AJCS 18(07):374-381 (2024) ISSN:1835-2707
https://doi.org/10.21475/ajcs.24.18.07.pne19
Assessment of genetic diversity of
Stephania rotunda
Lour. collected
in Northern Vietnam using RAPD and ISSR markers
Thi Thao Ninh
1, 2
, Tien Phat Nguyen
1
, Huyen Trang Dang
1
, Ha Duc
Chu
3
, Truong Son Dinh
1
, Xuan Canh Nguyen
1
, Thi Dung Pham
1
,
Thanh Hai Nguyen
1
, Thi Hue Nong
1
*
1
Faculty of Biotechnology, Vietnam National University of Agriculture,
Hanoi, Vietnam
2
Centre for Horticultural Science, Queensland Alliance for Agriculture
and Food Innovation, The University of Queensland, Australia
3
Faculty of Agricultural Technology, University of Engineering and
Technology, Vietnam National University, Hanoi, Vietnam
Abstract:
Stephania rotunda
Lour. is a valuable
traditional medicinal plant that is in danger of
extinction. This research aimed to examine the
genetic relationship of 32
Stephania rotunda
Lour.
accessions collected in Vietnam by using RAPD and
ISSR markers. 14 RAPD and 14 ISSR primers
successfully detected 163 loci of which 133 (80.19%)
were polymorphic and a total of 3047 scorable
bands were obtained. The 14 RAPD markers
produced 1346 scorable bands, 89.63% of which
were polymorphic while 14 ISSR primers resulted in
Submitted:
16/09/2023
Revised:
03/02/2024
Accepted:
24/04/2024
Full Text PDF
1701 bands, 70.76% of which were polymorphic.
The RAPD primers showed a mean PIC of 0.3 and Rp
value of 2.28 while the average PIC and Rp indexes
for ISSR markers were 0.20 and 1.47, respectively.
UPGMA dendrogram obtained from cluster analysis
of RAPD and ISSR combined data grouped 32
accessions into five clusters at 76% variation with
Jaccard's similarity coefficient varying from 0.586 to
0.951. Moreover, principal component analysis was
also used to determine genetic relationships among
32 collected accessions
.
The results showed high
genetic dissimilarity among 32
Stephania rotunda
Lour. Correlation analysis between the matrices of
similarity coefficient was measured
test. A moderate correlation value (
r
= 0.543)
between RAPD vs ISSR matrices, but the strong
correlation value between RAPD and pooled data (
r
= 0.930) and between ISSR and combined matrices
(
r
= 0.814) were obtained, suggesting a
combination of RAPD and ISSR markers appears to
be productive in studying the genetic variance in
Stephania rotunda
Lour. Our results could provide a
solid foundation for further conservation
management and breeding programs of
Stephania
rotunda
Lour.
Keywords: ISSR; genetic diversity;
Stephania rotunda
Lour
.
; RAPD.
Abbreviations: ISSR_Inter-Simple Sequence Repeats; PCA_Principal
Coordinate Analysis; PIC_Polymorphism Information Content
RAPD_Random Amplified Polymorphic DNA; Rp_Resolving Power;
UPGMA_Unweighted Pair-Group Method with Arithmetic. Average
Introduction
Stephania rotunda
Lour. (
S. rotunda
) is a medicinal creeper plant
belonging to the large genus
Stephania
of Menispermaceae with about
60 species, most of which are mainly distributed in Southeast Asia
(Nguyen, 2003; Luo et al., 2008; Desgrouas et al.
,
2014). In this genus,
37 species in China, 15 species in Thailand, and about 16 species in
Vietnam have been recorded (Lo, 1978; Nguyen, 2003; Hu et al., 2008;
Vu et al., 2019). As a folk traditional medicine, it has been used for
the treatment of a wide range of illnesses including headache, asthma,
fever, and diarrhea (Semwal and Semwal, 2015). Phytochemical
analyses identified at least 40 types of alkaloids in various parts of
this plant
(Desgrouas et al., 2014).
In Vietnam
, S. rotunda
is commonly found in a wide range of regions,
especially in calcareous mountain areas. With its rich biodiversity,
Vietnam is a part of South-Central China that is one of 25 biodiversity
hotspots listed by Myers et al.
