen et al., 2007), Artemisia herba-alba (Haouari and Ferchichii, 2008), Rheum tanguticum (Hu et al., 2010), and Arrhenatherum elatius (Meng et al., 2011). Several studies concerning the genetic diversity of species within the ginger family have involved analysis of nuclear or plastid DNA sequences as well as molecular markers. The phylogeny of species within the Zingiberaceae has been revised on the basis of DNA sequences of the nuclear internal transcribed spacer (ITS) and plastid matK region (Kress et al, 2002). Analysis of the plastid matK sequence and ITS in 108 species, including 72 members of the genus Alpinia (Kress et al., 2005), revealed ambiguity in prior classifications of members of the genus Alpinia (Schumann, 1904; Smith, 1990) and a need to recognize new generic boundaries in the Alpinieae. Globba was distinguished from the related genera using Parsimony and Bayesian analyses involving ITS and plastid trnK-matK sequences, and the four species of Mantisia were formally transferred into Globba, even though this change was not consistent with morphological criteria for classification (Williams et al, 2004). A Brazilian germplasm collection of turmeric was characterized using simple sequence repeat markers and demonstrated significant differences 2

between and within the Brazilian groups of accessions (Sigrist et al., 2011). Most studies of A. oxyphylla have focused primarily on the pollination biology, chemical composition, and pharmacology of the species (Wang et al., 2005; Xu et al., 2009). Nevertheless, there is no information available about how genetic variability within the species relates to its natural distribution. In the present study, we investigated the extent of genetic diversity and genetic structure of A. oxyphylla natural populations by analyzing 163 accessions collected from almost all original areas in Hainan Island. Our findings may provide important theoretical guidance for the conservation and genetic improvement of A. oxyphylla germplasm resources.

MATERIALS AND METHODS Plant Material Over a 2-yr period, we collected a total of 163 accessions of A. oxyphylla from seven locations, mainly in Hainan, which represent the original distribution of the species within China. The sample included nine accessions collected from the SouthChina Botanical Garden in Guangdong province. We used Alpinia officinarum Hance as the outgroup species. The natural geographic distribution of all accessions in the seven locations is shown in Fig. 1, and the accession numbers corresponding to each site are shown in Table 1. Fresh young leaves were collected and stored on ice before DNA extraction.

Inter-Simple Sequence Repeat Primers Screening The sequences of primers used for ISSR analysis were obtained from the University of British Columbia, Canada, and the primers were synthesized at the Shanghai Sangon Biological Engineering Technology Co. Ltd., Shanghai, China. For initial screens to identify the most promising primers, we screened 95 ISSR primers in 20-μL PCR mixtures each testing two random template DNAs from the accessions. From these initial results, we selected 20 primers with high polymorphism and reproducibility for amplification reactions involving each of the accessions in isolation.

DNA Extraction and Polymerase Chain Reaction Amplification Genomic DNA was extracted using the improved cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). DNA quality was evaluated using 0.8% agarose gel electrophoresis and an ultraviolet spectrophotometer, and all DNA samples were diluted to 25 ng/μL before use for PCR amplification. Amplification reactions were performed in a volume of 20 μL, containing 25 ng template DNA, 2.5 mM Mg 2+, 0.2 mM dNTP mixture, 0.8 mM primer, and 1.0 U Taq polymerase (Biocolors Co. Ltd.). Polymerase chain reaction was performed using a T1 thermocycler (Biometra), with initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 45 sec, 41–59°C for 45 sec, and 72°C for 90 sec, followed by

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a final extension at 72°C for 10 min. Amplification products were separated electrophoretically on 2% agarose gels in 0.5X TBE buffer, using a 100-bp DNA ladder (MBI Fermentas) as a DNA molecular weight marker. Gels were stained using Goldview (SBS Genetech Co., Ltd.) at a voltage of 4 V/cm for 1 h in an electrophoresis unit (Bio-Rad) and visualized using a Tanon 4100 imaging system.

Data Statistics and Analysis Unequivocally scorable and consistently reproducible amplified DNA fragments ranging between 200 bp and 1200 bp in size were transformed into a binary character matrix (1 = presence, 0 = absence). Assuming that populations were in Hardy-Weinberg equilibrium at these ISSR marker loci, genetic diversity parameters of each geographic population were calculated using the software package POPGENE32 (Yeh et al., 2000), including the percentage of polymorphic loci (PPL), observed number of alleles, effective number of alleles, Nei’s (1973) gene diversity, Shannon’s information index, total gene diversity, intrapopulation gene diversity, the coefficient of gene differentiation, and gene flow. Analysis of molecular variance (AMOVA) (Excoffier et al., 1992) was performed using WINAMOVA 1.55 software (Stewart and Excoffier, 1996) to estimate variance components for the ISSR patterns and to partition the total variance into intrapopulation and interpopulation. Significance of variance components was tested after 1000 permutations. On the basis of the binary character matrix, the genetic relationship was studied by means of cluster analysis and principal component analysis (PCA) using NTSYS-pc2.1 software (Rohlf, 2002). The evolutionary relationship was estimated by using the neighbor-joining (NJ) program in Mega 5.0 (Tamura et al., 2011) by genetic distance.


