Chinese Journal of Oceanology and Limnology Vol. 31 No. 3, P. 609-616, 2013 http://dx.doi.org/10.1007/s00343-013-2064-8
Population genetics of Sargassum horneri (Fucales, Phaeophyta) in China revealed by ISSR and SRAP markers* YU Shenhui (于深辉)1, 2, CHONG Zhuo (崇卓)1, ZHAO Fengjuan (赵凤娟)3, YAO Jianting (姚建亭)1, DUAN Delin (段德麟)1, ** 1
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
Graduate University of Chinese Academy of Sciences, Beijing 100049, China
3
Shandong Key Laboratory of Eco-environmental Science for Yellow River Delta, Binzhou University, Binzhou 256600, China
Received Sep. 10, 2012; accepted in principle Oct. 21, 2012; accepted for publication Dec. 23, 2012 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2013
Abstract Sargassum horneri is a common brown macro-alga that is found in the inter-tidal ecosystems of China. To investigate the current status of seaweed resources and provide basic data for its sustainable development, ISSR (inter simple sequence repeat) and SRAP (sequence related amplified polymorphism) markers were used to analyze the population genetics among nine natural populations of S. horneri. The nine studied populations were distributed over 2 000 km from northeast to south China. The percentage of polymorphic loci P% (ISSR, 99.44%; SRAP, 100.00%), Nei’s genetic diversity H (ISSR, 0.107–0.199; SRAP, 0.100–0.153), and Shannon’s information index I (ISSR, 0.157–0.291; SRAP, 0.148–0.219) indicated a fair amount of genetic variability among the nine populations. Moreover, the high degree of gene differentiation Gst (ISSR, 0.654; SRAP, 0.718) and low gene flow Nm (ISSR, 0.265; SRAP, 0.196) implied that there was significant among-population differentiation, possibly as a result of habitat fragmentation. The matrices of genetic distances and fixation indices (Fst) among the populations correlated well with their geographical distribution (Mantel test R=0.541 5, 0.541 8; P=0.005 0, 0.002 0 and R=0.728 6, 0.641 2; P=0.001 0, 0.001 0, respectively); the Rongcheng population in the Shandong peninsula was the only exception. Overall, the genetic differentiation agreed with the geographic isolation. The fair amount of genetic diversity that was revealed in the S. horneri populations in China indicated that the seaweed resources had not been seriously affected by external factors. Keyword: Sargassum horneri; population genetics; ISSR; SRAP; markers
1 INTRODUCTION Sargassum horneri (Turner) C. Agardh is abundant in northwestern Pacific seas (Yoshida, 1983) and is one of the most common seaweeds in China (Tseng and Lu, 2000). In marine ecosystems, Sargassum forms algal beds and is an inhabitant of the lower littoral zone. For artificial seaweed breeding, wild S. horneri populations have been collected extensively and this, together with coastal pollution and herbivore grazing, has caused the natural resources to become depleted, leading to sea desertification or “Isoyake” areas (Fujita, 2010; Nagai et al., 2011). Currently, S. horneri is one of the main candidates for seaweed bed reconstruction in Japan, the Republic of Korea,
and China (Yamauchi, 1984; Choi et al., 2003; Sun et al., 2009). Therefore, to protect this species, an understanding of the status of Sargassum populations is needed and research on the population genetics of wild S. horneri in China is urgently required. The genus Sargassum, especially S. horneri, has been studied in detail. Yoshida et al. (1998, 2001) studied the life histories of autumn-fruiting and spring-fruiting types of S. horneri populations, and
* Supported by the National Natural Science Foundation of China (Nos. 40618001, 40976085), the Chinese Academy of Sciences and Guangdong Provincial Joint Projects (No. 2009B091300086), and Public Science and Technology Research Funds Projects of Ocean ** Corresponding author:
[email protected]
