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African Journal of Agricultural Marketing ISSN: 2375-1061 Vol. 11 (3), pp. 001-006, March, 2023. Available 
online at www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 
 

Full Length Research Paper 

 

Inter-population variation of chromosome and 

RAPD markers of Suaeda nudiflora (Willd.) Moq. a 

mangrove species in India 
 

S. N. Jena and A. B. Das* 
 

Cytogenetics Laboratory, Regional Plant Resource Centre, Bhubaneswar 751015, Orissa, India. 
 

Accepted 10 November, 2022 
 
Inter-population genetic diversity in Suaeda nudiflora, a mangrove, was investigated through RAPD and 
chromosome analysis among five Indian populations. Somatic chromosome numbers 2n=36 reported for the first 
time in population (Pop) I and Pop-II whereas in Pop-III, 2n=36 and 40 chromosomes. Pop-IV showed 2n=54 
numbers from high saline environment while Pop -V showed 2n=40 chromosomes. 102 RAPD bands were 
polymorphic out of a total 182 amplicons with an average of 3.64 bands per population per primer suggests 
genetic divergence in inter-population level. The dendogram based on the RAPD analysis showed two broad 
groups suggesting ecotypic adaptability in different saline habitat. The maximum overall relatedness of the pop-
II with rest of four populations was 57% indicated by mean similarity while pop-IV revealed minimum mean 
similarity of 49% suggesting new cytotypes formation. The probable mechanism of overcoming high salinity 
stress by maintaining polyploidy (2n=54) is discussed. 
 
Key words: Genetic divergence, genetic polymorphism, RAPD markers, somatic chromosome, Suaeda nudiflora. 

 
INTRODUCTION 

 
Mangroves have common needs to adapt to adverse 
environmental conditions associated with regular seawa-
ter inundation, for which individual species have develop-
ped different strategies and characteristics (Duke et al., 
2002). Spatial patterns in genetically adaptive traits indi-
cate that some populations survive more successfully 
under changing environment conditions (Dodd and Rafii 
2002). Assessment of the genetic differences between 
populations of the same species gives a measure of the 
extent to which such populations are generally isolated 
from each other, while genetic comparison of different 
species shows information about the extent of divergence 
and potentially, when such divergence took place 
(Hogarth 1999). The extent of genetic difference among 
different populations is an important measure of the 
diversity of that area. Therefore, there is an obvious lack  
 
 
 
*Corresponding author’s. E-mail: a_b_das@hotmail.com. 
1
Present address: Conservation Biology & Molecular 

Taxonomy, National Botanical Research Institute, Lucknow, 
India. 

 
 
 
 
of knowledge concerning the genetic characteristics wit-
hin and between populations of mangrove species as well 
as genetic relationship in mangrove ecosystems.  

Suaeda nudiflora, a tropical halophytic grass that toler-
ates high temperature, generally found on the moist soil 
of river sea side ward fringe which always remain water-
logged with high and low tides. The plant species in the 
mangrove forests are constantly under environmental 
stress due to high saline conditions, extreme temperature 
and high salt deposition on the mud flat, therefore have 
adapted themselves to these frequent and fluctuating 
changes. S. nudiflora is dioecious with no vegetative 
propagation. Moreover, it is insect-pollinated; gene flow is 
expected to decrease considerably with distance. This 
unique plant, which has a versatile form with branches 
spreading on the soil surface, has a high food value for 
people along the coastal belt. However, under altered 
ecological and physical conditions in mangrove ecosys-
tem, discernible changes were reported in genetic consti-
tution of S. nudiflora (Tomlinson 1986, Jena et al., 2002) 
besides its morphology. Until recently, with exception of 
occasional reports on somatic chromosome analysis 
(Jena et al., 2002) most of the studies that investigate S. 



2 

 

 
 
 

 
Table 1. Genotypes of S. nudiflora from different populations of Bhitarkanika mangrove forest, Orissa, India, with their physical 

characteristics and chromosome number. 
 

