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Global Research journal of Natural Science  
& Technology (GRJNST)  
Volume: 04 - Issue 2 (2026), 2072  
ISSN P: 2790-7643 ISSN E: 2790-7651  
www.grjnst.net  
https://doi.org/10.53762/grjnst.04.02.23  
Genetic Diversity Analysis of Indigenous Flora Using Molecular Markers  
Received: 27 March 2025. Accepted: 3 April 2026. Published: 27 April 2026  
Sohail Raja jatoi  
Institute of plant SciencesUniversity of Sindh Jamshoro  
sohail.raja333@gmail.com  
Sobia Sattar  
Institute of plant Sciences University of Sindh Jamshoro  
sobiasattararain@gmail.com  
Kosar parveen  
Institute of plant Sciences University of Sindh Jamshoro  
kosarparveen422@yahoo.com  
Imran Ali Bhurt  
Institute of plant Sciences University of Sindh Jamshoro  
emranali007007@gmail.com  
Sadia Talpur  
Institute of plant Sciences University of Sindh  
sadiatalpur.ss@gmail.com  
GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643  
Article ID: 2072  
https://doi.org/10.53762/grjnst.04.02.23  
Copyright © 2026 GRJNST. This article is published under an Open Access model. It is made available to the public under the terms of the Creative  
Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use and distribution  
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Abstract: The adaptability, survival and sustainable use of indigenous plants depend  
upon its genetic richness. This research focused on the diverse need for the  
assessment of genetic diversity of local flora of Sindh through the aid of molecular  
technique. The genetic structure and conservation of various indigenous plant  
species from different ecological zones were studied. This study explores genetic  
diversity patterns inferred from molecular marker techniques to illuminate  
population differentiation and ecological adaptation. It also draws attention to the  
need for incorporating novel genetic tools for biodiversity assessment in less  
developed areas. The objective of this study was to evaluate and compare the  
genetic diversity of indigenous plant populations from various ecological zones of  
Sindh using RAPD and SSR markers. The samples of leaves were taken from  
different ecozones coastal, desert and irrigated zone of Sindh. Genomic DNA was  
extracted using the CTAB-modified method and PCR amplification was carried out  
using RAPD and SSR primers. Agarose gel electrophoresis was performed on  
amplified products and binary-coded genetic data were scored for statistical  
analysis with the use of POPGENE, NTSYS, and PAST software. High  
polymorphism (78.17% RAPD; 81.25% SSR) indicated large genetic diversity  
among populations of the two species, as shown from the results. The highest  
genetic diversity was noted in desert regions such as Tharparkar, and comparatively  
lower was observed in irrigated regions. Cluster and PCoA analyses further  
indicated that populations clustered according to ecological affinities. The authors  
conclude that the genetic diversity within species of the native flora of Sindh is  
considerable but not evenly distributed. It suggests the prioritization of genetically  
rich populations for in-situ conservation and advises embedding molecular data on  
genetic diversity into national biodiversity management programs for effective  
conservation planning.  
Keywords: Plants, Genetic, Molecular, Flora, Ecozones  
Introduction  
Research on plant genetic diversity has gained renewed attention for its potential to provide  
insight into evolutionary history, ecological adaptation and conservation of biological  
resources over time. Indigenous plants form an important genetic resource for ecosystem  
functioning as well as a source of raw material for agriculture, medicine and industrial  
purposes (Zhao et al., 2023). The use of molecular markers is a method that has  
revolutionized genetic variation assessment, quantifying levels at the DNA level with high  
accuracy and precision. Molecular approaches are less environmental context dependent and  
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yield more reproducible datasets than classical morphological and biochemical methods  
(Nybom, 2004). Together, these advances have allowed researchers to undertake more  
accurate genetic structure, population dynamics and phylogenetic investigations. With global  
anthropogenic decline of biodiversity, measuring the genetic composition of a plant species  
on demand has never been more needed. Genetic diversity is significant for its direct purpose.  
This has implications for conservation biology, and sustainable development (Borah et al.,  
2021). Gradient analysis of genetic So, variability within and between plant populations is  
also useful to identify unique genotypes which are under threat and need immediate  
protection. It helps to devise new strategies for conservation too: each life requires both in  
situ and ex situ conservation(X. Zhang et al., 2024). At a larger scale, it is all about genetic  
diversity critical in the enhanced capacity of plants to respond to evolving environmental  
changes, climate included. change, pests and diseases. Plant breeding programs also use  
molecular marker techniques. to identify those crops that have the potential to be resilient for  
negative biotic or abiotic conditions(Mondini et al., 2009). As that the use of molecular tools  
to study biodiversity gives not only theoretical knowledge, but also the potential to apply that  
knowledge in agriculture and environmental management. Pakistan has been bestowed with  
diverse ecological zones from coastal to hilly areas of a variety of natural native plant  
species. As such, this diversity is an irreplaceable national blessing that has ecological,  
economic and cultural value. High growing population & urbanization, deforestation &  
climate variability.  
