Journal of Survey in Fisheries Sciences 7(2) 123-132 2021 Microsatellite genetic differentiation between two populations of European ( glanis) in Iran

Behmanesh Sh.1; Amiri A.2; Yarmohammadi M.3; Golshan M.4*

Received: April 2020 Accepted: October 2020

Abstract In the present study the population genetic structure of European catfish in the Anzali Lagoon and Aras Lake were examined using microsatellite markers. Sixty fin clip samples of Silurus glanis from two regions were collected and for genetic analysis 6 microsatellite loci were used to assess the population genetic structure of S. glanis. There were significant differences based on average number of alleles per locus and

heterozygosity between two populations (p<0.01). The observed heterozygosity (Ho)

for each population (He) per locus was from 0.297 to 0.733 in Aras lake and Anzali lagoon samples, respectively.The Analysis of molecular variance (AMOVA) indicated that the proportion of the genetic variation attributed to differences among populations

of the S. glanis was highly significant for both FST and RST (FST = 0.165, RST = 0.38, p<0.001). Excess or lacks of heterozygosity was observed but most of used microsatellite loci in selected areas were at Hardy-Weinberg equilibrium. Our finding showed the two populations are genetically separated, therefore fisheries management and restocking program of this species especially in Anzali Lagoon is recommended.

Keywords: European Catfish, Genetic population, microsatellite markers

Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] 1-Inland Waters Aquaculture Research Center, Iranian Fisheries Science Research Institute, Agricultural Research Education and Extension Organization (AREEO), Bandare Anzali, Iran 2- Department of Biology, Faculty of Fisheries Sciences Science, Ahvaz branch, Islamic Azad University, Ahvaz, Iran 3-International Sturgeon Research Institute, Agricultural Research Education and Extension Organization (AREEO), Rasht, Iran 4-Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Tehran, Iran *Corresponding author's Email: [email protected] 124 Behmanesh et al., Microsatellite genetic differentiation between two populations of European …

Introduction Although, allozyme (Triantafyllidis et Diversity plays important roles in al., 1999a) and mitochondrial DNA populations due to provide necessary (mtDNA) (Triantafyllidis et al., 1999b; spectrum of genotypes for adaptive Krieg et al., 2000) were examined to response to change environmental study population genetic of S. glanis, conditions (Webster et al., 2018). neither of them were able to reveal high Heterozygous individuals usually are levels of genetic variability to separate superior to less heterozygous wild and hatchery populations. Krieg et individuals in characteristics such as al. (1999) reported population growth, fertility, and disease resistance differentiation among different species (Beardmore et al., 1997). of family using microsatellite Microsatellites are known as a simple markers. Triantafyllidis et al. (2002) sequence repeats (SSRs), simple showed that the genetic diversity was sequence length polymorphisms much higher than previous allozyme (SSLPs), or short tandem repeats and mtDNA studied on this species by (STR), are regions of DNA that exhibit using 10 microsatellite markers in short repetitive sequence motifs. These different areas. Quan et al. (2006) motifs are often composed of 1-6 base examined population structure and pair repeat sequences. Microsatellites genetic variation of northern sheatfish have been used as genetic markers to (S. soldatovi) in wild and farmed assess population genetic structure, populations using microsatellite

broodstock identify for management in markers. Results showed that Ho and He hatcheries, evolutionary biology, and other heterozygosity parameters conservation biology and genetic were not different between populations. mapping (Triantafyllidist et al., 2002). Bahrami Kamangar et al. (2015) European catfish has global distribution investigated the genetic diversity and as a native or an introduced species genetic structure of four populations of (Cucherousset et al., 2018). Silurus Silurus glanis by microsatellite DNA glanis is well known as an economic markers. species and an important for S. glanis is one of the native species aquaculture industry (Rees et al., 2017). of Iran where mainly distributed in There are large number of studies have Anzali Lagoon and Aras Lake, north been conducted on reproductive cycles, and northwest of Iran. Due to genome manipulation and biology of overfishing and poor fisheries Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] gametes of S. glanis (Legendre et al., management, the capture production 1996; Smitherman et al., 1996; has been declined over the last decades. Varkonyi et al., 1998; Brzuska and Thus, the present study aimed to assess Adamek, 1999; Bolliet et al., 2001; genetic diversity of Iranian populations Kumar et al., 2017; Zibiene and Zibas, of S. glanis in Anzali Lagoon and Aras 2019; Linhart et al., 2020) but study of Lake regions using microsatellite DNA population genetic structure is rare. markers. Journal of Survey in Fisheries Sciences 7(2) 2021 125