Population Source of collection  Physical Characteristics Chromosome 

  Soil pH Salinity (ppt) Soil type number (2n) 

Pop-I Hansina Bhitargada 6.4-6.9 3-16 Clayey/fine sand 36 

Pop-II Chitta Kolha Rajnagar 6.3-7.0 5-18 fine silt/clay 36 

Pop-III Dangamal Righagarh 6.8-7.5 7-20 clayey, compact 40,36 
Pop-IV Gupti 6.9-7.8 10-23 Sandy and clayey 54 
Pop-V Talchua 6.7-7.6 6-21 Dark-clay soil 40 

 
 
nudiflora used morphological characteristics of mangro-

ves.  
Molecular markers, unlike morphological markers, are 

stable and have been found to be very useful in popula-
tion studies (Aitkin et al., 1994) they have been used to 
quantify accurately the extent of genetic diversity within 
and between population (Chalmers et al., 1992, Waugh 
and Powell 1992). The application of these markers in 
assessing intra-specific variation in mangrove species 
have been recently studied (Parani et al., 1997, Lakshmi 
et al., 1997). Among the various DNA marker systems, 
RAPDs have been used extensively for a variety of pur-
poses, including population studies (Powell 1992). It, in 
particular, has found widespread application due to their 
technical simplicity and the availability of large numbers 
of arbitrary primers that saturate the genome.  

In this study, five populations from different ecotypes of 
S. nudiflora growing in Bhitarkanika forest in India were 

genetically investigated. To examine the level of genetic 
variation of their diversity, chromosome analysis and 
RAPD markers were used. To our knowledge, this is the 
first genetic investigation done on S. nudiflora 

 
MATERIALS AND METHODS 
 
Plant Material 
 
Five different populations (thirty individuals per populations) of 
Suaeda nudiflora (Willd) Moq. were collected from Bhitarkaniaka 
mangrove forest of eastern coast of India (latitude and longitude of 

20
0
 40'N, 86

0
 52'E) for the present study. Bhitarkanika is a single 

compact patch of estuarine forest in Orissa about 192.9 sq. Km and 
is the second largest compact patch after Sundarbans of West 
Bengal in main land of India that is crossed by countless creeks, 
rivers and waterways. The physical characteristics of the study sites 
are given in Table 1. From each study site, root tips and young 
leaves were collected for chromosome and DNA isolation respect-
ively. Roots were fixed and preserved in 70% ethanol for further 

investigation and young leaves were stored in a –85
0
C. 

 
Chromosome Preparation 
 
Root tips were collected in the field and put in 0.05M oxiquoline 
solution and left at room temperature for 3 hrs and subsequently 
fixed in 1:3 ratio (acetic acid : ethanol) for over night then soaked in 
45% acetic acid for 20 min stained in 2% acetic-orcine:1NHCl (9:1) 
for over night. A minimum of 10 roots from different plant/ popula-
tion were squashed in 45% acetic acid and observed under micros-
cope for chromosome count and photography. 

 
 
Isolation of DNA 
 
5 g of young leaf tissue was ground under liquid nitrogen and 
suspended in 20 ml of CTAB buffer (2% Cetyl Trimethyl Ammonium 
Bromide, 100 Mm Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl and 

1% -mercaptoethanol). The suspension was incubated at 60
0
C for 

30 min. The DNA was extracted in chloroform- isoamyl alcohol 
(49:1) and centrifuged at 5000 g for 20 min. The aqueous phase 
was taken up and DNA was precipitated with two volumes of chilled 
ethanol. The DNA was hooked out and dried with vacuum 
concentrator and a trace amount of TE buffer (10 mM Tris-HCl + 
1mM EDTA, pH 8.0) was added to dissolve the DNA. The DNA 

again purified then treated with RNase at 37
O

C for 1h followed by 
chloroform: isoamyl alcohol extraction and ethanol precipitation in 
the presence of 0.3 M sodium acetate (pH 5.2). The DNA was 
spooled out, washed in 70% ethanol, air dried and dissolved in TE 
buffer. DNA concentration was estimated using Versafluor TM 
Fluorometer (Bio-Rad, USA) using Hoechst 33258 as the fluro-

metric dye. The DNA was diluted to final concentration of 25ng l
-1

 
using TE buffer and used as template DNA for RAPD analysis. 
Leaves were collected and bulked from different plants for each 
population and replicated three times for DNA isolation. 