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found that distinct gradients in the physical conditions affected the seasonal growth patterns of populations in Hiroshima Bay, Japan. Ang (2006) pointed out that seasonal changes in water temperature were critical to determining phenological patterns of Sargassum spp. Subsequently, Zhao et al. (2008b) reported that the optimal growth of S. thunbergii germlings occurred at 25°C with 44 μmol photons/(m2·s) of light. Choi et al. (2008) described the physiological differences of S. horneri in germling and adult stages. Pang et al. (2009) investigated the sexual reproduction and seedling production of S. horneri in a tank culture. With the development of various molecular tools, the population genetic variability of Sargassum has attracted more and more attention. Wong et al. (2007) discriminated between S. binderi, S. oligocystum, and S. baccularia using a random amplified polymorphic DNA (RAPD) approach. Uwai et al. (2009) studied the phylogeography of the S. horneri/filicinum complex in Japan using the mitochondrial cox3 gene. Cheang et al. (2010) revealed low genetic variation among introduced and indigenous S. muticum using the ITS2 and rbc genes and the TrnW–TrnI intergenic spacer. Hu et al. (2011) revealed the phylogeographic heterogeneity of S. horneri from the northwestern Pacific in relation to the late Pleistocene glaciation and tectonic pattern based on the cox3 and rbcL genes. In addition, ISSR (inter simple sequence repeat) markers that reflected variations in the regions between adjacent reversed microsatellites have been applied successfully to investigate the genetic variability of seaweed for which the genomic DNA sequence was unknown (Zietkiewicz et al., 1994; Wang et al., 2005, 2008). Moreover, SRAP (sequence related amplified polymorphism) markers for detecting polymorphisms within open reading frames (ORFs) were developed by Li and Quiros (2001). The ORFs are amplified using forward primers from the GC-rich exon regions and reverse primers from within AT-rich intron or promoter regions (Li and Quiros, 2001). To date, there are only a few reports of the application of SRAP markers to study algae (Qiao et al., 2007). Here, we used ISSR and SRAP markers to study the population genetics of S. horneri with the aims of assessing the genetic structures and differentiations of S. horneri populations, and exploring the correlation between genetic diversity and geographical distributions of the Sargassum
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N 40° 38°
YT
DL RC
QD
36° 34° 32°
SS
30° LX ZJ NJ
28° 26° 24°
FJ
22° NZ 20° 110°
112°
114°
116°
118°
120°
122°
124° E
Fig.1 Map showing the areas of coastal China from which the nine S. horneri populations were collected Please see Table 1 for a description of the population codes.
populations in China. This study should provide a basis for the future sustainable development of Sargassum.
2 MATERIAL AND METHOD 2.1 Sample collection and DNA extraction Nine S. horneri populations from five provinces of China—Liaoning, Shandong, Zhejiang, Fujian, and Guangdong—were examined (Fig.1; Table 1). From each of the populations, 22 algal fronds that were two meters apart were collected and dried with silica gel, and then stored at -20°C until used. An S. fusiforme (Harvey) Setchell population was used as the outgroup to investigate the population genetic variability of S. horneri. About 0.3 g of each frozen alga sample was cleaned and grated in liquid nitrogen. The DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. About 50 ng/μL DNA was obtained from each sample.
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Table 1 Details of the S. horneri and S. fusiforme populations those were used in the study Population code
Species
Geographical position
Date of collecting
DL
Sargassum horneri
Lüshun, Dalian, Liaoning; 38°47′N, 121°14′E
July 6, 2007
YT
S. horneri
Daqin Island, Yantai, Shandong; 38°19′N, 120°49′E
July 27, 2005
RC
S. horneri
Lidao, Rongcheng, Shandong; 37°13′N, 122°36′E
July 18, 2008
QD
S. horneri
Badaguan, Qingdao, Shandong; 36°03′N, 120°20′E
July 13, 2005
SS
S. horneri
Shengsi Island, Zhoushan, Zhejiang; 30°45′N, 122°26′E
May 11, 2008
LX
S. horneri
Luxi Island, Wenzhou, Zhejiang; 27°59′N, 121°10′E
May 23, 2008
NJ
S. horneri
Nanji Island, Wenzhou, Zhejiang; 27°30′N, 121°05′E