The expansion of habitat fragmentation and the unsustainable use of species have increased  
many plant species at risk of genetic erosion. Though, studies on genetic diversity using  
extensive molecular techniques are non-existent in Pakistan. A retrospective has signaled for  
the future–on the kinds of conversational strategies largely classical with most failing  
incorporate consider that genetic diversity-is and the exist in plant populations. The  
magnitude of these shortcomings underscores the compelling need for new molecular tools to  
advance conservation of native plants across the nation. Although molecular marker  
technologies have been used worldwide, few studies concerning the genetics of native flora  
are available, especially in developing countries like Pakistan. It restricts the capacity of  
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formulating effective conservation and management measures driven by specific empirical  
data. This study aimed to address the lack of adoption of molecular marker techniques in  
studying genetic diversity for indigenous plant species due to limited understanding needed  
for conservation planning. Develop an accurate genotyping dataset for regional flora using  
modern molecular marker systems. Such an ambition is to develop a genetic archive that will  
inform conservation and breeding initiatives. Besides, the national biodiversity policies must  
incorporate these research results to guarantee sustainable use and conservation of plant  
genetic resources.  
Literature Review  
Genetic diversity is fundamental to the ability of native plant species to adapt to  
environmental change and survive extinction. This molecular diversity can be accurately  
measured by molecular markers, and it would aid the conservation in trees and sustainable  
use of local plants (Hasnain & Mehvish, 2020). The literature displays a quick evolution from  
basic PCR-based markers to genome-great methods with severa case studies in wild and  
endangered plants.  
The part of Genetic Diversity in Native and Endangered Plants  
It also underpins the conservation of wild and endangered species because genetic variation  
determines plants' ability to respond to climate, pests, and habitat change. Remarkably, taxa  
that are threatened or endemic often display high levels of within-population diversity but  
different degrees of population differentiation which reflect geographic structure, gene flow  
and or exploitation Marker-based data on patterns of diversity have becoming a common  
component in priority setting for in situ and ex situ conservation and breeding.  
Different Types of Marker Systems and What Defines Them  
S.No.  
Marker Type/Use  
Features and Applications  
Related Studies  
SSR/microsatellites  
Co‑dominant,  
highly (Mondini et al.,  
polymorphic;  
preferred 2009; Nam et al.,  
for population structure, 2021; Rai, 2023)  
core collections, cultivar  
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ID  
Dominant, low prior info; (Goswami et al.,  
RAPD, ISSR, AFLP, SRAP, SPAR  
rapid  
screening  
of 2021; Nam et al.,  
wild/endemic  
often  
species, 2021; Szabo et  
combined  
for al., 2021)  
robustness  
Gene‑targeted (SCoT, CBDP, iPBS, Focus on functional or (Hasnain  
&
R‑ISSR)  
retrotransposon  
regions; Mehvish, 2020;  
El-  
useful for fine structure Porth  
&
and trait‑linked variation  
Kassaby, 2014;  
Zen et al., 2004;  
Y.  
Zhang  
&
Maginn, 2014)  
NGS/SNP & RAD‑seq derived  
High‑throughput,  
(Courregelongue  
genome‑wide;  
used  
to & Pons, 2024;  
develop dense SSR/SNP Haque,  
panels in non‑model, rare Nam et al., 2021;  
2025;  
species  
Zhao  
et  
al.,  
2023)  
Genetic Diversity in Indigenous Plant Populations: Patterns  
In fact, most variation (65–90%) is found within populations for many wild and indigenous  
species. Moderate to high levels of among-population differentiation (e.g. Gst/Fst ≈ 0·16–  
0·40) are typically associated with geographic isolation, habitat fragmentation and gene flow  
restriction (Haque, 2025; Nam et al., 2021). Conservation based on habitat-selection is  
urgently needed rather than the presumption of severe genetic erosion for some endangered  
taxa which exhibit unexpectedly high diversity and relatively large gene flow.  
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Methodological Considerations and Trends  
Comparative studies show that dominant markers (RAPD, AFLP, ISSR) provide broadly  
similar estimates but lower levels of diversity compared with SSRs, which also indicate  
higher within-population heterozygosity and finer structure. Cluster resolution and  
discriminating unique breeding/restoration genotypes can be achieved by using multiple  
marker systems in combination or integrating morphology/phytochemistry with DNA data  
(Hu et al., 2024; Pandey et al., 2023).  