 
PCR-RAPD Analysis 
 
RAPD profiles were generated by using single decamer random 
oligonucleotide primers (Operon Technologies, Alameda, USA) in 
polymerase chain reaction (PCR) following the standard protocol of 
Williams et al. (1990). Primer sequence is shown in Table 2. Ampli-
fication reaction mixture of 25 l for each polymerase chain reaction 
(PCR) contained 25 ng of genomic template DNA, 200M of each 
dNTP, 25ng of primer, 0.5 unit of Taq DNA Polymerase (Bangalore 
Genei Pvt. Ltd., Bangalore, India) and 10x PCR assay buffer (50 
mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 9.0). The reaction 
mixture was carried out in a Gene AmpPCR 2400 thermal cycler 
(Perkin Elmer, USA) in the following temperature cycles: holding at 

94
0
C for 5 min at start, followed by 44 cycles of 92

0
C for 1 min, 

40
0
C for 1 min and 72

0
C for 2 min and a final additional extension 

at 72
0
C for 15 min. PCR- products were stored at 4

0
C and electro-

phoretically separated in 1.5% agarose gel in 1×TAE buffer and 
visualized by ehidium bromide staining. To determine the size of the 
polymorphic fragments, Gene Ruler 100 bp DNA ladder plus (MBI 
Fermantas, Lithuania) was used as size standard. The gel was 
visualized under UV light and photographed for documentation. 

 
RAPD Data Scoring and Analysis 
 
In RAPD analysis, the presence or absence of the bands was taken 
into consideration and the difference in the intensity of the band 
was ignored. For all populations, bands on RAPD gels were scored 
as (1) when present or (0) when absent. Jaccard’s similarity coeffi-
cient values (Jaccard, 1998) were calculated for each pair wise 
comparison between genotypes and similarity matrix was constru- 



3 

 

              

Table 2. RAPD primers, their nucleotide sequence and number of RAPD bands generated from five different populations of S. nudiflora of Bhitarkaniaka   
               

 Primer Primer Sequence Total No. Pop-I   Pop-II Pop-III   Pop-IV  Pop-V  

  (5'--3') of band P U P U P U P U P U  

 1. OPA-05 AGGGGTCTTG 16 2 0 3 0 3 0 2 0 1 0  

 2. OPA-08 GTCACGTAGG 22 1 2 1 0 2 0 2 1 2 1  

 3. OPA-11 CAATCGCCGT 19 0 0 0 0 3 1 2 0 2 1  

 4. OPA-14 TCTGTGCTGG 17 1 0 1 0 2 0 0 1 2 0  

 5. OPD-02 GGACCCAACC 13 1 0 0 0 1 0 2 0 3 1  

 6. OPD-08 GTGTCCCCCA 27 2 0 3 0 2 0 4 2 4 0  

 7. OPD-12 CACCGTATCC 15 3 0 2 0 2 1 1 0 1 0  

 8. OPN-04 GACCGACCCA 24 0 0 0 0 3 0 2 0 4 0  

 9. OPN-11 TCGCCGCAAA 14 2 0 1 0 2 0 3 0 1 0  

 10.OPN-15 CAGCGACTGT 15 1 0 2 0 3 0 2 0 2 0  
 

Pop-Popualation, P-Number of polymorphic band, U-Number of unique band.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1a-1c. Somatic chromosome numbers in different population of S. nudiflora collected from Bhitarkanika mangrove forest of Orissa. 

Somatic cell of Pop-I showing 2n=36 (1a) collected from Hansia bhatighar having salinity 3-16 ppm, Pop-V showing 2n=40 (1b) 
chromosomes collected from Talchua area having salinity 7-20ppm, Pop-IV with 2n=54 (1c.) collected from Gupti with salinity 10-23 ppm. 



4 

 

 
 
 

 
ted. This matrix was subjected to unweighted pair group method for 
arithmetic average analysis (UPGMA) to generate a dendrogram 

using average linkage procedure. NTSYS-pc software (Rohlf, 1993) 
was used for analysis. 
 

 

RESULT 

 

Chromosome Analysis 

 

Somatic chromosomes that were counted from all the 
collected populations from different saline zones showed 
very interesting results. The only population (Pop-IV) 
grown in Gupti having high salinity showed 2n=54 chro-
mosomes (Table 1). The other two Pop-I and Pop-II 
showed 2n=36 chromosomes and the rest Pop-III showed 
2n=36 mixed with 2n=40 chromosomes (Table 1, Figures 
1a- c). However, the cells bearing 36 chromo-somes are 
not statistically significant. We attempted meiotic study in 
these population for better understanding of chromosome 
pairing behavior and natural ploidy form-ation were 
unsuccessful because of its very short dura-tion of 
metaphase period in the field condition. 
 

