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  • Full Length Research Article
  • Date Received: 16-12-2024
  • Date Revised: 18-01-2025
  • Date Published: 31-03-2026
Genetic Relationships Among Local Oscillatoria Isolates from Freshwater Sources Using Inter-Simple Sequence Repeat (ISSR) Technique
Safa Ghassan Hameed1, Harith Kamil Buniya1
  1. Department of Biology, College of Education for Pure Sciences, University of Anbar, Ramadi, Iraq

Abstract

Background: Molecular characteristics are good markers for distinguishing between closely related species. Molecular taxonomy has been significantly improved by DNA-based strategies with increasing accuracy and efficiency in identifying species through the use of PCR-based methods. We used inter-simple sequence repeat (ISSR) markers in this research to compare and examine the genetic profiles of several Oscillatoria species from native freshwater environments.

Methods: Algal samples were taken from natural freshwater environments in the autumn season (September–November 2022). To reduce contamination, DNA was obtained from unialgal cultures of Oscillatoria spp. Then, ISSR analysis was done to assess genetic diversity and phylogenetic relationships between the isolated strains.

Results: Seven species were identified: Oscillatoria acuta (OS1), O. princeps Vaucher (OS2), O. annae (OS3), O. margaritifera (OS4), O. proteus Skuja (OS5), Oscillatoria sp. (OS6), and O. sancta (OS7). O. annae and Oscillatoria sp. were most similar and were found to have the greatest genetic similarity (index = 0.6598), indicating that a close evolutionary relationship exists. The two with the lowest similarity were O. acuta and O. proteus Skuja (index = 0.4330), indicating greater genetic divergence.

Conclusion: Our results support the use of ISSR markers in determining genetic diversity and phylogenetic relationships among Oscillatoria spp. This method is promising for improving the molecular taxonomy of cyanobacteria.

Keywords

Genetic Diversity, Oscillatoria, Phylogenetic analysis, Cyanobacterial taxonomy, ISSR markers

Introduction

Oscillatoria is a common and highly distributed genus of filamentous cyanobacteria (blue-green algae) that inhabits freshwater environments [1,2]. It has unbranched filaments made up of regular cells in one row, usually housed in a soft mucilaginous sheath. Oscillatoria is unlike most other cyanobacteria in that it does not form heterocysts or akinetes in regular conditions, although environmental stress will induce the production of one or the other [3,4]. One of its most distinguishing characteristics is its ability to perform anoxygenic photosynthesis—photosynthesis that does not produce oxygen—and to control its buoyancy through the use of gas vesicles to move vertically through the column of water in order to minimize shading by deeper cells [3].

Ecologically, Oscillatoria is an important primary producer in aquatic ecosystems, generating oxygen and cycling nutrients [4]. It is part of the food web as a major component, supporting several aquatic invertebrates and insect larvae. Specific species also play a role in nitrogen fixation, fixing nitrogen from the air and making it bioavailable, especially in nitrogen-deficient environments [5]. Yet, with excess nutrient loading, Oscillatoria will grow and reproduce in excess, producing so-called harmful algal blooms (HABs). These blooms cause a deterioration in the quality of the water, destabilize ecosystems, and are dangerous for aquatic life [5-7].

In addition to its ecological functions, Oscillatoria has been of interest due to its biotechnological potential. The organism has been used in wastewater treatment, biofuel production, and the biosynthesis of bioactive compounds. At the structural level, its filaments have regular cellular morphology with the terminal cells tending to have a characteristic cap-like thickening known as the calyptra. It reproduces by fragmentation that is brought about by necridia (dead cells with jelly-filled cavities) and specific hormogonia [5,8].

For the identification of genetic diversity in Oscillatoria, the current research employs Inter-Simple Sequence Repeat (ISSR) analysis, a molecular tool capable of detecting polymorphisms and investigating genetic diversity. ISSR is employed in genetic mapping, fingerprinting, and biodiversity due to its good reproducibility and power of discrimination [9,10]. ISSR markers have been used in very few studies from Iraq to examine the diversity of cyanobacteria [11]. Interestingly, Maulood et al. (2013) observed that there were 81 Oscillatoria species in Iraqi aquatic ecosystems, indicating the commonality of the genus in Iraq [12].

This study examined the genetic diversity and phylogenetic relationships of Oscillatoria spp. obtained from aquatic environments in Iraq’s Anbar Governorate using ISSR markers. This study aimed to improve understanding of the taxonomy of cyanobacteria and ecological versatility in the underexplored region.