Table 1. Population of Alpinia oxyphylla examined in the inter-simple sequence repeat analysis. Code GZL DLS BC QXL MG WZS GD

Population Ganzhaling, Hainan Diaoluoshan, Hainan Bacun, Hainan Qixianling, Hainan Maogan, Hainan Wuzhishan, Hainan South China Botanical Garden, Guangdong

Sample size Latitude Longitude 30 18 30 17 34 25 9

N

E

18.39 18.66 18.74 18.70 18.60 18.78 23.19

109.66 109.92 109.74 109.70 109.51 109.52 113.36

RESULTS Genetic Diversity within Populations Twenty ISSR primers selected for an initial screen of 95 ISSR primers for their ability to reproducibly identify polymorphisms were used to detect the genetic diversity of all accessions of A. oxyphylla. From a total of 402 amplified bands generated across the 163 accessions, 365 polymorphic bands were identified. The polymorphism rate was 90.80%. The number of polymorphic bands associated with each primer range from 32 (for primer UBC843) to 9 (for primer UBC822). As indicated in Table 2, the polymorphism percentage ranged from 100% (for primers UBC807, UBC808, UBC810, UBC814, UBC842, and UBC843) to 64.7% (for primer UBC880). The sizes of amplified fragments ranged from 200 bp to 1200 bp. Parameters of intra- and intergroup genetic diversity among the seven geographic populations of A. oxyphylla,

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Figure 1. The geographical location for seven different populations of Alpinia oxyphylla Miq in Hainan and Guangdong province. GZL, Ganzhaling; DLS, Diaoluoshan; BC, Bacun; QXL, Qixianling; MG, Maogan; WZS, Wuzhishan; GD, South China Botanical Garden, Guangdong.

Table 2. Attributes of the amplified inter-simple sequence repeat analysis (ISSR) primers used to generate ISSR markers from 165 samples of Alpinia oxyphylla.


Primer name

Sequence (5’-3’)

UBC807 UBC808 UBC809 UBC810 UBC811 UBC812 UBC813 UBC814 UBC822 UBC840 UBC842 UBC843 UBC844 UBC857 UBC864 UBC873

(AG)8T (AG)8C (AG)8G (GA)8T (GA)8C (GA)8A (CT)8T (CT)8A (TC)8A (GA)8YT (GA)8YG (CT)8RA (CT)8RC (AC)8YG (ATG)6 (GAC A)4

UBC880 UBC881

(GGA GA)3 GGG(TGG GG)2 TG HVH (TG)7 CAT GGT GTT GGT CAT TGT TCC A

UBC891 UBC899

Annealing temp.

No. of loci amplified

No. of polymorphic loci

% polymorphic loci

°C 50 53 55.5 50.5 51 50 50 53 50 53.8 55 51 55 52 48 53.5

17 25 22 14 26 18 24 27 11 11 15 32 17 19 15 31

17 25 18 14 25 13 20 27 9 10 15 32 16 17 12 31

100 100 81.82 100 96.15 72.22 83.33 100 81.82 81.82 100 100 94.12 89.47 80.00 100

54 58

17 18

11 15

64.71 83.33

58 53.5

22 21

20 18

90.91 85.71

402

365

90.80

Total

Table 3. Parameters of intra- and intergroup genetic diversity among the seven populations of Alpiniae oxyphyllae by intersimple sequence repeat markers. n = number of genotypes per population; #loc P = number of polymorphic loci; PPL = percentage of polymorphic loci; na = observed number of alleles; ne = effective number of alleles (Kimura and Crow [1964]); H = Nei’s (1973) gene diversity; I = average genetic diversity index; Ht = total gene diversity; Hs = intrapopulation gene diversity; Gst = coefficient of gene differentiation; Nm = estimate of gene flow from Gst [Nm = 0.5 (1-Gst)/Gst]. Population n GZL DLS BC QXL MG WZS GD Total Std. dev Mean Std. dev

30 18 30 17 34 25 9 163 –

loc_P PPL

#

200 191 238 198 247 242 46 365 –

na

ne

H

I

% 49.75 1.4975 1.2439 0.1453 0.2225 47.51 1.4751 1.2319 0.1385 0.2123 59.20 1.5920 1.2848 0.1724 0.2654 49.25 1.4925 1.2298 0.1390 0.2143 61.44 1.6144 1.2768 0.1677 0.2594 60.20 1.6020 1.2676 0.1648 0.2561 11.44 1.1144 1.0746 0.0430 0.0636 90.80 1.9080 1.3418 0.2129 0.3373 – ±0.2894 ±0.3322 ±0.1720 ±0.2339

