The present study is a cross-sectional descriptive study. Chemicals were bought from Sigma Aldrich. BIO-RAD T100 thermal cycler was used for gene sequencing. Results were analyzed by the help of software “Bioedit”.

Sampling

A total of forty-five fish (Acanthopagrus latus) from three different areas of Baluchistan (Gaddani, Dam and Kund Malir) were collected aseptically. Blood sample (1mL) was collected from caudal vein by using vacuum tubes with EDTA (ethylenediaminetetraacetic acid) as an anticoagulant. The samples were stored in ice container at -20^oC and brought to the laboratory of molecular Genetics, Faculty of Veterinary and Animal Sciences, Lasbella University of Agriculture, Water and Marine Sciences, Uthal, for further analysis.

DNA Extraction  

DNA (deoxyribonucleic acid) was extracted from samples by using inorganic method as suggested by previous study [7]. Before the DNA extraction, blood samples were frozen at temperature of -70°C for 20 to 30 minutes and at -20°C for twenty -four hours. The blood samples were thawed before extraction of DNA. Tris EDTA buffer (Tris HCL 10mM, EDTA 2mM) was added in 1ml blood samples and mixed inverting three to four times. All centrifugation in further stages were done at 25°C. All the samples centrifuged for the 12 minutes at 4000 rpm and after that discarded the supernatants. Pellets were produced at the bottom and dissolved smoothly. Once again TE buffer was added. All the supernatant was discarded leaving 0.5ml.Pallets were added in 3000 µL TSE Buffer (Buffer A1) (Tris HCL, EDTA, NaCl)  and 200µL of 10% SDS & 20µL proteinase K. Proteinase K was used for protein digestion in the blood. After that , samples were incubated in water bath at 40°C for 24 hours. After 24 hours, the incubation was checked for the total digestion of the pellets. The resultant tubes were places in ice and 500µL NaCl was added . After mixing, tubes were placed again placed on ice for 10-15 minutes, centrifuged at 4000 rpm for 10 minutes to shot down the protein sample and salt. The supernatant was poured in a 15mL falcon tube and isopropanol with the same volume was added and the tubes were slightly overturned until the DNA became observable. The tubes were stored at room temperature for 10 minutes. The tubes were spun to attach the DNA on tube and the supernatant was carefully discarded. 2mL of 70% ethanol was used to wash DNA. DNA was centrifuged at 4000 rpm for 10 minutes and ethanol was discarded. DNA pellet was dried in air at 37°C for 24 hours. The next day,  400µl  TE buffer was added to dissolve  DNA. To inactivate nucleases, the tubes were placed in a water bath at 70°C for 1 hour. Tubes were left to cool off at room temperature. DNA was shifted into cap of 2.0 ml autoclaved tubes and stored at -20 °C.

Conformation and quantifying of DNA

For the conformation, quality and quantity of DNA, gel electrophoresis was used. 0.6 g of agarose gel powder was weighed and transferred into a beaker having 100ml IX TAE buffer. The solution was heated in oven for two minutes. After 2 minutes, 0.6µL ethidium bromide was added in the solution for  DNA staining.  The solution was left on gel plate for 30 minutes at room temperature for solidification. When the gel got solidified, it was transferred into electrophoresis tank which was filled with IX TAE buffer. The level of TAE buffer was kept 1cm above the gel. 3µL of tracing agent: 3X Bromophenol blue was added in  2µLDNA and was loaded in the wells carefully. Finally, the gel was observed under UV trans illuminator. The quality and quantity of DNA sample was analyzed and single compact pink florescent bands were observed.

Primer Designing

For the amplification of mtDNA D-loop the specific primers were designed for this study by using the software (Primer3 Input-0.4.0) following the sequence of D-loop region Mt DNA (>AB916800.1) available on NCBI (http://www.ncbi. nlm.nih.gov. Sequence and product size of the specific primers are given in (Table 1). For successful result of the PCR, both primers were amplified(64°C to 54°C) and were used in thermo cycler. The temperature at which primer showed best results was selected. The subsequent PCR were carried out at optimized annealing temperatures. Polymerase chain reactions were carried out in a 25 μL reaction mixture containing, PCR Buffer(5x) 2.5 μL, Taq Polymerase 0.5 μL, dNTPs 0.5 μL, MgCl2 2 μL, Primer Forward 1 μL, Primer Reverse 1 μL, DNA 3 μL, Distal Water 14.2 μL. The amplification conditions for primers of the mitochondrial DNA D-loop were as follows: denaturation at 94°C for 5 min; followed by 35 cycles of denaturation at 94°C for 30 sec; annealing at 57°C for 30 sec; and extension at 72°C for 45 sec; with a final extension at 72°C for 10 min, on a Thermocycler. The PCR product was subjected to electrophoresis for detection amplified DNA.

Gel Electrophoresis and UV Trans-illumination

The Agarose gel electrophoresis method was used for the visualization of DNA and PCR product. This method was performed for the confirmation of the presence of PCR product. For the making 1.8% gel, 1.2gm agarose gel was weighed on weighing balance and mixed with 60ml of 1X TAE buffer into a sterilized beaker and transferred into microwave oven for two minutes at 100ºC for fully dissolving the agarose gel. Then taken out the beaker from oven and 0.6µL of Ethidium bromide was added in the beaker for the staining of DNA in the agarose gel. To make wells, combs were carefully fixed in the gel casting tray and then mixture was poured carefully and gently into it. For the solidification of gel casting tray was kept in a equalized place. Then gel was taken from the gel casting tray and kept in gel electrophoresis. The samples and ladder was loaded in corresponding wells. Then power supply was provided to the gel for electrophoresis. 200 Amperes 100 Volts were used to run the sample for 30 minutes. At the end, for the observation of DNA bands, gel was visualized in UV illuminator. The DNA bands of the samples were differentiated with base pair standard marker for the identification.