(2000). However, the biodiversity of
S.
rotunda
plants in these regions has been critically endangered by a
chaotic history. Moreover, the indiscriminate overexploitation
of
Stephania
plants has resulted in the endangerment of many species.
In Vietnam,
S. rotunda
is classified as Class IIA for the species that is
threatened with extinction listed in Decree 84/2021/ND-CP
(Vietnamese Government, 2021). Thus, it is important to provide an
efficient and reliable strategy for the conservation and development of
this species. Molecular analysis and assessment of genetic diversity
can provide useful information about taxonomic identification,
evolution amongst the species as well as the molecular background of
different natural phenomena (Csillery et al., 2010). However, the
knowledge of this species is still limited in Vietnam.
Fig 1. (A) RAPD banding profile obtained with primer OPA-01 and (B)
ISSR banding profile obtained with primer UBC-811. Lane M: 1kb
ladder, lane from 1 - 32 were PCR products from accessions in terms
of No. given in Table 1.
Table 1. Amplification of 14 RAPD markers in 32
Stephania rotunda
Lour. accessions.
Primer
Sequence
Tm
(
o
C)
Total
No. of
loci
Polymorphic
loci
Polymorphism
(%)
Total
No.
of
band
s
PIC
Rp
OPA-01
GAGGCCCTTC
34
8
8
100.00
143
0.3
0
3.44
OPA-02
TGCCGAGCTG
34
8
7
87.50
101
0.2
7
1.81
OPB-01
GTTTCGCTCC
32
8
8
100.00
66
0.1
9
1.88
OPB-04
GGACTGGAGT
32
7
5
71.43
102
0.2
0
1.88
OPC-03
GGGGGTCTTT
32
8
7
87.50
168
0.2
9
3.25
OPC-08
TGGACCGGTG
34
7
7
100.00
116
0.3
2
3.25
OPD-01
ACCGCGAAGG
34
4
3
75.00
55
0.2
5
1.44
OPE-04
GTGACATGCC
32
4
3
75.00
66
0.0
0.38
9
OPE-07
AGATGCAGCC
32
5
5
100.00
135
0.2
5
1.56
OPN-03
GGTACTCCCC
34
6
6
100.00
82
0.4
0
3.75
OPO-01
GGCACGTAAG
32
6
5
83.33
50
0.1
6
1.13
OPO-02
ACGTAGCGTC
32
10
10
100.00
128
0.2
7
3.50
OPS-05
ACAGGTGCGT
32
6
6
100.00
66
0.2
9
2.38
OPR-12
TTTGGGGCCT
32
4
3
75.00
68
0.3
5
2.25
Total
91
83
1254.76
1346
3.6
3
31.8
8
Average/
Primer
6.5
5.93
89.63
96.1
4
0.3
0
2.28
The examination of the genetic dissimilarity of medicinal plants plays
a vital role in the conservation and utilization of plant genetic
materials. Both morphological and DNA markers can be utilized to
determine the genetic variance within and among plant individuals or
populations. The morphological markers are cheap and simple, but
they have demerits since the phenotypes are greatly influenced by
environmental conditions and the developmental stages of the plants.
In contrast, molecular markers, also known as DNA markers, including
random-amplified polymorphic DNA (RAPD), inter-simple sequence
repeats (ISSRs), amplified fragment length polymorphism (AFLP),
single nucleotide polymorphism (SNPs), and sequence tag sites (STSs),
etc. are stable, detectable in every part of the plant, unaffected by
environmental factors, and exhibit greater levels of polymorphism
(Semwal and Semwal, 2015). RAPD and ISSR have been successfully
effective in revealing the diversity in the DNA of many medicinal
plants in particular, such as ginseng (Wei
et al., 2014),
Nilgirianthus
ciliates
(Ramakrishnan et al., 2019),
Podophyllum hexandrum
(Naik et
al., 2010),
Justicia adhatoda
(Kumar et al., 2014), medicinal plants of
Solanaceae
family (Singh et al., 2022).
This study is the first effort to evaluate the genetic diversity among
the 32
S. rotunda
accessions grown in various
geographical locations of Vietnam by utilizing RAPD and ISSR markers.
will facilitate the effective conservation,
management, and development of
S. rotunda
and other endangered
medicinal plants.