Ht

Hs

0.2151 ±0.0310

0.1387 ±0.0133

Gst 0.3553

Nm 0.9074

as determined using the POPGENE software, are listed in Table 3. It seems that there is a higher genetic diversity for all natural populations with an average genetic diversity index of 0.3373 ±0.2339, and an average Nei’s gene diversity of 0.2129 ±0.1720. Among the seven populations, the 4

PPL ranged from 11.44% (GD) to 61.44% (MG), and the genetic diversity index ranged from 0.0636 (Guangdong [GD]) to 0.2654 and 0.2594 (Bacun [BC] and Maogan [MG]), showing that the MG and BC populations were characterized by more genetic diversity than other populations. The lowest level of diversity was found among the GD accessions.

Genetic Structure and Diversity between Populations The total genetic diversity of A. oxyphylla was 0.215, and the genetic diversity within a population was 0.139. A coefficient of genetic differentiation of 0.355 indicated that 35.5% of the total genetic variation for A. oxyphylla occurs within populations, and the majority (approximately 64.5%) occurs between populations. There is a low level of gene flow (0.907) between populations. Nei’s genetic distance between every pair of populations was estimated to range from 0.044 (for MG and Ganzhaling [GZL]) to 0.203 (for Wuzhishan [WZS] and GD), and the genetic identity ranged from 0.816 (for WZS and GD) to 0.957 (for GZL and MG) (Table 4). This indicated that the longest genetic distance lies between the WZS and GD populations, and the shortest genetic distance lies between the MG and GZL populations. There is no clear correlation between genetic distance and geographical distance. Analysis of molecular variance showed a significant

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Cluster Analysis An unweighted pair group method with arithmetic averaging (UPGMA) tree obtained from data generated using 365 ISSR markers in all accessions spanning the seven natural populations is shown in Fig. 2. Rooting from the outgroup species A. officinarum separated all accessions into eight distinct groups with a genetic distance value of 0.40. Accessions from the same geographic population were grouped together with several crosses. The eight groups were WZS, Qixianling (QXL), GD, BC, MG1, MG2, Diaoluoshan (DLS), and GZL. One accession, MG 30, is unique relative to the other groups. Some of the accessions from the BC population were classified as part of the QXL group, and only one sample (DLS18) from the DLS population was classified as part of the BC group. The accessions from the MG population were divided into two groups, MG1 and MG2, and two other samples, QXL17 and WZS3, clustered within the MG1 group. Some accessions from the GZL population clustered as part of the DLS group. Together, the results suggest that genetic diversity within A. oxyphylla populations are primarily influenced by geographical distribution, and there is minimal gene flow between A. oxyphylla. The availability of accessions from different areas is thus very important for genetic conservation and future genetic improvement.

Evolutionary Relationship among Populations The evolutionary relationships of the seven populations studied are represented in Fig. 4. The much more extensive genetic distance of GD accessions was farthest from other populations, suggesting that Guangdong is likely the site of origin of the species in China. The division of samples from Maogan into two groups likely results from the variation caused by environmental impact and longterm evolution. Principal coordinate analysis indicated that besides the outgroup species A. officinarum, accessions from the WZS group showed independent distribution, accessions from QXL, DLS, GZL, and BC groups located into a larger surrounding area, and accessions from GD and MG groups traced to an area between the WZS and the main area (QXL, DLS, GZL, BC) (Fig. 3). The PCA based on a bi-dimensional space of genetic structure revealed genetic differences between populations. crop science, vol. 52, july– august 2012

Table 4. Nei’s genetic identity (above diagonal) and genetic distance (below diagonal) among populations. GZL, Ganzhaling; DLS, Diaoluoshan; BC, Bacun; QXL, Qixianling; MG, Maogan; WZS, Wuzhishan; GD, South China Botanical Garden, Guangdong. Populations GZL DLS BC QXL MG WZS GD

GZL

DLS

**** 0.047 0.065 0.082 0.044 0.123 0.162

0.955 0.937 0.922 0.957 0.884 **** 0.940 0.917 0.944 0.886 0.062 **** 0.936 0.949 0.886 0.087 0.066 **** 0.928 0.859 0.058 0.052 0.075 **** 0.911 0.121 0.122 0.152 0.094 **** 0.171 0.163 0.184 0.154 0.203

BC

QXL

MG

WZS

GD 0.850 0.843 0.850 0.832 0.858 0.816 ****

Table 5. Results of analysis of molecular variance. MSD, mean squared deviation. Source of variation

Degrees of freedom

MSD

Variance component

P†

Interpopulations Intrapopulations

6 158

443.228 34.070

17.705 (34.20%) 34.070 (65.80%)

Crop Science Proofing Instructions

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