Methods

Sample Collection
Fresh algal samples were obtained from freshwater sources in Ramadi, Anbar Governorate, western Iraq, during September–November 2022. Following sampling, the samples were transported directly to the Department of Biology laboratory to maintain viability. Laboratory maintenance involved incubation under conditions simulating natural light–dark cycles and a natural temperature regime.

Sample Preparation and Cultivation
Upon laboratory arrival, specimens underwent sequential rinsing with sterile distilled water to remove adherent particulate matter and external contaminants. Subsequently, cleaned samples were aseptically transferred to sterile 15 mL conical tubes containing fresh distilled water for preservation and further processing. A defined washing/picking protocol was repeated until unialgal cultures were obtained. A serial dilution method was employed to establish unialgal cultures. These cultures were incubated under controlled light/dark conditions for two weeks.

Genomic DNA Extraction
Genomic DNA was extracted from the algal samples using a commercial genomic DNA extraction kit (Geneaid, Taiwan), following the manufacturer’s protocol. The quality of DNA was assessed via agarose gel electrophoresis using a 1% (w/v) agarose gel at a constant voltage of 65 V for 1 hour. DNA bands were visualized under ultraviolet (UV) light. DNA purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Scientific), and DNA purity was evaluated using the A260/A280 ratio, with purity values ranging between 1.6 and 2.0.

ISSR Amplification
ISSR-PCR reactions were conducted on DNA extracted from seven Oscillatoria species. The ISSR-PCR reactions were prepared according to the protocol described by Araújo et al. (2023) [13], using the following components:

1. PCR PreMix (commercially prepared).

2. ISSR primers (as listed in Table 1), supplied by Integrated DNA Technologies (IDT), Korea.

3. DNA template (25–50 ng per reaction).

PCR amplification was performed using primers with sequences and annealing temperatures listed in Table 1. The amplified products were analyzed using agarose gel electrophoresis, and banding patterns were documented for genetic analysis.

Results

Genomic DNA Extraction
Electrophoresis performed on 1% (w/v) agarose gel confirmed the successful extraction of genomic DNA from all seven Oscillatoria species. Each sample exhibited an intact high-molecular-weight genomic DNA band. DNA purity, as determined by a NanoDrop spectrophotometer (A260/A280 ratio), ranged from 1.6 to 2.0, indicating acceptable quality for molecular analysis.

ISSR Amplification Results
Seven ISSR primers were utilized to assess the genetic variation among the Oscillatoria isolates. All primers generated bands, with polymorphism varying among primers, indicating high levels of genetic diversity. The amplification patterns revealed the number of binding sites (bands), their molecular size range, and the proportion of identical and polymorphic bands across the samples.

Primer SA5 produced the highest number of bands (47), with a molecular size range of 350–3000 bp, while SA1 yielded the fewest bands (33), ranging from 200–2000 bp. Unique and absent bands were also observed across species, with primer-specific band variations detailed in Table 2 [13,14].

Overall, 280 total bands were recorded across all primers. Of these, 21 (7.5%) were identical, while 259 (92.5%) were polymorphic, highlighting substantial interspecies genetic variation.

Species-Specific ISSR Patterns
Species-wise analysis (Table 3) indicated that Oscillatoria margaritifera (OS4) displayed the highest number of bands (45), while Oscillatoria acuta (OS1) showed the fewest (33). The largest amplified product (3000 bp) appeared in OS2, OS4, and OS5, while the smallest fragments (200 bp) were identified in OS2, OS3, and OS4.

OS5 exhibited the highest number of unique ISSR bands (9), followed by OS4 (8), OS1 and OS6 (6 each), OS3 (4), OS2 (3), and OS7 (2). Absent bands were detected in OS4 (1), OS6 (2), and OS7 (1). These distinct banding profiles support the presence of genetic differentiation among the isolates.

Genetic Affinity Estimation
Genetic similarity among the species was calculated using the NTSYSpc 2.10e software based on the Nei similarity coefficient. The highest genetic similarity was recorded between OS3 (O. annae) and OS6 (Oscillatoria sp.) with a similarity index of 0.6598. The lowest similarity was observed between OS1 (O. acuta) and OS5 (O. proteus Skuja), with a similarity index of 0.4330, as shown in Figure 2.