Results
RAPD analysis
A total of 91 loci were yielded by 14 RAPD primers with a mean of 6.5
loci per primer (Table 1, Fig 1A). The percentage of polymorphism was
from 71.43% for primer OPB-04 to 100% for primers OPA-01, OPB-01,
OPC-08, OPE-07, OPN-03, OPO-02, and OPS-05 with an average of
89.63% polymorphism per primer. The Polymorphism Information
Content (PIC) value of 14 RAPD markers varied from a lowest of 0.09
(OPE-04) to a highest of 0.40 (OPN-03) with a mean of 0.3. The
resolving power (Rp) value was lowest for primer OPE-04 (0.38) and
highest for primer OPN-03 (3.75) with an average of 2.28 per primer
(Table 1).
The Jaccard similarity index resulting from RAPD data ranged from
0.478 between accession 8 and accession 13 to 0.956 between
accession 29 and 30, with an average of 0.735 (Supplemental Table 1).
At 73.5% similarity, the 32
S. rotunda
Fig 2.
coefficients of 32
Stephania rotunda
Lour. accessions based on RAPD
data analysis.
Fig 3.
coefficients of 32
Stephania rotunda
Lour. accessions based on ISSR
data analysis.
Table 2. Amplification of 14 ISSR markers in
Stephania rotunda
Lour.
accessions.
Primer
Sequence
Tm
(
o
C)
Total
No. of
loci
Polymorphic
loci
Polymorphism (%)
Total
No.
of
bands
PIC
Rp
UBC-807
(AG)
8
T
50.0
5
4
80.00
95
0.06
0.31
UBC-808
(AG)
8
C
50.0
7
5
71.43
103
0.13
1.06
UBC-811
(GA)
8
C
50.0
8
7
87.50
162
0.36
4.75
UBC-812
(GA)
8
A
49.0
5
3
60.00
102
0.33
2.5
UBC-813
(CT)
8
T
50.0
5
3
60.00
128
0.20
1.5
UBC-823
(TC)
8
C
50.0
6
4
66.67
158
0.24
2.13
UBC-824
(TC)
8
G
50.0
5
4
80.00
140
0.19
1.25
UBC-827
(AC)
8
G
52.4
4
4
100.00
80
0.28
1.5
UBC-848
(CA)
8
RG
53.0
4
3
75.00
94
0.32
2.13
UBC-864
(ATG)
6
45.0
4
4
100.00
107
0.27
1.31
UBC-873
(GACA)
4
45.0
4
2
50.00
118
0.13
0.63
UBC-888
(CA)
7
BDB
47.0
4
2
50.00
125
0.04
0.19
UBC-889
(AC)
8
DB
47.0
5
2
40.00
152
0.08
0.5
UBC-891
(TG)
7
HVH
47.0
6
3
50.00
137
0.1
0.81
Total
72
50
1701
2.75
20.56
Average/primer
5.14
3.57
70.76
121.5
0.20
1.47
*
R = A/G; B = non-A; D = non-C; H = non-G; V=non-T.
Table 3. Relative efficiency of molecular markers for determining
polymorphism in
Stephania rotunda
Lour. accessions.
Parameters for marker efficiency
Molecular marker system
RAPD
ISSR
Combined RAPD and
ISSR
Number of cultivars
32
32
32
Total number of primers
14
14
28
Total number of loci
91
72
163
Total number of polymorphic loci
83
50
133
Polymorphism (%)
89.63
70.76
80.19
Total number of scorable bands
1346
1701
3047
Polymorphism information
content (PIC)
0.30
0.20
0.23
Resolving power (Rp)
2.28
1.47
1.87
Table 4. Matrix comparisons of Mantel test between markers.
Comparison
Matrix
correlation
(
r
)
p-value
(two-
tailed)
Alpha
RAPD vs. ISSR
0.543
< 0.0001
0.05
RAPD vs. combined RAPD and
ISSR
0.930
< 0.0001
0.05
ISSR vs. combined RAPD and
ISSR
0.814
< 0.0001
0.05
Fig 4.
coefficients of 32
Stephania rotunda
Lour. accessions based on RAPD
and ISSR data analysis.
accessions were separated into four main clusters as shown in Fig 2.