Materials and methods some modifies in regents and annealing Sample collections and DNA extraction temperature. Six microsatellite loci for Sixty individuals of S. glanis were S. glanis were used and all of the loci obtained from two selected sites, Anzali have been described by Krieg et al. Lagoon (37°28′22″N 49°27′44″E) and (1999). Detailed information of these Aras Lake (39°05′28″N 45°24′08″E) microsatellite loci such reference, through trap and fin clips were taken forward and reverse primers, fragment and placed in 100% ethanol size and annealing temperature was immediately. Total DNA was extracted described in Table 1. The PCR from each individual using standard amplification was performed in a final SDS proteinase-K digestion; phenol: volume of 25 µL, containing 100 ng chloroform: isoamylalcohol extraction template DNA, 10 mM forward and and ethanol precipitation as described in reverse primers, 200 µm of dNTPs, 0.5 Hillis et al. (1996). The quality and u/µL of taq DNA polymerase (Cinagen,

quantity of extracted DNA and Iran), 50 mM MgCl2, 10X PCR buffer Polymerase chain reaction (PCR) was and distilled water using BIOER examined by 1% agarose gel thermal cycler (XP cycler gradient, 96 electrophoresis and nanodrop plus, Bioer, ) under the following spectrophotometery (ND 1000, USA) conditions: initial denaturation of 4 min respectively. at 94ºC (primary denaturation) followed by 30 cycles of 30 s denaturation at PCR profiles and primer sequences 94ºC, 60 s at the respective annealing The PCR amplifications were done temperature, and 60 s extension at 72ºC, according to Krieg et al. (1999) with finishing with 10 min at 72oC as the elongation period.

Table 1: Information of 6 polymorphic microsatellite loci used in the present study. Annealing Locus Primer (5’-3’) Size (bp) Reference temp. (ºC) F-CCACTTATGCACCTGAAGG Sgl33INRA R-GGCCAATTAAACAGGTACAG 150-200 58 F-CCAATTTACCTCAGACTACTTCTG Sgl5fINRA R-GCACGTGCAAAGTCCTG 125-210 55 F-CTTTGGTGAGTCAGAAACACG Sgl695INRA R-GCACTACTGGTAGATGCT 180-250 56 Krieg et al. (1999) F-CTGCTCAATCAAAGTTGGTTC Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] Sgl7159INRA R-CAAACTAAGTTCAGCCAGGC 220-300 55 F-GTGAATGTGCTTTAAGGGC Sgl7eINRA R-GTTCATGGTGTCACTGCG 200-300 59 F-GGCTGTATGTTAAGTTATTTTCAG Sgl7fINRA 220-300 60 R-CTGAGCAGTGGCCAGAATG

126 Behmanesh et al., Microsatellite genetic differentiation between two populations of European …

Gel electrophoresis and statistical (Cleaver Scientific Ltd, UK). Gel run analyses was carried out at 120 V until the The PCR products were separated by loading buffer reached the bottom of electrophoresis through 6% (w/v) the plate. After electrophoresis, the gel polyacrylamide gel (29:1 acrylamide: was stained with a silver nitrate bis acrylamide; 1x TBE buffer) using a protocol (Fig. 1). Cleaver gel electrophoresis system

Figure 1: Polyacrylamide electrophoresis patterns of Silurus glanis in the locus Sg133INRA.

