Cross-species amplification of Clupeidae microsatellite DNA markers in common kilka, Clupeonella cultriventris from the Caspian Sea

Document Type: Research Paper

Authors

Islamic Azad University of Tonekabon

Abstract

Common kilka Clupeonella cultriventris (Nordmann, 1840) is a brackish water and small pelagic fish species and is one of the most abundant fishes that live gregariously in the Caspian Sea. A total of 60 specimens of adult common kilka were sampled from two seasons. Fifteen pairs of microsatellites previously developed for A.sapidissima, C. pallasi, C.harengus, and S. pilchardus were tested for cross-species amplification on the common kilka. In this study, only five primer pairs were used successfully. Analyses revealed that the average of alleles per locus was 14.4. The average observed and expected heterozygosity was 0.153 and 0.888, respectively. All loci significantly deviated from H–W equilibrium. These results together with significant Rst. values for genotypic differences support the existence of different genetic populations along the Caspian Sea coast (Guilan Province).

Keywords


Cross-species amplification of Clupeidae microsatellite DNA markers in common kilka, Clupeonella cultriventris from the Caspian Sea

 

M. Norouzi1*, M. H. Samiei1

1-Dept of Marine Biology and Fisheries Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran.

 

* Corresponding author’s E-mail: mnoroozi@toniau.ac.ir

(Received: Dec. 02.2014   Accepted: May. 03.2015)

ABSTRACT

Common kilka Clupeonella cultriventris (Nordmann, 1840) is a brackish water and small pelagic fish species and is one of the most abundant fishes that live gregariously in the Caspian Sea. A total of 60 specimens of adult common kilka were sampled from two seasons. Fifteen pairs of microsatellites previously developed for A.sapidissima, C. pallasi, C.harengus, and S. pilchardus were tested for cross-species amplification on the common kilka. In this study, only five primer pairs were used successfully. Analyses revealed that the average of alleles per locus was 14.4. The average observed and expected heterozygosity was 0.153 and 0.888, respectively. All loci significantly deviated from H–W equilibrium. These results together with significant Rst. values for genotypic differences support the existence of different genetic populations along the Caspian Sea coast (Guilan Province).

Keywords: Population genetics, South Caspian Sea, microsatellite, Clupeonella cultriventris

 

INTRODUCTION

Common kilka, Clupeonella cultriventris, that belongs to the family Clupeidae lives in the Caspian Sea, and feeds on zooplankton and crustaceans such as copepods and cladocerans (http://www.fishbase). They spawn in spring. The Caspian stocks of common kilka face challenges resulting from overfishing and the invader Ctenophore Mnemiopsis leidyi (Velikova et al., 2012; Rowshantabari et al., 2012; Nasrollahzadeh, 2010). Microsatellites are abundantly distributed across the genome, demonstrate high levels of allele polymorphism, and can be easily amplified by PCR (Sekar et al., 2009). Microsatellite genotypes are particularly helpful to identify the genetic structure in closely related populations, regardless of whether they are in an evolutionary equilibrium (Chistiakov et al., 2005). In addition, primers designed for one species can often be used for other related species (Chistiakov et al., 2005). However, the development of new species-specific microsatellites by traditional methods requires substantial amount of time and money. One possible shortcut on the path of describing new microsatellite makers is to cross-amplify markers from other species, based on the fact that the microsatellite flanking regions may be conserved in closely related species (Hulák et al., 2010).

 

MATERIALS AND METHODS

This study represents the population genetic analysis of common kilka in the South Caspian Sea (Guilan Province, Anzali Port). A total of 60 specimens of adult common kilka were sampled from a single sampling location, but the fish were caught during different seasons (spring and summer) and preserved in 95% ethanol. Genomic DNA was extracted from the fin tissue using High Pure PCR Template Preparation Kit (Roche Applied Science, Germany) according to manufacturer’s instructions. The quality and concentration of DNA were assessed by 1% agarose gel  electrophoresis and spectophotometry (CECIL model CE2040)

 

Table 1. Microsatellite loci used for cross-species PCR amplifications in common kilka, GenBank Accession no., Reaction consistence, Cycling condition, PCR Amplification.

Locus

 

GenBank

Accession no.

Reaction consistence

Cycling  condition

PCR Amplification

Refrence

Cpa6

AF309801

0.2 mM each dNTPs; 0.4µM each primer; 200 ng template DNA;  0.4 U/HotStarTaqTM DNA polymerase; 1x HotStarTaqTM PCR buffer ; 4.5 mM MgCl2

95 oC/10m [95oC/30s;52 oC/2m ;72 oC/1m]2

[95oC/1m;51 oC/1m ;72 oC/1m]38  72 oC/5m

Yes

 

Cpa8

AF309804

0.2 mM each dNTPs; 0.4µM each primer; 200 ng template DNA;  0.4 U/HotStarTaqTM DNA polymerase; 1x HotStarTaqTM PCR buffer ; 4.5 mM MgCl2