Materials and Methods  
Genetic Diversity among selected indigenous plant species collected from main ecological  
zones of Sindh, Pakistan. Sample sites were like Coastal belts of Thatta, Badin, desert areas  
of Tharparkar and Umerkot, docile irrigated plains of Hyderabad and Sukkur. These regions  
show ecological variation across the province. It was collected during periods of active  
vegetative growth by way of field surveys, ensuring that ample viable biological material was  
available. To account for intra-population variability, healthy young leaf tissues were  
collected from several individuals of each species. Every sample was assigned to its own ID  
code. Handheld GPS (Global Positioning System) records geographic coordinates. We  
recorded habitat variables including soil type, vegetation type, and moisture level. Samples  
were initially preserved in silica gel for DNA integrity during transport.  
The experimental work was done in molecular laboratories at University of Sindh, and Sindh  
Agriculture University. Laboratory Setup Centrifuge Micropipette set Vortex mixer Water  
bath Laminar flow cabinet Thermal cycler Electrophoresis unit UV transilluminator Gel  
documentation system The genomic DNA was extracted following a modified CTAB  
protocol appropriate for the presence of secondary metabolites in plant tissues. Using liquid  
nitrogen, the leaf samples were ground to a fine powder. Cell lysis with CTAB extraction  
buffer. Purification of DNA was by chloroform–isoamyl alcohol. Cold isopropanol was used  
for DNA precipitation. Pellets were washed with ethanol, dried and resuspended in TE buffer.  
DNA quality was determined and visualized on 1% agarose gel [8]. Measurement of DNA  
concentration was done using UV–Visible spectrophotometer by means of 260/280 nm ratio.  
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Selected for downstream processing were samples still showing high molecular weight DNA  
intact integrity following lysis. Molecular analysis based on Random Amplified Polymorphic  
DNA markers Simple Sequence Repeat markers. Reaction mixture contains template DNA,  
primer mix, dNTPs, MgCl₂), buffer solution and Taq DNA polymerase. PCR amplification  
was performed in thermal cycler The amplification program was as follows: an initial  
denaturation step at 95 degrees C for 5 min, followed by cyclic denaturation, primer  
annealing and strand extension (25 cycles of 30 s at 95 degrees C, 30 s at the determined Tm  
minus ^C, and 90 s at 72 degrees C), together with a final extension phase of 72°C for  
another ^start time>min. TAE Buffer System Agarose Gel Transfection Amplified fragments  
of DNA DNA banding was done with safe nucleic acid dye. The visualization was carried out  
using a UV transilluminator. The gel images were captured using a gel documentation  
system. For band scoring binary approach applied as 1 for presence, 0 for absence. Genotype  
calling was performed based on a binary data matrix. Statistical analyses were performed  
using POPGENE, version 1.32. Genetic parameters were estimated as percentage  
polymorphism (PP), genetic diversity (He: Nei’s gene diversity) and Shannon’s information  
index. Computing genetic similarity matrix, NTSYS version 2.1 Cluster analysis used  
Unweighted Pair Group Method with Arithmetic Mean algorithm followed. Genetic  
relationships among populations are shown by dendrogram construction. PAST version 4.03  
for Principal Coordinate Analysis was used for multivariate analysis. Genetic dispersion  
patterns were represented by graphical plots. All procedures were performed according to  
standard molecular biology protocols. Experiments were designed to match laboratories’  
facilities available in different locations of Sindh region. Reproducibility, reliability, regional  
applicability for genetic diversity assessment of indigenous flora was ensured using Method.  
Results and Discussion  
The 3 selected indigenous plant species from Sindh were subjected to genetic analysis, which  
showed a significant level of polymorphism in most of the populations sampled. Scorable  
bands with 20 RAPD primers added up to a total of 142 all, including 111 polymorphic band,  
which give the average of polymorphism equal to (78.17%). As reported (Pandey et al., 2023;  
Porth & El-Kassaby, 2014), the SSR analysis with eight primer pairs generated a total of 64  
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alleles with an average of 5.33 alleles per locus. The comprehensive marker performance is  
summarized in (Table 1), exhibiting differences in allele number and polymorphism among  
the loci. The high degree of polymorphism reflects the fair amount of genetic variation  
between the populations studied, particularly those from environmentally challenged regions  
like Tharparkar.  
Table 1. Summary of Molecular Marker Analysis  
Marker  
Type  
No. of  
Primers  
Total  
Bands/Alleles  
Polymorphic  
Bands  
%
Mean Alleles  
per Locus  
Polymorphism  
RAPD  
SSR  
20  
12  
142  
64  
111  
52  
78.17%  
81.25%  
—
5.33  
Table 2 indicates large differences in genetic diversity indices across sampling regions.  