Microsatellite alleles were identified by examine the partition of variance their size in base pairs (Peakall and between and within two populations. Smouse, 2006). Allele lengths were estimated by comparison with a 100 bp Results DNA marker ladder (Fermentas GmbH, PCR amplification Germany). Measures of genetic Eight microsatellite loci were diversity were determined for each successfully amplified where two sets population including, number of alleles (Sgl310INRA and Sgl325INRA)

per locus, observed heterozygosity (Ho), showed monomorphic bands in two

expected heterozygosity (He) and populations and six sets produced deviations from Hardy-Weinberg polymorphic bands (Table 2). Locus equilibrium (HWE) between pairwise Sgl5fINRA in samples of Anzali loci. The genetic distance between Lagoon showed highest number of populations of two areas was estimated alleles (12) and locus Sgl7eINRA in using the Nei’s standard genetic samples of Aras Lake presented the distance index (Nei, 1972). The lowest (4) (Table 2). pairwise differentiation between populations of the two areas was also Genetic variations of microsatellite loci

Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] characterized using pairwise estimates within populations of θ values of Fst were tested for A total of 88 unique alleles were found significant departure. All calculations across the six loci in the two

were conducted using GENALEX populations. Number of alleles (NA),

version 6. Analysis of molecular the observed (Ho) and expected (He) variance (AMOVA) was used to heterozygosity of 6 polymorphic primers per population are shown in Journal of Survey in Fisheries Sciences 7(2) 2021 127

Table 3. These parameters were in the (9.3), Ho (0.639) and He (0.589) in range of 4-12, 0.197-0.807 and 0.226- Anzali Lagoon were significantly

0.733, respectively. The mean of NA higher than Aras Lake (p<0.01).

Table 2: The genetic polymorphism of Anzali Lagoon and Aras Lake population. (NA, number of alleles; Ho, observed heterozygosity; He, expected heterozygosity) Locus Parameters Anzali lagoon Aras lake NA 10 6 Sgl33INRA Ho 0.795 0.289 He 0.702 0.425

NA 12 6 Sgl5fINRA Ho 0.486 0.350 He 0.385 0.318

NA 8 5 Sgl695INRA Ho 0.807 0.197 He 0.733 0.380

NA 9 5 Sgl7159INRA Ho 0.621 0.395 He 0.640 0.226

NA 8 4 Sgl7eINRA Ho 0.610 0.274 He 0.505 0.297 A 9 6 Sgl7fINRA Ho 0.515 0.201 He 0.569 0.436

Mean NA 9.3 ± 3.28 5.3 ± 2.18 Mean Ho 0.639 ± 0.055 0.284 ± 0.155 Mean He 0.589 ± 0.031 0.324 ± 0.098

Table 3: The computed genetic differentiation parameters. Populations Genetic difference (Fst) Genetic distance Anzali Lagoon and Aras Lake 0.165 0.38

Population genetic differentiation Hardy–Weinberg equilibrium tests The results of AMOVA indicated (HWE) significant differentiation in pair-wise All 6 loci used in this study were tested for deviation from Hardy–Weinberg FST between two populations. The Fst equilibrium. The results showed that was 0.165 which showed significant significant departures from HWE were

Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] differences between populations. detected using the probability test Genetic distance calculated based on (p<0.05). Of the six markers assessed Nei (1972). Both computed parameters for HWE, the loci Sgl695INRA and presented significantly differences Sgl7eINRA in Anzali Lagoon and between populations (p<0.01). Sgl5fINRA locus in Aras Lake showed excess heterozygosity. Sgl7159INRA in Anzali Lagoon and Sgl7eINRA and 128 Behmanesh et al., Microsatellite genetic differentiation between two populations of European …