95 oC/10m [95oC/30s;52 oC/2m ;72 oC/1m]2

[95oC/1m;51 oC/1m ;72 oC/1m]38 72 oC/5m

Yes

Miller et al.(2001)

Cpa100

AF309790

2mM MgCl2

[58 oC]40

No

 

Cpa104

AF309791

0.2 mM each dNTPs; 0.4µM each primer; 200 ng template DNA;  0.3 U/HotStarTaqTM DNA polymerase; 1x HotStarTaqTM PCR buffer ; 4.5 mM MgCl2

95 oC/10m [95oC/30s;51.5 oC/45s ;70 oC/45s]2

[95oC/40s;51.5 oC/45s ;72 oC/45s]38  72 oC/5m

Yes

 

Cpa107

AF309792

1mM MgCl2

[49 oC]40

No

 

Cpa120

AF309795

1mM MgCl2

[61 oC]40

No

 

Cpa134

AF309798

1mM MgCl2

[61.2 oC]40

No

 

Cpa125

AF309796

0.2 mM each dNTPs;  0.2µM each primer; 200 ng template DNA;  0.3 U/HotStarTaqTM DNA polymerase; 1x HotStarTaqTM PCR buffer ; 4.5 mM MgCl2

95 oC/10m [95oC/30s; 59oC/40s ;70 oC/45s]2

[95oC/40m;59 oC/40s ;72 oC/45s]38 72 oC/5m

Yes

Miller et al.(2001)

AsaC051

EF014992

0.2 mM each dNTPs;  0.2µM each primer; 200 ng template DNA;  0.3 U/HotStarTaqTM DNA polymerase; 1x HotStarTaqTM PCR buffer ; 4.5 mM MgCl2

95oC/10m [94oC/30s;54 oC/40s ;72 oC/2m]38  72 oC/5m

Yes

 

AsaC059

EF014993

1.25mM MgCl2

[58 oC]40

No

Julian and Barton,2007

AsaC249

EF014994

2.5mM MgCl2

[59 oC]40

No

 

AsaC334

EF014995

2.5mM MgCl2

[56 oC]40

No

 

SAR1.12

EF012617

2.5mM MgCl2

[49 oC]40

No

Gonzalez and Zardoya, 2007

1235

AF304362

2.5mM MgCl2

[58 oC]40

No

McPherson et al., 2001

1014

AF304360

2.5mM MgCl2

[58.5 oC]40

No

 

             

 

 

 

and stored at −20°C until use. Fifteen pairs of microsatellites previously developed for American shad (Alosa sapidissima), Pacific herring (Clupea pallasi), Atlantic herring (Clupea harengus), and sardine (Sardina pilchardus) were tested for cross-species amplification on the common kilka. For all primer pairs, amplification was performed in a reaction volume of 25 µL containing 0.2 mM of dNTPs, 0.2–0.4 µM of each primer, 200 ng of template DNA; 0.3–0.4 unit of HotStarTaqTM DNA polymerase; 1×HotStarTaqTM PCR buffer, and 2.5–4.5 mM MgCl2 (Table 1). PCR products were separated on 10% polyacrylamide gels (29:1 acrylamide: bis-acrylamide; 1×TBE buffer) followed by silver staining. Gels were run at 40 mA for 14h. Alleles were sized using Uvitec software, and each gel contained an allelic ladder (100bp) to assist in consistent scoring of alleles.

Data analysis via codominant data were computed in the GenAlex 6 software (Peakall and Smouse, 2006) and Arlequin 3.5 (Excoffier and Lischer, 2010).

RESULTS

Of the 15 pairs of microsatellite primers, 10pairs did not show any flanking sites on the common kilka genome. Five pairs of primers (Cpa6, Cpa8, Cpa104, Cpa125 and AcaC051) were amplified successfully and they showed polymorphic pattern in the 60 individuals assayed. All microsatellite primers that were able to produce DNA bands displayed a characteristic disomic banding pattern.

The average number of alleles found in the seasons was 14.4 (±1.7) and ranged at each locus from 5 (AcaC051) to 21 (Cpa8) alleles. The effective number of alleles (Ne) per locus ranged from 4 to 17.8, with an average of 10.7 (±1.03). Allelic richness per locus and population ranged from 7.4 to 23.5 (Cpa104, 23.5; Cpa6, 16.9; Cpa8, 13.4; Cpa125, 17; and AcaC051, 7.4). All sampled populations contained a significant number of private alleles, none of which was found in other seasons (Table 2). The Nm and Fst via frequency, ranged from 25.14 to 5.64 and from 0.010 to 0.026, respectively (Tables 2, 3).

 

Table 2. Number of privet allele, actual size (bp) and Allele frequency in spring and summer seasons.