Cluster I contained most of the accessions including 1, 5, 2, 3, 4, 11,
12, 16, 17, 31, 28, 29, 30, 18, 20, 22, 23, 21, 19, 14, 13, 15, 24, and
27. Cluster II consisted of accessions 25 and 26. Cluster III had only
one accession 32. Cluster IV was composed of five accessions
including 6, 7, 9, 10, and 8 (Fig 2).
ISSR analysis
The 14 ISSR markers produced a total of 72 amplification loci with an
overall mean of 5.14 loci per primer (Table 2, Fig 1B). UBC-811 primer
gave the maximum loci (8) while UBC-827, UBC-848, UBC-864, UBC-
873, and UBC-888 primers exhibited the smallest number of loci (4.0).
The percentage of polymorphism was from 40.00% (UBC-889 primer)
to 100% (UBC-827 and UBC-864 primers) with a mean of 70.76%
polymorphism per primer. The lowest PIC index of 0.04 was for
primer UBC-888 whereas the maximum PIC index of 0.36 was found
in UBC-811 primer with a mean PIC value of 0.20 per primer. The
mean Rp index of 14 ISSR primers was 1.47 with a
maximum index of 4.75 for UBC-811 primer and the smallest index of
0.31 for UBC-807 primer (Table 2).
The Jaccard similarity index based on ISSR data ranged from 0.583 to
0.972 with a mean of 0.794 (Supplemental Table 2). Accessions 28
and 29 exhibited the biggest similarity value (0.972) and accessions 9
and 12 showed the least similarity (0.583). The dendrogram displays
the construction of five main clusters of 32
S. rotunda
accessions at
the coefficient value of 79.4% (Fig 3). Cluster II is the largest cluster
with 23 accessions including 2, 3, 31, 17, 18, 20, 23, 28, 29, 30, 4,
13, 5, 16, 14, 15, 19, 22, 21, 27, and 24. Cluster III consisted of five
accessions 6, 7, 10, 8, and 9. Each cluster I and IV had only accession
1 and 32, respectively. Cluster V contained accessions 25 and 26.
Cluster VI comprised two accessions 11 and 12.
Combined RAPD and ISSR analyses
A combination of 14 RAPD and 14 ISSR primers generated 80.19%
polymorphic bands with the mean PIC and Rp indexes of 0.23 and
1.87, respectively (Table 3). The Jaccard similarity
Fig 5. PCA analysis of 32
Stephania rotunda
Lour. accessions using
F1, F2 are the main components of the PCA biplot contributed 62.88%
of the total variation in genetic relationship among 32 accessions.
Each point represents as accession number.
Table 5. Detail of the
Stephania rotunda
Lour. accessions collected
from different regions of Vietnam used in this study.
Accession
No.
Collection site
Laditude/longtitude
Altitude
(m)
1
Vinh Tien, Kim Boi, Hoa Binh
20°43'00"N,
105°25'13"E
425
2
Vinh Tien, Kim Boi, Hoa Binh
20°42'53"N,
105°26'02"E
479
3
Vinh Tien, Kim Boi, Hoa Binh
20°43'35"N,
105°26'40"E
299
4
Vinh Tien, Kim Boi, Hoa Binh
20°43'36"N,
105°26'47"E
359
5
Vinh Tien, Kim Boi, Hoa Binh
20°43'49"N,
105°26'22"E
440
6
Dao Duc, Vi Xuyen, Ha Giang
22°43'28"N,
104°58'16"E
175
7
Dao Duc, Vi Xuyen, Ha Giang
22°43'15"N,
104°57'22"E
146
8
Dao Duc, Vi Xuyen, Ha Giang
22°43'14"N,
104°34'16"E
126
9
Cao Bo, Vi Xuyen, Ha Giang
22°45'00"N,
104°51'56"E
873
10
Cao Bo, Vi Xuyen, Ha Giang
22°44'28"N,
104°52'46"E
689
11
Cao Son, Luong Son, Hoa Binh
20°48'44"N,
105°29'27"E
242
12
Cao Son, Luong Son, Hoa Binh