Sgl7fINRA loci in Aras Lake presented Anzali Lagoon was higher than Aras lack of heterozygosity. The other loci in Lake and the level of genetic diversity two populations were at HWE test was significant higher (p< 0.01) (Table 4). between two populations. High level of heterozygosity in samples of Anzali Table 4: The genetic deviation test (HWE) of Lagoon can be evidence of obtained S. glanis population. Locus Anzali Aras Lake alleles. It is worth mentioned that Lagoon numerous rivers reach Anzali Lagoon Sgl33INRA 0.0 0.0 and high heterozygosity could be Sgl5fINRA 0.0 -0.02 mating between probable populations in Sgl695INRA -0.1 0.0 these rivers.The present and previous Sgl7159INRA 0.205 0.0 Sgl7eINRA -0.018 0.155 studies (Bahrami Kamangar and Sgl7fINRA 0.0 0.035 Rostamzadeh, 2015) on the population genetic of S. glanis corroborate that Discussion there was genetic differentiation among Microsatellites are well known as two populations of S. glanis. Obtained suitable genetic markers and powerful alleles and heterozygosity parameters in tools for population genetic studies by the present study were similar to the their high level of allelic variability previous studies, so that these (Krieg et al., 1999; Dudu et al., 2008). microsatellite loci could be used as S. glanis is an economic species which genetic markers to identify other become popular fish with high populations of this family. consumption in most countries. In the Based on the AMOVA analyses, present study we used six specific significant genetic differentiation was microsatellite loci to assess the found among both populations of population differentiation in two areas, Anzali Lagoon and Aras Lake (p<0.01). Anzali Lagoon and Aras Lake from The Fst index based on AMOVA test is north part of Iran and all of these loci a useful relative measurement of were polymorphic. genetic differentiation among The present study found that the populations, changes from 0 to 1 number of alleles per locus was in the (Ballox and Lugan-Moulin, 2002; Quan range of 4-12 and Anzali Lagoon et al., 2006). High value of Fst indicates presented higher number of alleles (9.3 larger discrepancy and vice versa. It is in average) compared to Aras Lake (5.3 worth to mention that there are large Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] in average). Low number of alleles in number of reports in different species Aras lake samples may indicate using Fst parameter that all others found bottleneck effect due to reduce of various values of Fst and significant stocks, water pollution and destruction differentiations between studied or degradation of habitat (Selkoe et al., populations (Norouzi et al., 2008; 2006). The computed heterozygosity for Chakmehdouz et al., 2011). In the other study between four populations of Journal of Survey in Fisheries Sciences 7(2) 2021 129

S.glanis in different part of Iran, environmental degradation and Anazali Lagoon population showed a overfishing could be the reasons of low level of diversity (Bahrami deviation from HWE. In recent years Kamangar and Rostamzadeh, 2015). In habitat deterioration and overfishing of

the present study the computed Fst this species in these areas are increased value between two populations was that could be a reasonable explanation remarkable. Sampling areas are of observed deviation from HWE in this completely isolated basins and there is study. In the future, these factors could no migration. This level of be inbreeding and diminishing the differentiation among S. glanis population size in selected areas. populations could be due to a mutation In conclusion, the findings of this rate and gaps between allele sizes. study indicated that there are genetic Since geographical distance of selected differentiations in Anzali Lagoon and

area and significant values of Fst and Aras lake S. glanis. Significant variance genetic distance, it is suggested that two in microsatellite allele frequency populations are genetically provide evidence that Anzali Lagoon differentiated. populations in the south Caspian Sea It is worth to mention that when the basin is genetically structured. Thus the value of the genetic deviation index is protection of habitat areas and prevent closer to zero, the population is close to of harvest for conservation of gene Hardy–Weinberg equilibrium (Quan et pools should be applied. al., 2006). Our results were similar to those observed in other species of Acknowledgements catfish, in which significant amounts of The authors would like to thank Mr. population structure were documented Nahrevar and Mr. Nowrouzi for (Triantafyllidis et al., 2002). In the providing Silurus glanis samples. present study three loci in both populations were at HWE as well as References excess or deficient of heterozygous Bahrami Kamangar, B. and (departure from HWE). There are Rostamzadeh, J., 2015. Genetic several possible causes of deviation diversity and population genetic from Hardy–Weinberg equilibrium test structure of Wels (Silurus glanis such as shortage of samples, using of Linnaeus, 1758) in the northwest of nonspecific primers, population sub- Iran. Environmental Biology of Downloaded from sifisheriessciences.com at 11:34 +0330 on Thursday September 30th 2021 [ DOI: 10.18331/SFS2021.7.2.10 ] structuring, low level of polymorphism fishes, 98, 1927-1934. and the most probable is presence of Ballox, F. and Lugon-Moulin, N., null alleles in inheritance of 2002. The estimate of population microsatellite loci (Paetkau and differentiation with microsatellite Strobeck., 1995; Sekino et al., 2003). markers. Molecular Ecology, 11, Quan et al. (2006) reported that 155-165. 130 Behmanesh et al., Microsatellite genetic differentiation between two populations of European …

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