DOI: https://doi.org/10.21203/rs.3.rs-1839509/v1
Freshwater muscles are highly impacted by many human activities. As a result, it is considered as one of the most threatened ecosystem worldwide. Studying genetic diversity is crucial for any further research on conservation efforts. In the present study, inter simple sequence repeat (ISSR) marker analysis was employed to study genetic diversity between 8 randomly selected specimens of bivalve. Total of 53 bands were produced from the 10 primers. Of these 37 bands were polymorphic, resulted in 69.8% polymorphism. Some primers showed more polymorphic bands than the others. For example, primer ISSR M17 produced no polymorphic bands. While primer ISSR M1 produced 3 polymorphic bands (100% polymorphism). Cluster analysis based on similarity matrix obtained showed that the 8 bivalve specimens can be clustered into two groups, one group containing specimens 2 and 6 and the other group represents specimens 4, 5, 1, 3, 7, and 8. These data may suggest that the bivalves in Abohomos, Behera Governorate may have more than one species. The implication of these data on the conservation of the bivalve is explained. The conclusion is that the bivalve population have enough genetic diversity. These populations are adapted to the environment they lives in.
Little is known about the biology of freshwater bivalves in Egypt, except the information provided on the anatomy of one species by who studied ultrastructural of the digestive tubules of the freshwater mussel Caelatura parreyssi from the Nile River (Soliman 2001; Awad 2018).
The freshwater bivalves are eaten in other parts of the world such as Asian countries where it is used as a supplemental protein source (Ibrahim et al., 1999).
Most mussels have excellent ecological adaptability, and the different characteristics of shell shape (various shell morphologies) confuse nomenclature and classification. Recently, molecular techniques have been used to perform molecular phylogenetic analyzes of 70 species of Unionidae to complement their morphological and behavioral properties (Lopes-Lima et al., 2017). As a result, they phylogenetically divided the Unionidae into 6 subfamilies and 18 phyla. The three newly described tribes were Chamberlainiini, Cristariini, and Lanceolariini. Finally, a new classification system for Unionidae was provided (Lopes-Lima et al., 2017). The application of molecular technology in the study of freshwater mussels changed the understanding of evolutionary relationships between strains of freshwater mussels and presented the first molecular phylogeny of freshwater mussels (Graf and Cummings 2006; Graf and Cummings 2007). Our understanding of the evolution of freshwater mussels and their comprehensive classification has been greatly refined (Pfeiffer and Graf 2013; Pfeiffer and Graf 2015). In addition, freshwater mussels (especially from Asia) have recently been the focus of attention. Using molecular approaches, our understanding of the species-level diversity and distribution has improved (Zieritz et al., 2018). In the present study, we aim at providing an insight into the freshwater bivalve biodiversity present in Abohomos, Behera Governorate. A field survey was conducted in May 2017 and the material was identified at least to genus level based on shell morphology. ISSR technique was used in order to study the genetic diversity between the eight specimens. The produced genetic data were used to assess the genetic variation among the individuals and whether these individuals may potentially form a species flock.
Bivalve collection: Total of 8 samples were collected randomly from a freshwater lake in Abohomos, Behera Governorate.
Inter Simple Sequence Repeat (ISSR): The current molecular biology work as well as data analysis were done at the Alexbiotechnology Molecular Biology Service Lab (Sidi Gaber, Alexandria, Egypt). The DNA was extracted from bivalve’s soft tissue using a standard phenol-chloroform extraction(Aranishi and Okimoto 2006 ; Taggart et al., 1992). DNA Quantification and purity measurement was done using Nano Drop ND-200 Spectrophotometer (AOSHENG, China) and was used as well to quantify the DNA present in all of the samples. The quality or purity of the elution in terms of the presence of humic acids (indicated by the absorbance ratio at 260 nm/230 nm) and protein contaminants (indicated by the absorbance ratio at 260 nm/280 nm) was also assessed using the ND-200 Spectrophotometer. The primers used in this study were synthesized by Jena Bioscience Company (Germany). The sequence of these primers and the annealing temperature for each primer is presented in table 130.
PCR Amplification Conditions: PCR Reactions were performed in 200 µL micro-centrifuge tubes containing template DNA, primers, heat stable Taq DNA polymerase (FastGene, Taq ReadyMix PCR, Nippon Genetics, Germany) and PCR-grade water (Jena Bioscience). PCR reactions were performed using a thermocycler (TC-3000, Techne). The PCR reaction volume was 25 ul containing 50 ng of genomic DNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 250 µM each of dNTP, 3 mM MgCl2, 0.6 µM primer, 1 U of Taq polymerase. PCR-ISSR cycles conditions were as the following: 1 cycle of denaturation at 94oC for 4 min, 40 cycles of denaturation at 94oC for 1 min, annealing at 49 -50oC for 1.30 min, extension at 72oC for 3 min and a final extension at 72oC for 5 min a final hold at 20oC (Obeed et al., 2008).