20°48'32"N,
105°30'25"E
224
13
Cao Son, Luong Son, Hoa Binh
20°48'32"N,
105°31'28"E
125
14
Cao Son, Luong Son, Hoa Binh
20°48'33"N,
105°30'45"E
241
15
Cao Son, Luong Son, Hoa Binh
20°48'41"N,
105°32'11"E
382
16
Tu Son, Kim Boi, Hoa Binh
20°43'44"N,
105°23'53"E
348
17
Tu Son, Kim Boi, Hoa Binh
20°43'26"N,
105°23'34"E
453
18
Tu Son, Kim Boi, Hoa Binh
20°43'12"N,
105°25'19"E
454
19
Tu Son, Kim Boi, Hoa Binh
20°42'50"N,
105°25'19"E
614
20
Tu Son, Kim Boi, Hoa Binh
20°44'52"N,
105°23'46"E
375
21
An Binh, Lac Thuy, Hoa Binh
20°24'02"N,
105°45'40"E
71
22
An Binh, Lac Thuy, Hoa Binh
20°23'59"N,
105°45'47"E
142
23
An Binh, Lac Thuy, Hoa Binh
20°23'47"N,
105°45'47"E
70
24
Nam Ke, Muong Nhe, Dien Bien
22°06'03"N,
102°38'43"E
526
25
Muong Thin, Tuan Giao, Dien
Bien
21°37'05"N,
103°22'00"E
871
26
Muong Thin, Tuan Giao, Dien Bien
21°37'03"N,
103°21'56"E
833
27
Tan Uyen, Tan Uyen, Lai Chau
22°04'34"N,
103°39'49"E
764
28
Doan Ket, Da Bac, Hoa Binh
20°56'00"N
105°02'55"E
810
29
Doan Ket, Da Bac, Hoa Binh
20°55'28"N
105°01'59"E
1054
30
Doan Ket, Da Bac, Hoa Binh
20°55'40"N
105°02'35"E
896
31
Doan Ket, Da Bac, Hoa Binh
20°56'31"N
105°02'36"E
944
32
Quang Thanh, Thanh Hoa
19°45'45"N,
105°46'56"E
4
value based on pooled RAPD and ISSR data analysis ranged from 0.586
to 0.951 with a mean of 0.762. The highest similarity value was
between 29 and 30 accessions (0.951) whereas accessions 9 and 13
exhibited the least similarity of 0.586 (Supplemental Table 3). The
UPGMA dendrogram based on combining data grouped 32
S. rotunda
accessions into five different groups at 76.2% similarity. Cluster I
contained 22
accessions including 1, 5, 2, 3, 4, 13, 14, 15, 16, 17, 22, 18, 20, 23,
28, 29, 30, 31, 21, 19, 24, and 27. Cluster II had two accessions 11
and 12. Cluster III comprised accessions 25 and 26. Cluster IV had
only one accession 32. Cluster V consisted of five accessions including
6, 7, 9, 10, and 8 (Fig 4).
The Mantel test and Pearson's correlation (
r
) exhibited a moderate
coefficient of 0.543 (p<0.0001) between ISSR and RAPD markers
whereas genetic matrics of RAPD or ISSR makers and integrated data
showed a strong correlation with
r
= 0.930 and 0.814 (p<0.0001),
respectively (Table 4). The results indicated that the efficiency of
genetic variation analysis of RAPD to 32
S. rotunda
accessions is
higher than that of ISSR.
Principal component analysis (PCA) based on the combined RAPD and
ISSR data displayed the genetic relationships of
S. rotunda
accessions
in two-dimensional space accounted for 45.71% and 17.17% of the
total genetic varia difference. The PCA data separated 32
S. rotunda
into major clusters that appeared similar to the tree diagram
generated from the cluster analysis (Fig 5). Accessions 1, 12, 11, 13, 5,
14, 15, 3, 4, and 2 were grouped into one cluster whereas accessions
16, 19, 17, 21, 23, 18, 30, 31, 22, 29, 28, and 20 were in the same
cluster. Accessions 6, 7, 8, 9, and 10 were gathered together in one
cluster, and accessions 24, 25, 26, and 27 were in one group.
Unsurprisingly, accession 32 was isolated into one group from the
remaining accessions (Fig 5).