Sequencing of PCR Products

For the sequencing of the products, after preparation products the samples were sent to CAMB (Center of Applied and Molecular Biology) University of Punjab Lahore for Sequencing of the samples.

Analysis of Results

DNA sequencing was analyzed by the help of software manually using Bioedit software version [8]. Blast2 software was used for the sequences against the normal sequences. 

Figures & Tables

Acanthopagrus latus (Yellowfin sea bream) is one of the most important and common specie found in Indian and pacific warm coastal belts [8,9]. It is found in coastal belts of Baluchistan and Sindh, Pakistan with diverse phenotypic characteristics. Acanthopagrus latuswhich is locally called as Dandhia is available throughout the year in the fish markets of Balochistan and Sindh with reasonable prices and widelyused as a sea food by the local population. It is a commercially significant specie and a high protein source [3,10]. A.latus is also used and processed in the feed mills as feed for the poultry industry. It is a high concern that overfishing in the gulfs of Persian and Omanhas resulted insevere decline in the population of A.latus [8].  During the current study,it was observed that A.latusis being exploited in the costal belts of Baluchistan which may cause a decline in the population of the A.latus in the region. This concern has been raised by International Union for Conservation of Nature (IUCN) in Pakistan. Food and Agriculture Organization (FAO) of America suggests that  A.latus and related species that belong to family Sparidae are threatened or near-threatened to be extinctdue to overfishing [11]. Current study was performed on A. latus found in the costal belt of Balochistan. The prime objective of the study was to analyze the morphological (phenotypic) and genetic characteristics using the mitochondrial DNA D-loop region.A total of forty-five fish A. latus from three different areas of Baluchistan (Gaddani, Dam, and Kundmalir) were collected aseptically.

The phenotypic characteristics revealed four types of Acanthopagrus named as type A with flat dark body and gray fins, type B with slightly long body with yellow and white color, the color of fins was observed light gray except pectoral fins which were yellow and light gray. Type C was flat and had a predator type of body. The color of the body was white, yellow and red. The dorsal fins were light gray, pectoral fin was yellow and light gray, pelvic and anal fins were yellow and caudal fin was gray. Type D had a flat body with light gray color. Dorsal and pectoral fins were light gray, pelvic and anal fins were yellow and the caudal fin was gray with slightly yellow color at the dorsal end.Having such a diverse variety of the Acanthopagrusis not surprising asits diverse morphological characteristics have been previously classified in various species including A.arabicus, a novel specie found in Middle East, costal belts of Iran and Pakistan [12]. Study carried out by Orrel et al reported species with variable color formation ranging from red or pinkto yellowor gray. It was also found with golden or silver reflection with dark spots, stripes or bars [10,12]. It has been also repotrted diverse color formation including white, yellow to strong vivid, black streaks at the base of anal fin. In their study they also reported high diversity among individuals and moderate diversity among population [2].

The genetic characterization was done by observing the mitochondrial DNA D-loop region in the current study. Mitochondrial DNA D-loop provides a good source of information for the management as well as the selective breeding or diversity of the sea fauna. Displacement region (D-loop) of mitochondrial DNA is a noncoding, hypervariable region controlling the replication and transcription of mitochondrial DNA [13]. It has widely been used in various species from human to fish for genetics characterization and variation analysis among the species. Mitochondrial DNA D-loop region is divided into three domains including left, center and right domains with high variable regions in the left and right domains. Few scientists also have reported 64 variable sites at mitochondrial DNA, D-loop region of A.latus including 60 variations at 5’ end, 3 at 3’ end and 1 variable at tRNA^Phe gene [14]. The current study revealed three variations;two heterozygous variations at 32 and 172 nucleotide positions (Adenine substituted by Thymine and Thymine substituted by Guanine) and one homozygous variation at 72 nucleotide position (an insertion of a Thymine).

The phenotypic characteristics revealed four different types of the A. latus depending on the color of the body and fins. They wereclassified as type A, B, C and D.All types show variation in the color of the body and color of dorsal, pectoral, pelvic, caudal, and anal fins. The genetic sequencing of the study revealed three genetic variations at mt DNA D-loop region. The heterozygous variations at 32 and 172 nucleotide positions were observed as Adenine was substituted by Thymine and Thymine was substituted by Guanine.  Whereas one homozygous variation at nucleotide position :72 was observed as an insertion of Thymine.

Author Contributions

Khalil Hasni and Abdul Hameed Baloch designed the experiments and performed experiments.Shazia Rahim and Muhammad Jan did sampling, performed the experiments, and wrote the initial draft of manuscript. Ahmed Nawaz performed experiments,Chandni Wajid did sampling and did phenotypic studies, Sonia Tariq did sampling, analyzed the data and wrote the manuscript, Muhammad Sharif Hasniidentified species, performed analysis. All authors commented and edited the manuscript.

Conflict of Interest

The authors declare that there is no conflict of interest.

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