c Indian Academy of Sciences
ONLINE RESOURCES
Application of sequence-related amplified polymorphism to genetic
diversity analysis in Limonium sinense
DING GE and ZHANG DAIZHEN∗
Chemical and Biological Engineering College, Yancheng Institute of Technology, Jiangsu Yancheng 224051,
People’s Republic of China
[Ge D. and Daizhen Z. 2015 Application of sequence-related amplified polymorphism to genetic diversity analysis in Limonium senense.
J. Genet. 94, e35–e38. Online only: http://www.ias.ac.in/jgenet/OnlineResources/94/e35.pdf]
Introduction
Limonium sinense, an endangered halophytic species endemic to China, mainly distributed in the salt marshes of Shandong, Jiangsu and Zhejiang provinces etc., it is an insectpollinated and outcrossing species. Due to the sporophytic
selfincompatibility (Erben 1979) and habitat deterioration
and human exploitation in recent years, it has been listed in
New Agricultural Plant Variety Protection of PRC in 2001
(Dong 2005).
The sequence-related amplified polymorphism (SRAP)
technique is a new molecular marker for amplifying open
reading frames, which was introduced by Li and Quiros
(2001). It has been effective for genetic analysis in different
species (Li and Quiros 2001; Song et al. 2010; Soleimani
et al. 2012). In the present study, SRAP markers were used
to investigate genetic diversity and population structure within
and among six natural populations of L. sinense, which could
provide further insight and develop useful strategies for its
conservation.
Materials and methods
Plant material
Data analysis
All clear and reproducible bands were scored as presence (1)
and absence (0). Genetic diversity indices were calculated
using PopGene ver. 1.31 (Yeh et al. 1999). Mantel test and
principal coordinate analysis (PCA) were performed using
NTSYS pc (Rohlf 2000).
Analysis of molecular variance (AMOVA) was performed to estimate variance components within and between
populations using Arlequin 3.1 software (Excoffier et al.
2005). Gene flow (Nm) between populations was calculated from ST (genetic differentiation values) using (Nm =
(1−ST )/4ST ) (Wright 1951).
Results and discussion
A total of 138 individuals from six natural populations of L.
sinense were evaluated (figure 1). The detailed locations are
given in table 1.
SRAP-PCR amplification
SRAP analysis was performed as described by Li and Quiros
(2001). Thirteen SRAP primer combinations were employed
using six forward primers and four reverse primers (table 2).
The amplifications were conducted under the following
∗ For correspondence. E-mail: [email protected].
conditions: 5 min at 94◦ C, five cycles of 94◦ C for 1 min,
35◦ C for 1 min, 72◦ C for 2 min, the following 30 cycles with
an annealing temperature of 50◦ C and the final extension of
5 min at 72◦ C. The PCR products were separated on 6%
denaturing polyacrylamide gel and SRAP bands were stained
using silver sequenceTM DNA staining reagents (Promega,
Madison, USA) (figure 2). The analysis was repeated at least
twice for each DNA sample.
A total of 256 reliable bands were scored using 13 SRAP
primer combinations with an average of 17 bands. Genetic
diversity indices are reported in table 3. The results indicated that genetic diversity at species level (percentage of
polymorphic loci (PPL) = 90.62%, H = 0.2634) was a little higher than the average level between populations estimated by Nybom (2004). Compared with other Limonium
species (Palacios et al. 1999; Suárez-García et al. 2009;
Palop-Esteban et al. 2011), L. sinense possesses high genetic
diversity at species level and lower genetic diversity at the
population level. It is known that most species of Limonium
Keywords. genetic diversity; sequence-related amplified polymorphism; Limonium sinense.
Journal of Genetics Vol. 94, Online Resources
e35
Ding Ge and Zhang Daizhen
Figure 1. Locations of six populations of L. sinense in China. BZ,
Binzhou; QD, Qingdao; BH, Binhai; SY, Sheyang; DF, Dafeng; JX,
Jiaxing.
are selfincompatible (Baker 1966; Palop-Esteban et al.
2011), which is an important factor in maintaining the high
genetic variability of species (Eduardo et al. 2001).
Genetic distances and geographical distances are listed in
table 4. Mantel test revealed no significant positive correlation between matrices of genetic distance and geographic distance (r = 0.630; P > 0.05). AMOVA revealed that 71.72%
of total genetic variation occurred within populations and
28.28% was attributed to among populations (table 5). It was
reported that 19.3% of total genetic diversity resides among
the populations of 29 outbreeding species (Bussell 1999). L.
sinense is an insect-pollinated, outcrossing species due to its
Figure 2. SRAP fingerprinting patterns amplified by primer combination M2+E3.
sporophytic selfincompatibility system (Erben 1979). Like
most Limonium species, it may disperse many thousands of
seeds by wind. The value of Gst was 0.3377, indicating that
the major proportion of the total variation existed within
populations which implied the same trend with AMOVA.
Table 1. Location of six populations of L. sinense used in this study.