 

RAPD Analysis 

 

The number of amplification products ranged from 13 to 
25 for different populations and polymorphism ranged 
between 48% to 61%. A total of 182 amplification 
products were observed out of which 102 (56%) were 
polymorphic. RAPD profiles of five populations shared a 
number of common bands for all primers. The average 
number of amplification product obtained with one primer 
was 18.2% and 56.04% of the products were polymorph-
hic. Population specific polymorphic bands varied from 25 
in Pop-III and Pop-V to 13 in Pop-II (Table 2). Where as 
percentage of polymorphism ranged from 48.38% in Pop-  
I to 52% in Pop-II and 55.55% in Pop-V to 60%-97% in 
Pop-IV. RAPD profile of five populations showed varia-
tions in banding pattern when amplified by OPA-08, OPA-
11, OPA-14 and OPN-04 separately (Figures 2a-c). In 
OPA-08, two DNA markers of 1344bp and 709bp were 
found to be unique to Pop-V in contrast to 453bp and 
450bp unique bands to Pop-I while 1000bp and 340bp 
DNA fragments were observed in Pop-IV and Pop-V 
respectively. DNA marker with 2410bp was found a 
unique band to Pop-V while 1000bp DNA band was 
unique to Pop-III in OPA-11. For the primer, two DNA 
bands of 820bp and 330bp in Pop-III and Pop-V were 
also noted. The DNA bands with 1420bp were found as 
marker band in Pop-III, Pop-IV, and Pop-V whereas DNA 
band of 1120bp was found as marker in Pop-III and Pop-
IV. DNA fragment, having 910bp was a good marker 
which was not only present in Pop-IV using OPA-14 
primer while 300bp DNA fragment was unique to Pop-IV. 
In addition, in Pop-IV showed two marker bands 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. RAPD amplification profiles of five populations of S. 
nudiflora using OPA-8 & OPA-11(a), OPD-8 (b) and OPN-4 (c) and 
marker DNA (M) Gene Ruler 100bp DNA ladder plus (MBI 
Fermantas, Lithuania) from left to right showing major marker 
RAPD fragments i.e. 450bp in Pop-I, 300bp and 1000bp in Pop-III 
and 1344bp in Pop-V amplified by OPA-8 primers (arrow heads). 
OPA-11 primer produced marker bands of 330bp, 2134bp and 
2410bp in Pop-II, Pop-I and Pop-V respectively. OPD-8 primers 
showed 850bp and 976b in Pop-I, 630, 980, 2400 bp in Pop-IV and 
OPN-4 amplified 1064bp, 1300bp, 1730bp in Pop-III and 1820 bp in 
Pop-IV (arrow heads).  
 

 

of 630bp and 908bp in OPD-08 primer. In the same 
primer, 2000bp, 1650bp and 1200bp DNA fragment were 
found markers bands in Pop-IV and Pop-V while DNA 
marker of 976bp was found in Pop-I and Pop-II in 
contrast to 1233bp DNA marker in Pop-I, Pop-II and Pop-  
III. With OPN-04 primer, 1300bp and 1064bp DNA bands 
were found as marker to Pop-III and Pop-V whereas 
2200bp DNA fragment was found to only Pop-IV and 
Pop-V. Three populations (Pop-III, Pop-IV, Pop-V) had a 
common DNA marker band of 1730bp. The maximum 
genotype-specific polymorphism (62.56%) was found in 
Pop-V followed by Pop-III (50%) and Pop-IV (37.5%) 
while in Pop-II and Pop-I there was the minimum number 
of polymorphic bands (4 and 6 respectively). The genetic 
distance was maximum between Pop-I and Pop-IV (Table 
3). 

 

Cluster analysis 
 
Pair wise comparisons were made for the RAPD profiles 

obtained through the use of 10 random primers in the 

representative samples of all five genotypes of different 
populations of Bhitarakanika. The DNA characteristics of 



5 

 

 
 
 

 
Table 3. Genetic distance between the investigated population 

of S. nudiflora using RAPD analysis. 
 

Pop-I Pop –II Pop- III Pop-IV Pop-V 

Pop- I 100     

Pop- II 16.6 100    

Pop- III 50.0 42.4 100   

Pop- IV 63.4 57.2 46.9 100  

Pop- V 61.3 55.2 35.5 38.8 100 
 

 

all the populations of S. nudiflora showed a single tree 
with major two branches. Pop-I and Pop-II clustered 
together with a similarity coefficient of 0.83 and made one 
branch of the tree while the rest three population formed 
the other branch of the tree where pop-III and pop-V are 
sister group. Among these three population, Pop-III and 
Pop-V showed a closer affinity with 64% of similarity 
while the Pop-IV showed a very less close similarity with 
Pop-III (53%) and Pop-V (61%). The highest value of 
mean similarity coefficient 0.57 was found in Pop-II, follo-
wed by Pop-III (0.56). The lowest value of mean similarity 
coefficient was recorded for the genotype collected from 
Pop-IV. 