Discussion
DNA markers such as ISSRs, RAPDs, AFLPs, SSRs, and SNPs have been
demonstrated to be practical in examining genetic dissimilarity in
Lee et al., 2012; Liu et al., 2018; Bi et al.,
2021; Singh et al., 2022). In this study, the genetic diversities of 32
S.
rotunda
accessions collected from different geographical zones in
Vietnam were analyzed through RAPD and ISSR molecular markers. A
large number of studies previously showed that RAPD markers provide
a higher capacity for detecting polymorphism and genetic diversity
than ISSR markers (Ninh et al., 2022; Gupta et al., 2008; Verma et al.,
2017) while other research indicated that ISSR markers have more
efficiency than RAPD in identifying polymorphism in many plant
species (Zietkiewicz et al., 1994; Pham et al., 2021). In medicinal plant
research, Hamouda (2019) has shown that the percentage of
polymorphism among 14 collections of
Silybum marianum
populations in Egypt identified by RAPD markers was 73.2% whereas
by ISSR markers was 79.3%. However, Baruah et al. (2017) revealed
that 90.68% of bands generated by ISSR primers showed
polymorphism among
Cymbopogon
species while this number in the
case of RAPD primers was lower (88.62%). In our study, the RAPD
primers are more efficient, detecting 89.63% polymorphism in 32
accessions of
S. rotunda
, compared to ISSR which detected 70.76%
polymorphism. The higher PIC value observed in RAPD markers also
added strength to the above observation. Similarly, Bui et al. (2022)
also found that the mean PIC index of RAPD (0.72) was higher than
that of ISSR primers (0.64) in an important medicinal plant in Vietnam,
Pseuderanthemum latifolium
.
The resolving power (Rp) is a measure of the ability of a molecular
marker to differentiate among individuals (Prevost and Wilkinson,
1999). In this study, Rp value of RAPD markers (2.28) was higher than
that of ISSR markers (1.47) (Table 3) suggesting a higher power of
RAPD for the discrimination of genetic diversity among
S. rotunda
accessions. Supporting these findings, our UPGMA clustering analysis
showed that the dendrogram generated by RAPD and pooled
RAPD+ISSR data grouped 32 accessions of
S. rotunda
in similar
clusters except that accessions 11 and 12 were separated into one
group in the combined data-generated dendrogram (Fig 3 and Fig 4).
Moreover, the correlation test showed that RAPD gave a stronger
correlation coefficient (
r
= 0.930) than ISSR (
r
= 0.814) with pooled
data (Table 4). According to Ninh et al. (2022) and Chowdhury et al.
(2002), the ISSR system produced more complex marker patterns and
was more reproducible than the RAPD approach meaning that ISSR is
advantageous when discriminating closely related accessions.
Nonetheless, based on higher polymorphism, PIC, Rp, and correlation
coefficient values, the RAPD method was slightly more effective than
ISSR in the evaluation of genetic variation in
S. rotunda
. This is
because the target sequences in the genome detected by the two
marker systems were different. While RAPD primers are distributed
along the genome, ISSRs are found only between microsatellite
sequences (Bachmann, 1997; Landergott et al., 2001; Penner, 1996).
The similarity coefficient was from 0.478 - 0.957 in RAPD, 0.583 -
0.972 in ISSR, and 0.586 - 0.951 in joined data which indicated high
genetic diversity among 32
S. rotunda
accessions collected from
different geographical areas in Vietnam. Different environmental
conditions such as temperature, relative humidity, sunlight, rainfall,
wind, etc. may be the main contributors to the high genetic variation
among the
S. rotunda
populations. Supporting our observations,
previous studies showed similar results in medicinal plants, such as
Caragana microphylla
(Huang et al., 2016) and
Nilgirianthus ciliates
(Ramakrishnan et al., 2019) wherein climate factors affect genetic
variability. It was found that
S. rotunda
accession 28 was closely
related to accession 29 based on the similarity matrix obtained from
pooled data analysis. This finding was not surprising since these two
accessions were both collected from Doan Ket, Da Bac, Hoa Binh
province. The last similarity value was obtained between accessions 8
and 13, accessions 9 and 12, and 9 and 13 according to RAPD, ISSR,
and pooled data analyses (Supplemental Tables 1, 2, and 3). These
results can be attributed to the fact that these accessions were
collected from different geographical areas such as accession 8 and 9
was collected in Ha Giang province whereas accession 12 and 13 were
collected from Hoa Binh province.