Population Individual
code
code
BZ
QD
BH
SY
DF
JX
1–6
7–30
31–57
58–84
85–111
112–138
Sample Longitude Latitude
size
(E)
(N)
Location
Binzhou, Shandong province
Qingdao, Shandong province
Binhai, Jiangsu province
Sheyang, Jiangsu province
Dafeng, Jiangsu province
Jiaxing, Zhejiang province
6
24
27
27
27
27
118◦ 54
120◦ 18
119◦ 83
120◦ 30
120◦ 48
120◦ 46
37◦ 17
36◦ 14
34◦ 13
33◦ 34
32◦ 59
30◦ 27
Table 2. Sequence of SRAP primers used in this study.
Forward primer
Reverse primer
Me1: 5 -TGAGTCCAAACCGGATA-3
Me2: 5 -TGAGTCCAAACCGGAGC-3
Me4: 5 -TGAGTCCAAACCGGACC-3
Me5: 5 -TGAGTCCAAACCGGAAG-3
Me6: 5 -TGAGTCCAAACCGGAAA-3
Me8: 5 -TGAGTCCAAACCGGACT-3
Em1: 5 -GACTGCGTACGAATTAAT-3
Em2: 5 -GACTGCGTACGAATTTGC-3
Em3: 5 -GACTGCGTACGAATTGAC-3
Em4: 5 -GACTGCGTACGAATTTGA-3
Journal of Genetics Vol. 94, Online Resources
e36
Genetic diversity of Limonium sinense by SRAP
Gene flow number (Nm) was 0.633 which indicated there
was limited gene exchange between populations. Limonium
seeds are very light and dispersed by wind for long distance,
this may be important in promoting gene flow. However, in
fact, gene flow of L. sinense is still at low level because the
seed germination rate was low (Zhou et al. 1998). PCA analysis of 138 individuals is shown in figure 3. It is obvious
that populations JX and DF were distinct while the remaining
populations formed two clusters. In natural conditions, millions of seeds mature in September or October. Flowing seawater will help accelerate the spread of seeds, but this may
also account for the overlapping of individuals from different populations and the lack of correlation between genetic
and geographic distances.
L. sinense is a perennial halobiotic plant with brightly
coloured flowers and are of medicinal and ornamental
values. It can also be used for resistance to wind and sand,
and transformation of salt and alkali soil with a high application value. The seed germination rate of Limonium was low
due to lack of contact with soil because of the seeds being
packed by the calyx (Zhou et al. 1998; Wang et al. 2010).
This may be a biotic reason to result in decreasing of this
species. In addition, the decline in this species in recent years
may be attributed to harvesting for medicinal use and habitat
destruction by urbanization (Dong 2005).
Table 3. Genetic diversity parameters of L. sinense populations
revealed by SRAP.
Population code
PPL
Na
Ne
H
I
BZ
QD
BH
SY
DF
JX
Population average
Species-level value
34.38
56.25
46.88
37.50
53.12
50.00
46.36
90.62
1.3438
1.5625
1.4688
1.3750
1.5312
1.5000
1.4634
1.9062
1.2431
1.4214
1.3161
1.2507
1.3323
1.3491
1.3188
1.4337
0.1424
0.2337
0.1822
0.1424
0.1930
0.1948
0.1814
0.2634
0.2079
0.3381
0.2677
0.2094
0.2863
0.2849
0.2657
0.4082
Na , observed number of the alleles; Ne , effective number of alleles;
I, Shannon’s information index; H, Nei’s gene diversity.
Table 4. Nei’s genetic distances and geographic distances between
L. sinense populations. Nei’s genetic distances are given below
the diagonal and geographic distances (km) are given above the
diagonal.
Pop ID BZ
BZ
QD
BH
SY
DF
JX
QD
BH
SY
DF
–
210.754 346.023 481.333
0.1746
–
240.580 333.951
0.2067
0.1157
–
144.940
0.2509
0.0825
0.0977
–
0.1528
0.1259
0.0668
0.0839
0.2106
0.1287
0.1200
0.1461
JX
586.003 800.968
445.268 667.903
241.412 455.157
111.317 333.951
–
222.634
0.1315 –
Table 5. AMOVA analysis among and within L. sinense populations using SRAP markers.
Source of variation
d.f.
Sum of
square
Mean
square
Variation
component
Total
variation (%)
P value
Among populations
Within populations
5
132
139.7073
373.8796
27.941
2.832
1.1166
2.8324
28.28
71.72
<0.001
<0.001
3
19
20
JX
0.36
7
8
18
14
13
10
11
22
23
6
124
125
21
BZ+QD
1
2
114
9
33
91
92
87
0.21
136
137
117
135
25
26
16 15
17
12
24
51
37
38
35
34
48
BH+SY
52
53
0.37
46
47
0.19
-0.08
60
116
115
58
59
67
68
31
32
106
107
97
98
62
61
39
103
104
42 65
41
40
64
85 27 56
9386
55 100
66
101
63
96
29 30
28
84
49
50
120
45
69
36
43
44
Dim-2 0.01
-0.17
88
89
99
109
110
102 132
119
118
105
57
94 128
95
127
54
123
74
73
75 108
79
80
72
77
76
78
130 129
131
111
90
82
83
DF
81
126
4
5
0.07
112
113
138
133
134
70
71
121
122
-0.35
-0.23
-0.36
-0.16
0.03
Dim-1
0.23
0.43
Figure 3. Relationships of individuals of L. sinense based on 3D plot of PCA by SRAP markers.