 

DISCUSSION 
 
A DNA based diagnostic assay like RAPD is able to 
identify genotypes directly and can therefore help miti-
gate complications arising from earlier cytological and 
morphological studies. The utility of combined previous 
cytogenetics studies (Jena et al., 2002) and RAPD mar-
kers in resolving phylogenetic patterns in S. nudiflora is  
 
 
 
 
 

 
Clade 1 

 
 

 
Clade 2 

 
 
 
 
Figure 3. UPMGA phenogram of five populations of Suaeda 

nudiflora from five different habitats of Bhitarkanika mangrove forest 

of Orissa, India. 
 

 

clearly demonstrable. Rapid genetic differentiation is 
likely among population groups with divergent chromo-

somes . Between adjacent geographically defined cytoty-
pes of S. nudiflora, there was a significant polymorphism. 

  
  

 
 

 

Remarkably high individual gene diversity has been 
observed between populations as evedinced by chromo-
some records in different populations of S. nudiflora 
(Figures 1a-c). Since 1930, investigators have tried to 
associate the numerical chromosome variation found in 
plants with the environment and to relate the different 
cytotypes occupying different niches in terms of tempera-
ture, luminosity, humidity etc. (Bennet 1987). Intra-speci-
fic chromosome variation was extensive and informative 
to conservation biologists. Although we have assumed 
earlier the existence of different cytotypes of this species 
(Jena et al., 2002), it has been now better understood 
with chromosomal and RAPD data. RAPD data support 
the existence to defined cytotypes for adaptation of differ-
rent populations at various environmental conditions. 
Gene diversity between populations was more prominent 
in the gel figures, where each genotype from each 
population has been amplified with the same primer. In 
addition to population, cytogenetic data and RAPD data 
at inter-population levels have proved to be extremely 
instructive in developing a better understanding of diverg-
ence. In particular, there are highlighted distinct popula-
tions and local groups within same species, which are not 
only genetically distinct but are confined to geographically 
restricted and unique plant communities. In the present 
study we showed the variability of the RAPD banding 
pattern in S.nudiflora, which was evident by chromosome 
number, although morphologically they are not distingui-
shable. Our findings confirm genetic divergence among 
populations belonging to different biological units. Such 
molecular techniques are useful even to delimit species, 
especially morphologically similar taxa. Ecological (habi-
tat) comparison is also powerful method to recognize 
different biological units with similar morphology, espe-
cially when they are sympatrically distributed. The obser-
ved inter-population divergence for populations with diff-
erent geo-location could be attributed to the adaptability 
with the fluctuating micro-climatic conditions with different 
degrees of temperature, light tolerance, and salinity 
gradient (Dawson et al., 1993). 

UPGMA dendogram (Figure 3) shows too clear clades, 
where pop-III, pop-V and pop-IV are grouped together 
(clade1), whereas pop-I and pop-II are grouped in clade  
2. Among populations, genetic variations are relatively 
high suggesting that the populations are largely isolated 
from each other with gene flow. Local selection and 
restricted gene flow between the genotypes has been 
contributed more to the limited genetic variability of this 
species. Thus, it seems likely that fragmentary process 
will accelerate in this species, which appear to be an 
inherently slow group to respond in an evolutionary 
sense. Since the genotypes were physically isolated, the 
genetic content of the individuals that originally colonized 
the locations might be one of the causes of divergence.  

In conclusion, it is observed that though S. nudiflora 

does not show significant morphological variations, the 

present investigation using both chromosomal data and 



6 

 

 
 
 

 

molecular markers reveals that substantial inter-popula-
tion variation does exist that confirm the existence of 

cytotypes and genotypes for better adaptation of this 
species in ecotype level in different magnitude of adverse 

condition in Bhitarakanika mangrove forest, Orissa, India. 

 

ACKNOWLEDGEMENT 
 
The authors are very much grateful to Prof. J. Dolezel, 
Head, Molecular Cytogenetis and Cytometry Laboratory, 
Institute of Experimental Botany,National Academy of 
Science in Czech Republic, Olomouc, Czech Republic for 
his careful editing and valuable suggestions during the 
preparation of the manuscript. We are grateful to the 
Director, Institute of Physics, Bhubaneswar, DST, Govt. 
of India for providing liquid nitrogen free of cost. We 
would like to acknowledge the Ministry of Environment, 
Government of India for the financial support [Grant No. 
3/7/2000-CS (M)] to carry out this work. 

 
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