Dec. 30, 2008
FJ
S. horneri
Zhangpu, Zhangzhou, Fujian; 23°59′N, 117°49′E
Apr. 3, 2010
NZ
S. horneri
Naozhou, Zhanjiang, Guangdong; 20°51′N, 110°34′E
Apr. 14, 2010
ZJ
S. fusiforme
Dongtou Island, Wenzhou, Zhejiang; 27°52′N, 121°11′E
May 17, 2008
Table 2 ISSR and SRAP primers used for the analyses ISSR
Primer (5'3')*
ISSR
Primer (5'3')*
SRAP
Primer (5'3')
807
(AG)8T
844
(CT)8RC
Em1
GACTGCGTACGAATTAAT
808
(AG)8C
848
(CA)8RG
Em2
GACTGCGTACGAATTTGC
810
(GA)8T
849
(GT)8YA
Em3
GACTGCGTACGAATTGAC
811
(GA)8C
851
(GT)8YG
Em4
GACTGCGTACGAATTTGA
815
(CT)8G
855
(AC)8YT
Em5
GACTGCGTACGAATTAAC
823
(TC)8C
859
(TG)8RC
Em6
GACTGCGTACGAATTGCA
828
(TG)8A
864
(ATG)6
Me1
TGAGTCCAAACCGGATA
834
(AG)8YT
873
(GACA)4
Me2
TGAGTCCAAACCGGAGC
835
(AG)8YC
880
GGA(GAG)2AGGAG
Me3
TGAGTCCAAACCGGAAT
840
(GA)8YT
890
(GGAGA)3
Me4
TGAGTCCAAACCGGACC
841
(GA)8YC
Me5
TGAGTCCAAACCGGAAG
* Y=C/T; R=A/G
2.2 ISSR and SRAP analyses The ISSR reaction system consisted of 1× EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China), 1.25 μmol/L of each of the primers in turn, and 1.3 μL of DNA template in a final volume of 20 μL. In all, 21 ISSR primers were used (Table 2, selected from primer set #9, No. 801–900, University of British Columbia). The PCR procedure was 94°C for 6 min, and 35 cycles of 94°C for 30 s, 48°C for 30 s, 72°C for 90 s, and finally 72°C for 6 min in a Master Thermal Cycler (TaKaRa, Japan). The PCR products were separated in 1.0% agarose gel with D2000 as the size marker (Tiangen Biotech Co. Ltd.) and photographed using a digital imager (Bio-Rad, Hercules, California, USA). The SRAP reaction system consisted of 1× EasyTaq PCR SuperMix (TransGen Biotech), 0.2 μmol/L of each of the primers in turn, and 1.0 μL of DNA template in a final volume of 20 μL. In all, 26 SRAP
primers were selected for use (Table 2, Li and Quiros, 2001). The PCR procedure was 94°C for 6 min, 5 cycles of 94°C for 1 min, 35°C for 1 min, and 72°C for 1 min; then 30 cycles of 35°C increased to 50°C; and finally 72°C for 6 min. The PCR products were separated in 6.0% (w/v) polyacrylamide gel [acrylamide-bisacrylamide (29:1), 1× TBE] at 1 500V for 1.5 h with Trans DNA marker I (TransGen Biotech), and stained in 0.1% AgNO3 solution. 2.3 Data analyses The ISSR and SRAP loci visualized by eye were noted as either present (1) or absent (0). The data matrices composed of 0 and 1 were computed to obtain Nei’s genetic diversity (H), Shannon’s information index (I), gene flow (Nm) and gene differentiation (Gst) using the POPGENE 1.32 software (Nei, 1978; Slatkin and Barton, 1989; Yeh and Yang, 2000). Nei’s unbiased genetic distance and
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Table 3 Genetic diversities of the Sargassum horneri populations Population code
ISSR markers
SRAP markers
P (%)
H
I
P (%)
H
I
DL
80 (44.4)
0.170
0.251
152 (36.5)
0.138
0.206
YT
90 (50.0)
0.199
0.291
129 (31.0)
0.126
0.184
RC
60 (33.3)
0.133
0.195
120 (28.9)
0.112
0.166
QD
74 (41.1)
0.172
0.249
120 (28.9)
0.111
0.165
SS
50 (27.8)
0.107
0.157
143 (34.4)
0.153
0.219
LX
84 (46.7)
0.177
0.261
135 (32.5)
0.125
0.186
NJ
90 (50.0)
0.194
0.284
110 (26.4)
0.103
0.152
FJ
50 (27.8)
0.113
0.166
140 (33.7)
0.127
0.190
NZ
64 (35.6)
0.139
0.204
106 (25.5)
0.100
0.148
Average
73 (40.6)
0.156
0.229
128 (30.9)
0.122
0.179
Species level
179 (99.4)
0.378
0.554
416 (100.0)
0.372
0.541
P (%): number (and percentage) of polymorphic loci; H: Nei’s genetic diversity; I: Shannon’s information index
the percentage of polymorphic loci (P%) were estimated and a dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPMGA) clustering in the TFPGA 1.3 Program (Miller, 1997). AMOVA (analysis of molecular variance) and fixation indices (Fst) were determined by the Arlequin 3.1 program with 1 000 permutations (Excoffier et al., 2005).The Mantel test was conducted using the TFPGA program to analyze the relationship between the matrices of geographical distributions and Fst, geographical distributions and genetic distances, and Fst and genetic distances of the S. horneri populations, based on the ISSR and SRAP markers (Mantel, 1967).