The PCR products were analyzed by electrophoresis on 2% agarose-gel. The DNA size marker used was the 100 bp DNA Ladder (MWD100, Nippon Genetics, Germany). The PCR products were electrophoresed at 100V/30 min using DNA gel electrophoresis (Mupid–One, JAPAN) containing 1× TBE buffer (0.045 M Tris-borate, 0.001 M EDTA) and 0.5 µg/ml ethidium bromide for 6.5 h at 90 V, and finally examined using gel documentation system (Nippon Genetics, Europe) (Obeed et al., 2008)..
ISSR Data analysis: Only the reproducible and consistent bands were recorded manually for further analysis. Amplified products scored for band presence (1) or absence (0) and a binary qualitative data matrix was constructed. Fragments which could not be un-ambiguously recognized were not scored for analysis. Analysis of Molecular Variance (AMOVA) was performed to analyze genetic distance among samples using PAST program. A cluster dendrogram based on similarity matrix obtained with unweighted pair group method using arithmetic average (UPGMA) was constructed based on the Nei’s genetic distances for determining the genetic relationship among populations (Nei 1978).
The results shown here are based on DNA profiles using ISSR marker analysis of 10 primers (Table 1). The band area and the number of monotypic and polymorphic bands produced by each ISSR primer are also shown in Table 1. A total of 53 bands were generated from the 10 primers. Of these 37 bands, it was polymorphic. Some primers showed more polymorphic bands than others. For example, the primer ISSRM17 did not produce a polymorphic band. On the other hand, primer ISSR M1 produced three polymorphic bands (100% polymorphism).
Table (1): ISSR primer sequence, number of monomorphic band, number of polymorphic band and annealing temperature for each primer.
| Primer | Primer Sequence | Number of bands | Polymorphic Band | Polymorphism % | Annealing temp |
|---|---|---|---|---|---|
| ISSR M1 | 5- AGC AGC AGC AGC AGC AGC − 3 | 3 | 3 | 100 | 50o C |
| ISSR M2 | 5- ACC ACC ACC ACC ACC ACC − 3 | 0 | 0 | 0 | 50o C |
| ISSR M3 | 5- AGC AGC AGC AGC AGC AGC − 3 | 6 | 3 | 50 | 50o C |
| ISSR M8 | 5- ACA CAC ACA CAC ACA CAC − 3 | 9 | 7 | 78 | 50o C |
| ISSR M9 | 5- ACA CAC ACA CAC ACA CCG − 3 | 0 | 1 | 100 | 49o C |
| ISSR M12 | 5- GAC ACG ACA CGA CAC GAC AC- 3 | 9 | 9 | 0 | 50o C |
| ISSR M17 | 5- CAG CAC ACA CAC ACA CAC − 3 | 5 | 5 | 0 | 50o C |
| ISSR F2 | 5- AGA GAG AGA GAG AGA GCG − 3 | 6 | 2 | 33.3 | 50o C |
| ISSR F4 | 5- AGA GAG AGA GAG AGA GTG − 3 | 8 | 2 | 25 | 50o C |
| ISSR F9 | 5- GAA GAA GAA GAA GAA − 3 | 7 | 5 | 71 | 50o C |
| Total | 53 | 37 | 69.8 |
The cluster analysis of the eight samples based on the UPGMA is shown in Fig. 1. The phylogenetic tree analysis revealed that the specimens 2 and 6 were grouped in one cluster while the specimens 1, 5, 4, 8, 7, 8 and 3 are grouped in another cluster. On the other hand, both specimens 4 and 5 are closely related to each other. Similarly specimens 3 and 7 and specimens 2 and 6 are closely related specimens.
Table 2 shows the similarity and distance indicators for the 8 samples. This table shows the highest similarity and distance index with a genetic distance of 0.90927 between samples 4 and 5, and the lowest similarity and distance index with samples 2 and 3 and samples 2 and 7 at a distance of 0.11066. It shows that. On the other hand, the groups of Samples 2 and 3 and Samples 2 and 7 had the same similarity index at 0.11066. On the other hand, the similarity index between Samples 3 and 1 and Samples 3 and 6 had the same similarity index of 0.47247.
Table (2): Similarity indices of the eight specimens of bivalves under study.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
|---|---|---|---|---|---|---|---|---|
| 1 | 1 | |||||||
| 2 | 0.51814 | 1 | ||||||
| 3 | 0.47247 | 0.11066 | 1 | |||||
| 4 | 0.66243 | 0.30159 | 0.75984 | 1 | ||||
| 5 | 0.73352 | 0.33773 | 0.66139 | 0.90927 | 1 | |||
| 6 | 0.64228 | 0.80672 | 0.47247 | 0.66243 | 0.57608 | 1 | ||
| 7 | 0.33835 | 0.11066 | 0.84914 | 0.59755 | 0.4843 | 0.33835 | 1 | |
| 8 | 0.49048 | 0.48731 | 0.6988 | 0.62476 | 0.69305 | 0.60406 | 0.6988 | 1.00 |
Intersimple Repeat (ISSR) consists of semi-arbitrary markers amplified by the polymerase chain reaction (PCR) using primers complementary to the target microsatellite. ISSR does not require genomic sequence information. In addition, it produces polymorphic, highly polymorphic patterns and produces dominant markers. In addition, as a genotyping technique, ISSR-PCR is fast, inexpensive, based on region variation between microsatellites, and is technically simpler than many other marker systems. This method provides reproducible results and produces abundant polymorphisms in many systems (Abdel-Mawgood 2012; Wolfe et al., 1998).