Cluster analysis revealed two major clades: the first included OS3, OS6, and OS7, indicating closer genetic relationships among these species. The second clade comprised OS4 and OS5, which were more distantly related to OS1 (Oscillatoria acuta) and OS2 (O. princeps Vaucher). The dendrogram illustrating these genetic relationships based on ISSR data is shown in Figure 1 [15,16].

Discussion

The ISSR primers successfully amplified DNA sequences between simple sequence repeats, enabling discrimination among Oscillatoria species. All seven primers generated reproducible banding patterns, although variation among primers was observed, with certain bands absent in the relevant species reported in Table 3, likely due to the absence of complementary sequences in their DNA. Such absence is crucial in molecular taxonomy, as it highlights interspecific genetic variability [13].

A total of 280 bands were amplified, with 259 (92.5%) being polymorphic and 21 (7.5%) identical across species. Polymorphic bands provide valuable markers for distinguishing closely related taxa. Shared bands, observed predominantly with primers SA1 and SA2, indicate conserved loci identical across species, which can aid in the classification of unknown strains [14].
Among the primers, SA3 and SA4 produced the highest number of unique bands (5 each), while SA5 revealed two absent bands, further indicating its discriminatory potential. Primers SA6 and SA7 showed moderate variation, each producing two unique bands, and SA1 generated three. SA2 yielded no unique or absent bands, suggesting it targets highly conserved regions [14]

Species-wise, OS5 exhibited the highest number of unique bands (9), suggesting greater genomic divergence, followed by OS4 (8), OS1 and OS6 (6 each), OS3 (4), OS2 (3), and OS7 (2). Absent bands were detected in OS4 (1), OS6 (2), and OS7 (1), emphasizing their divergence at certain loci [14,15].

The dendrogram derived from ISSR data segregated the seven Oscillatoria species into two main clusters. Cluster I comprised OS3, OS6, and OS7, showing close genetic similarity. OS4 and OS5 were in Cluster II and were closer to each other than OS1 and OS2. The efficacy of ISSR in determining the relationships at the species level was further established by these patterns of clustering [15,16].

The patterns of observed genetic variability are consistent with previous observations by Mühlsteinová et al. (2018), where ITS sequencing revealed significant genetic diversity in Oscillatoria spp. The authors highlighted that application of morphology alone has its own shortcomings because genetically distinct strains tend to exhibit small phenotypic differences [17]. This trend highlights the need to use molecular protocols in the taxonomy of cyanobacteria.

Recent work by Gemmill et al. (2021) indicates ISSR markers possess greater resolving power to separate taxonomic boundaries, particularly in close genera with similar morphology [10]. The findings of the current work further support the view that ISSR analysis is effective in systematics research on cyanobacteria, complementing earlier work by Araújo et al. (2023) and Unat (2022) [13,14]. Due to the comparative dearth of ISSR-based research on Iraqi freshwater cyanobacteria, these findings are valuable additions to the literature and highlight the need for further genomic research in this area.

The research emphasizes the necessity of multi-locus molecular techniques to elucidate relationships in the Oscillatoriaceae. Such research may establish the need for taxonomic revisions or the designation of new genera [17].

The current study effectively utilized ISSR markers to assess the genetic diversity between seven Oscillatoria strains from freshwater ecosystems in Anbar Governorate. Cluster analysis provided two distinct groupings: O. annae (OS3), Oscillatoria sp. (OS6), and O. sancta (OS7), with the most comparable genetic affinities. The second group contained O. margaritifera (OS4) and O. proteus Skuja (OS5), with closer similarity to one another than to O. acuta (OS1) or O. princeps Vaucher (OS2). These results not only validate the usefulness of ISSR markers in phylogenetic research but also provide valuable reference data that can guide subsequent studies to explore functional genes that are important in ecologically relevant processes, such as toxin biosynthesis and nitrogen metabolism, thereby further explaining the role of these organisms in the environment.

Conclusion

Statement & Declarations

Conflict of Interest

The authors have no conflicts of interest.

Author Contributions

Conceptualization: Harith Kamil Buniya

Methodology, Software, Formal Analysis, Data Curation: Harith Kamil Buniya, Safa Ghassan Hameed

Writing of original draft: Safa Ghassan Hameed

Review, editing, and supervision: Harith Kamil Buniya

All authors have read and approved the final manuscript.

Acknowledgment

The authors express gratitude to the Department of Biology, College of Education for Pure Sciences, University of Anbar, for providing laboratory facilities to complete this research.

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