The dendrogram of the combined RAPD and ISSR data demarcated 32
S. rotunda
accessions into five main clusters. Cluster I contained 20
accessions collected from Hoa Binh (1, 5, 2, 3, 4, 13, 14, 15, 16, 17,
22, 18, 20, 23, 28, 29, 30, 31, 21, 19), one accession collected from
Muong Nhe, Dien Bien (24) and one accession collected from Tan
Uyen, Lai Chau (27). Cluster II comprised two accessions collected
from Luong Son, Hoa Binh (11 and 12). Two accessions collected in
Tuan Giao commune, Dien Bien province were in cluster III (25 and 26)
whereas all five accessions from Vi Xuyen commune, Ha Giang
province (6, 7, 9, 8, 10) were grouped in cluster V. Accession 32
collected from Thanh Hoa province singled out from the rest of the
accessions (Fig 4). It can be seen that the accessions collected in
different communes but belonging to the same provinces were
separated into one group. This could be the result of the plants
growing in similar climate conditions (Huang et al., 2016). The mixing
of accession 24 collected in Muong Nhe commune, Dien Bien province,
and accession 27 collected in Tan Uyen commune, Lai Chau province
in cluster I of 20 accessions collected from Hoa Binh province could
mean that the region-specific diversity does appear when the plants
are grown in different geographical zones or the environmental
conditions in these locations are moderately similar. The results
acquired by cluster analysis also were further supported by the
principal component analysis (PCA) that indicates the separation of
the 32 accessions based on their geographical areas. In wild
conditions, the genetic diversity of each
Stephania rotunda
Lour. was
successfully uncovered by ISSR and RAPD markers.
Materials and methods
Plant materials
Thirty-two accessions of
S. rotunda
were collected from different
locations in central and Northern areas of Viet Nam being maintained
at the Faculty of Biotechnology, Vietnam National University of
Agriculture. The list of
S. rotunda
accessions is presented in Table 5.
DNA extraction
Genomic DNA was isolated from fresh leaves of
S. rotunda
plants by
the standard CTAB (Cetyl Trimethyl Ammonium Bromide) method
(Doyle and Doyle, 1987). DNA concentration and purity were
measured by a Nanodrop Spectrophotometer (Thermo). The DNA was
stored at -20°C.
RAPD-PCR and ISSR-PCR amplification
Fourteen RAPD and 14 ISSR primers were used for PCR amplification.
The sequences and melting temperature (Tm) of each primer are
presented in Table 2 and Table 3. PCR reactions were conducted with
1 l of primer (10 M), 1.0 l DNA (10 ng/l), 10 l MyTaq Buffer
(Bioline, USA), and the final volume made up of 2 l with distilled
water. The PCR was run in ASTEC Thermal Cyclers (Gene Atlas, ASTEC,
Japan) using a program of initial denaturation at 95
0
C for 5 min, 35
cycles of 30 s denaturation at 95
0
C, 30 s annealing at Tm, and 90 s
extension at 72
0
C, followed by a 10 min final extension at 72
0
C. PCR
products were visualized on 1% agarose gel in 1×TAE buffer by
electrophoresis at 100 V for 30 min and photographed by Bio-image
System (BioRad, Germany).
Data analysis
Clear and reproducible bands amplified with ISSR and RAPD primers
were manually scored as presence (1) or absence (0).
The polymorphism information content (PIC) index for each primer
was measured according to et al. (2000): PIC = 2𝑓(1 − 𝑓)
where
is the proportion of the present bands and (1 − 𝑓)
is the
proportion of the absent bands.
The resolving power (Rp) of each primer was calculated as proposed
by Prevos and Wilkinson (1999): Rp = ∑BI . In which, BI (Band
Informativeness) equals 1 − (2 × |0.5
- p
|) where
p
is the frequency of
accessions carrying a particular band.
Dendrograms were produced using the unweighted pair-group
method with arithmetic average (UPGMA) on the basis of
similarity coefficient TREE program of NTSYS 2.1 software. Genetic
similarity matrices by ISSR, RAPD and combined ISSR and RAPD
software were used as input for principal coordinate analysis (PCA).
Estimation of correlation value between markers was done using a
Mantel test in XLSTAT 2018 package.
Conclusion
This is the first study on the genetic diversity analysis of the medicinal
plant
S. rotunda
Lour. using molecular markers. The findings of the
research provide important information for conservation and breeding
programs of
S. rotunda
Lour.
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