Maximum Nei’s gene diversity (H = 0.36) was recorded in Tharparkar populations, while the  
lowest value (H = 0.21) was investigated in Hyderabad. Shannon's information index varied  
between 0.32 to 0.51, giving a moderate to high level of genetic variation. Increased diversity  
in desert populations may relate to evolutionary or ecological adaptiveness to harsh  
environments, while reduced biodiversity in irrigated settings may indicate anthropogenic  
stress or habitat homogeneity.  
Table 2. Regions of Sindh Genetic Diversity Parameters  
Region  
Tharparkar 82.4%  
Umerkot 79.2%  
% Polymorphism Nei’s Gene Diversity (H) Shannon Index (I)  
0.36  
0.33  
0.51  
0.47  
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Region  
Thatta  
Badin  
% Polymorphism Nei’s Gene Diversity (H) Shannon Index (I)  
76.5%  
74.8%  
72.6%  
0.30  
0.28  
0.25  
0.21  
0.44  
0.41  
0.36  
0.32  
Sukkur  
Hyderabad 69.3%  
Genetic similarity coefficients were between 0.62 and 0.89, confirming divergence at the  
same time as relatedness of populations. UPGMA based cluster analysis drew three distinct  
clusters according to ecological zones in which the populations were found (Borah et al.,  
2021; Goswami et al., 2021). One cluster was composed of coastal populations from Thatta  
and Badin, indicating adaptation to saline environments. Individuals from arid regions of  
Tharparkar and Umerkot clustered with one another indicating genetic similarity associated  
with dry conditions. Irrigated populations from Hyderabad and Sukkur clustered separately,  
indicating decreased variability due to less variability associated with environment of  
uniform agricultural environments.  
The first three axes of principal coordinate analysis (PCoA) explained 67.4% of total genetic  
variation. Populations were clearly separated according to their ecological zones in terms of  
spatial distribution. Hyderabad populations exhibited more compact hyperecological clusters,  
indicating PLIN instead of Tharparkar populations which revealed broader dispersion thus  
representing higher intra-population diversity which is responsible for genetic uniformity  
(Bidyananda et al., 2024; Mondini et al., 2009; Szabo et al., 2021). These findings agree with  
the results of Table 2, which showed an overall lowest value of diversity indices for irrigated  
regions. The genetic variation observed could be significant for conservation planning of  
Sindh. Higher diversity populations, particularly from desert environments, should be  
prioritised for in-situ conservation because of their adaptive potential. On the other hand,  
lower diversity populations might need to be maintained ex situ to avoid further genetic  
depletion (Haque, 2025; Hasnain & Mehvish, 2020; Nam et al., 2021). The regional  
differences in diversity patterns highlight the impact of environmental stress, habitat  
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fragmentation and human processes on genetic structure. Evaluation Remarks: The findings  
indicate that RAPD and SSR markers have good potential for estimating genetic diversity  
under local lab conditions. Our results not only highlight genetic structure variation at the  
species level of indigenous flora in Sindh but also endorse incorporation of molecular data to  
inform biodiversity conservation strategies.  
Conclusion and Future Recommendations  
The high amount of genetic diversity found in native plant species of Sindh as shown by our  
study seems to be due to obviously the variation considering from one ecological zone to  
another. The molecular markers analysis performed using RAPD and SSR techniques  
revealed a very high level of polymorphism, which indicates that these methods are highly  
useful for the assessment of genetic diversity under local laboratory conditions. Detection of  
allelic diversity confirming population structure is more accurately defined using SSR  
markers that have higher resolution. The regional historical analysis shows that Tharparkar  
and Umerkot desert ecosystems have greater genetic variability, whereas irrigated areas like  
Hyderabad and Sukkur are found to be genetically less variable. The genetic structure of  
coastal brown trout was influenced by environmental stress, habitat heterogeneity and human  
activities which shaped this pattern. They collectively provide further evidence of geographic  
and environmental control on genetic differentiation between populations (as is evidenced by  
their ecological grouping shown in the cluster and PCoA analyses).  
Future studies should sample more broadly to include a greater diversity of species and  
under-represented habitats which would allow for a fuller genetic inventory of native flora.  
Novel genomic technologies (SNP genotyping, RAD-seq and whole-genome sequencing)  
should allow significant improvement in resolution with respect to adaptive variation and  
evolution. Although molecular research of several species is extensive in Pakistan,  
collaboration between research institutions, forestry departments and policy-making bodies  
should be designed to ensure that such genomic information is conveniently transferred for  
improved implementation into the sustainable conservation strategies across different regions  
in Pakistan.  
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