Journal of Genetics Vol. 94, Online Resources
e37
Ding Ge and Zhang Daizhen
For the special habitat of L. sinense, in situ conservation should be first recommended. For ex situ conservation,
we need to design and establish a germplasm bank for this
species. Those populations with higher genetic variability
should be given priority in the seed bank. Priority should be
given to populations with higher genetic diversity, such as
QD, JX and BH populations.
Acknowledgements
This work was funded by National Natural Science Foundation
of China (no. 41301050; 31000142) and sponsored by ‘Qing Lan
Project’ and ‘333 Project’ of Jiangsu province to Daizhen Zhang
and the Foundation of Yancheng Insititute of Technology.
References
Baker H. G. 1966 The evolution, functioning, and breakdown of
heteromorphic incompatibility systems I. The Plumbaginaceae
Evol. 20, 349–368.
Bussell J. D. 1999 The distribution of random amplified polymorphic DNA (RAPD) diversity amongst populations of Isotoma
petraea (Lobeliaceae). Mol. Ecol. 8, 775–789.
Dong B. H. 2005 Research on the conservation of Limonium sinense
in the coast of Jiangsu. Chin. Wild Plant Res. 24, 28–30.
Eduardo L. B., Joaa O. S. and George J. S. 2001 Selfincompatibility, inbreeding depression and crossing potential in
five Brazilian Pleurothallis (Orchidaceae) species. Ann. Bot. 88,
89–99.
Erben M. 1979 Karyotype differentiation and its consequences in
Mediterranean Limonium. Webbia 34, 409–417.
Excoffier L., Laval G. and Schneider S. 2005 Arlequin 3.01, an integrated software package for population genetics data analysis.
Evol. Bioinform. Online 1, 47–50.
Li G. and Quiros C. F. 2001 Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR
reaction, its application to mapping and gene tagging in Brassica.
Theor. Appl. Genet. 103, 455–461.
Nybom H. 2004 Comparison of different nuclear DNA markers for
estimating intraspecific genetic diversity in plants. Mol. Ecol. 13,
1143–1155.
Palacios C., Kresovich S. and González-Candelas F. 1999 A population genetic study of the endangered palnt species Limonium
dufourii (Plumbaginaceae) based on amplified fragment length
polymorphism (AFLP). Mol. Ecol. 8, 645–657.
Palop-Esteban M., Segarra-Moragues J. G. and González-Candelas
F. 2011 Polyploid origin, genetic diversity and population structure in the tetraploid sea lavender Limonium narbonense Miller
(Plumbaginaceae) from eastern Spain. Genetica 139, 1309–
1322.
Rohlf F. J. 2000 NTSYS-pc, Numerical taxonomy and multivariate
analysis system, version 2.1., New York, USA.
Soleimani M. H., Talebi M. and Sayed-Tabatabaei B. E. 2012 Use of
SRAP markers to assess genetic diversity and population structure of wild, cultivated, and ornamental pomegranates (Punica
granatum L.) in different regions of Iran. Plant Syst. Evol. 298,
1141–1149.
Song Z. Q., Li X. F., Wang H. G. and Wang J. H. 2010 Genetic
diversity and population structure of Salvia miltiorrhiza Bge in
China revealed by ISSR and SRAP. Genetica 138, 241–249.
Suárez-García C., Pérez de Paz Julia, Febles R. and CaujapéCastells J. 2009 Genetic diversity and floral dimorphism in Limonium dendroides (Plumbaginaceae), a woody Canarian species on
the way of extinction. Plant Syst. Evol. 280, 105–117.
Wang L., Liu Y., Hua L. Y. and Zhang L. L. 2010 Studies on
the resources characteristic and chemical components for wild
Limonium aureum (L.) Hill. Northern Horticul. 11, 217–218.
Wright S. 1951 The genetical structure of populations. Ann.
Eugenet. 15, 323–354.
Yeh F. C., Yang R. C. and Boyle T. 1999 POPGENE Microsoft
windowsbased freeware for population genetic analysis. Release
1.31. University of Alberta, Edmonton, Canada.
Zhou X. Z., Li J. T. and Cui G. J. 1998 The bionomics of Limonium
bicolor and its mass propagation. Hebei. J. Forestry Orchard Res.
13, 331–334.
Received 28 October 2014, in revised form 3 February 2015; accepted 5 February 2015
Unedited version published online: 6 February 2015
Final version published online: 9 June 2015
Journal of Genetics Vol. 94, Online Resources
e38