3 RESULT 3.1 Genetic diversity The 21 ISSR primers (Table 2) generated 180 bands (200–2 200 bp long) of which 99.4% were polymorphic. The average number of bands per primer was 8.6. The highest P% was detected in the YT and NJ populations. The highest H and I were in YT, while the lowest genetic diversity was in SS population (Table 3). The 26 SRAP primers (Table 2) generated 416 bands (100–600 bp long), all of which were polymorphic. The average number of bands per primer was 16.0. The highest P% was detected in the DL population. The highest H and I were in SS, while the lowest genetic diversity was in NZ population (Table 3).
3.2 Genetic differentiation Based on the ISSR markers, the genetic distances ranged from 0.119 (LX and NJ) to 0.519 (QD and ZJ), or from 0.119 (LX and NJ) to 0.342 (QD and FJ) when the outgroup ZJ was excluded (Table 4). Similarly, based on SRAP markers, the genetic distances ranged from 0.171 (LX and NJ) to 0.471 (RC and ZJ), or from 0.171 (LX and NJ) to 0.382 (RC and NJ) when ZJ was excluded. The Gst value based on the ISSR data (0.654) revealed a high amongpopulation variation (65.38%) and a low withinpopulation variation (34.62%). The Gst value based on the SRAP data (0.718) gave a similar result. The gene flow (Nm) values of 0.265 and 0.196 based on the ISSR and SRAP data, respectively, indicated a low genetic exchange among populations. The dendrogram was constructed using the ISSR marker data and UPGMA (Fig.2). The nine populations grouped into three clusters: the three Zhejiang populations (LX, NJ, and SS) formed the first group; the Liaoning (DL) and Shandong (QD and YT) populations formed the second group; and the Shandong (RC) and South China (NZ and FJ) populations formed the third group. As expected, the outgroup S. fusiforme ZJ population clustered with all three S. horneri groups finally. High bootstrap values were detected (>64.3). The dendrogram based on the SRAP marker data (Fig.3) was similar to the ISSR marker-based dendrogram and consisted of three groups with high bootstrap values (>69.8). The AMOVA results based on the ISSR data
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Table 4 Nei’s unbiased genetic distances (Nei, 1978) based on the ISSR (below diagonal) and SRAP (above diagonal) analyses No.
DL
YT
RC
QD
SS
LX
NJ
FJ
NZ
ZJ
DL
****
0.183
0.308
0.214
0.223
0.278
0.284
0.306
0.335
0.423
YT
0.172
****
0.345
0.197
0.277
0.259
0.247
0.316
0.365
0.415
RC
0.280
0.289
****
0.368
0.297
0.359
0.382
0.178
0.197
0.471
QD
0.131
0.171
0.311
****
0.251
0.253
0.270
0.329
0.340
0.402
SS
0.290
0.246
0.253
0.302
****
0.191
0.210
0.290
0.296
0.423
LX
0.191
0.204
0.264
0.200
0.203
****
0.171
0.309
0.360
0.438
NJ
0.250
0.223
0.294
0.238
0.194
0.119
****
0.335
0.346
0.460
FJ
0.318
0.328
0.180
0.342
0.292
0.330
0.322
****
0.210
0.403
NZ
0.305
0.240
0.160
0.307
0.205
0.244
0.237
0.244
****
0.445
ZJ
0.516
0.451
0.439
0.519
0.516
0.482
0.426
0.448
0.425
****
Table 5 AMOVA results based on the ISSR and SRAP data Source of variation
Degree of freedom
Sum of squares
Variance components
Percentage of variation
Among groups
3
2 292.923
10.986
27.86
Among populations within groups
6
2 415.046
17.909
45.42 26.72
ISSR markers
Within populations
188
1 980.784
10.536
Total
197
6 688.753
39.431
Among groups
3
7 295.601
36.801
35.85
Among populations within groups
6
7 025.264
52.904
51.53 12.62
SRAP markers
Within populations
188
2 436.710
12.961
Total
197
16 757.576
102.666
Fst
0.733*
0.874*
*: P