 

 

Cap6

Cap8

Cap104

Cap 125

Asac051

Total

Number of privet allele (actual size)

Spring

1(164)

1(144)

2(334,364)

3(216,230,248)

-

7

Summer

2(120,124)

1(188)

1(388)

1(260)

1(180)

6

 

Table 3. F-Statistics and Estimates of Nm over All Populations for each locus using 5 sets of  microsatellite primers

 

Cap6

Cap8

Cap104

Cap 125

Asac051

Average

Fst

0.025

0.016

0.013

0.026

0.010

0.018

Nm

9.66

15.08

19

9.4

25.14

05.64


Discussion

The results of the present study showed that at least five microsatellite primers could be used to investigate population genetics of species in the Caspian Sea. There is initial indication of population differentiation of common kilka stocks in the Caspian Sea. Because the common kilka is a shared stock between 5 Caspian countries, it is highly recommended to develop a joint research project on this species, which would cover the entire Caspian Sea. In summary, this study provides preliminary evidence for the existence of at least two differentiated populations in the South Caspian Sea (Guilan Province, Anzali Port). The existing private alleles and significant Fst and Rst confirm that spring and summer populations in Anzali Port. Probably, extra populations are present in the Caspian Sea; therefore, a comprehensive investigation using more samples and primer sets from the entire Caspian Sea may confirm this hypothesis.

ACKNOWLEDGMENT

The study was supported by Islamic Azad University, Tonekabon Branch and was performed in Molecular Genetic Lab. We would like to thank Dr. Ali Nazemi, Amin Ravai and Mohhamad Eskandari (Dept. of Biology).

 

Brown, B.L., Gunter, T.P., Waters, J.M. and Epifanio, J.M. (2000). Evaluating genetic diversity associated with propagation-assisted restoration of American shad. Conservation Biology. 14: 294–303.

Chistiakov, D.A., Hellemans, B., Haley, C.S., Law, A.S., Tsigenopoulos, C.S.,  Kotoulas, G., Bertotto, D., Libertini A. and Volckaert, F.A. (2005). A microsatellite linkage map of the European sea bass Dicentrarchus labrax L. Genetics. 170: 1821–1826.

Excoffier, L., Laval G. and Schneider, S. (2005). Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online. 1: 47-50.

Gonzalez, E. G. and Zardoya, R. (2007). Isolation and characterization of polymorphic microsatellites for the sardine Sardina pilchardus (Clupeiformes: Clupeidae). Molecular Ecology Notes. 7: 519–521.

Hulák, M., Kašpar, V., Kozák P., Bu¢ri, M., Filipová, L. and Petrusek A. (2010). Cross-species amplification of microsatellite markers in the invasive spiny-cheek crayfish (Orconectes limosus): assessment and application. Journal of Applied Genetics. 51: 73–78.

Julian, Sh. E., Barton, M. L., (2007). Microsatellite DNA markers for American shad (Alosa sapidissima) and cross-species amplification within the family Clupeidae. Molecular Ecology Notes. 7: 805-807.

Laloei, F., Rezvani Gilkolaei, S., Nirani, M. and Taqavi, M. T. (2006). The PCR-RFLP investigation of Clupeonella cultriventris from the South Caspian Sea, Iran. Iranian Scientific Fisheries Journal. 15: 119-128.

Miller, K. M., Laberee, K., Schulze, A. D. and Kaukinen K. H. (2001). Development of microsatellite loci in Pacific herring (Clupea pallasi). Molecular Ecology Notes. 1, 131-132.

McPherson, A. A., O’Reilly, P. T., McParland, T. L., Jones, M. W. and Bentzen P. (2001). Isolation of nine novel tetranucleotide microsatellites in Atlantic herring (Clupea harengus). Molecular Ecology Notes. 1: 31-32.

Nasrollahzadeh, A. (2012). Caspian Sea and its Ecological Challenges. Caspian Journal Environment  Science. 8 (1): 97-104.

Peakall, R. and Smouse, P.E. (2006). GENALEX 6: genetic analysis in excel. Population genetic software for teaching and research. Molecular Ecology Notes. 6: 288-295.

 Rowshantabari, M., Finenko, G.A., Kideys, A. E. and Kiabi, B. (2012). Effect of temperature on clearance rate, daily ration and digestion time of Mnemiopsis leidyi from the southern Caspian Sea. Caspian Journal Environment  Science. 10 (2): 157-167.

Sekar, M., Suresh, E., Kumar, N.S., Nayak, S.K., and Balakrishna, C. (2009).  Microsatellite DNA markers a fisheries perspective Part 1: The nature of microsatellites. Genetics and Biodiversity. pp. 27-29.

Velikova, V.N., Shaudanov, A.K., Gasimov, A., Korshenko, A., Abdoli, A., Morozov, B., Katunin, D. N., Mammadov, E., Bokova, E. B., Emadi, H., Annachariyeva, J., Isbekov, K., Akhundov, M., Milchakova, N., Muradov, O., Khodorevskaya, R., Shahifar, R., Shiganova, T., Zarbaliyeva, T. S., Mammadli, T., Velikova, V., Barale, V. and Kim, Y. (2012). Review of the environment and bioresources in the Caspian Sea ecosystem 2000-2010. CaspEco Report. P: 423.