Genetic variation is an important tool for assessing the biological potential of an organism. Populations with high genetic variation can cope well with environmental changes such as water fluctuations, temperature fluctuations, and epidemics (Alam et al., 2010). On the other hand, reducing genetic diversity may increase susceptibility to environmental changes and ultimately lead to species extinction (Evans et al., 2010). This reduction in genetic diversity can affect growth and reproduction (Dixon et al., 2008). Therefore, maintaining a high level of genetic diversity is very important for species conservation (Barroso et al., 2008). Individuals need to be able to withstand changes in the environment and grow well. Therefore, genetic monitoring is ideal for use in reproductive programs aimed at genetic conservation (storage). Molecular markers are a viable and useful tool for studying and monitoring is ideal for use in a reproduction program with the aim of genetic conservation (stocking). Molecular markers are a viable and useful tool for studying and monitoring the genetic status of both natural populations (Alam and Islam2005). In this study, the similarity between specimens 2 and 3 is 0.11, which means that the genetic distance between some specimens is high, and the genetic distance between these two specimens is 0.89. Similarly, the genetic distance between samples 6 and 7 is 0.67. This suggests that rivers may be inhabited by multiple species of shellfish and need to be categorized. However, some individuals are very similar and can be classified as one species as follows: B. Items 3 and 7 and items 4 and 5 on different pages.
The first study of Egyptian freshwater mussels involved two populations collected from the Nile in Giza Governorate, Egypt (Mandahl-Barth1988). He classified them as a species belonging to the Unionidae family. However, others separated them as two different species (Ibrahim et al., 1999). In addition, a randomly amplified DNA polymerase polymorphism chain reaction marker (RAPD-PCR) was used to determine the genetic distance (D) between the two Unionidae species (Sleem and Ali2009). He concluded that the high genetic distance between the two specimens (0.64) suggests that they are two different species (Sleem and Ali, 2009).
very similar group of individuals. In addition, copies 2 and 6 form the third branch. In general, the genetic diversity of freshwater mussels seems to be relatively high. This may be because they are made up of multiple species. For example, the RAPD technique was used on populations collected from three water bodies in India, and the genetic diversity value reached 0.99, indicating that there is high genetic diversity between the population and individuals of the same species. (Alam et al., 2010). Several other studies support the discovery of the presence of high genetic diversity in freshwater mussels. Using 19 microsatellite markers and 64 specimens, one study considers the freshwater mussels (Unionidae) in the Yangtze River basin of China to be one of the most diverse communities on earth (Liu). et al., 2017). Another study concludes that global estimates of freshwater mussel diversity are 840 species in 161 genera6. Regional diversity is Nearctica: 302 spp., Neotropica: 172, Afrotropica: 85, Palearctica: 45, Indotropica: 219 and Australasia: 33, and that largest family is the Unionidae, with 674 species (Graf and Cummings, 2007).
Genetic diversity is important for species to survive changes in the environment. The presence of high genetic diversity among aquatic populations could significantly contribute to increasing the potential for evolution to address habitat changes, the effects of pathogen infections, and other selectivity. There are (Freeland et al., 2011; Liu and Yao 2013; MacDonald et al., 2011). The results presented here showed that the samples collected at Avohomos in Behera province show moderate genetic diversity. They have slightly higher levels of genetic variation and therefore have slightly greater ability to potentially adapt to their environment. Meanwhile, other studies suggest that freshwater mussels are considered one of the most endangered animal groups in the world (Aldridge et al., 2007; Vaughn 2010). Similarly, it has been suggested that freshwater mussels are a vulnerable group and may face global decline (Gallardo2018). Later studies predicted a major distribution contraction of the endemic freshwater fauna and fragmentation of the remaining suitable habitat. Their research has led to future expeditions to monitor the conservation status of freshwater biodiversity. A summary of the national conservation status of mussels in East and Southeast Asian countries (Zieritz et al., 2018). The major threats to the demographics of these countries are pollution, deforestation, interaction with alien species, changes in land use, dam construction and mining, climate change, sediment accumulation, overfishing and alien species predation. It was indicated that the major threats to the population diversity in these countries were pollution, deforestation, interaction with nonnative species, land-use change, dam construction and mining, climate change, sediment accumulation, overharvesting, nonnative predators, hydrological alterations, and urbanization (Zieritz et al., 2018).
the authors declare that no fund, grants or other supports were received during the Preparation of this manuscript: authors contribution: Eman Radwan, Rasha El Nagar, Ahmed Abdelmawgood, Khaed Radwan and Amel Ghonim contributed to the study design, material preparation, data collection and analysis and writing and submitting the article. Ahmed Abdelmawgood and Eman Radwan, Amel Ghonim, Khaled Radwan, Rasha ElNagar commented on the last version of the manuscript. All authors read and approved the final manuscript.
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