Evolutionary Genetic Analysis of the cytochrome b gene variation in the Salmo trutta fario with other salmons

PCR amplification and direct sequencing of regions of the cytochrome b mitochondrial genes was carried out on Iranian populations of the salmo trutta fario species. Samples of salmo trutta fario were also examined and used as an out group in the phylogenetic analysis. Results based on 1192 bp indicated differentiation between fario morphs from Iran and other salmonids also Iran and other country had collected. Despite the large phenotypic differences within salmonids, very low genetic variation was found. On the basis of the cytochrome b sequences studied, Salmo carpio and Salmo fibreni, which have been described as good species, there were high homology between salmo trutta fario , salmo trutta and salmo trutta caspius (99%), but regards salmo salar the rate of homology 93%. Regards salmo trutta fario, in hatchery trout has not red colour strips time of hatchery in salmo trutta fario whereas has a bluish grey body colour, However, they are larger than Atlantic salmon, salmo trutta caspius and salmo trutta, more regular in shape, and less intensively pigmented, moreover there are red spots are always observed in populations, however, the shape and size of salmo trutta fario is different with salmo trutta caspius and salmo trutta but the among of homology were high (99%). The salmo trutta fario and salmo trutta caspius are living in the Rivers of North of Iran that connected to Caspian Sea; perhaps these species with themselves has been conjugated, and regards salmo salar is living in the Atlantic Ocean and other Sea and Rivers connected to it, that so far to salmo trutta fario populations in Iran. However we cannot report exactly that how much homology between sequences, because already there are not complete sequence in GeneBank regards salmo trutta caspius and salmo trutta fario.


INTRODUCTION
There are different methods for identifying and authenticating for fish and fishery product, specially, DNA methods and molecular testing for increasing products.(Gill, et al., 1997;Rasmussen et al., 2008).In related to, there are wide range of approached of varying technical sophistication and cost which exploit diagnostic polymorphism within mitochondrial DNA and nuclear DNA genomics.Different molecular methods including RFLP-PCR techniques, SSCP-PCR techniques also sequencing of DNA fragments (Carrera et al., 2000;Abercrombie et al., 2005;Lee et al., 2004), approaches have been successfully implemented to identify partially degraded and otherwise compromised products (Dalvin et al., 2010).In general, mitochondrial DNA transmitted maternal traits, mtDNA targeted methods have predominated in such studies, because of the general robustness and higher cellular copy number of mtDNA compared with nDNA.(Mackie et al., 1999).In this paper we used PCR and direct sequencing techniques to compare and analyzing segments of cytochrome b DNA sequence variation in the salmo trutta fario and observed among morphologically distinct salmo trutta fario, salmo trutt, salmo trutta caspius and salmo salar populations.Regards to cytochrome b, Bernatchez et al., (1992), studied on the variation of it gene, also, they studied segments of the mitochondrial control region, with this results, we can discussed the congruence in phylogenetic relationships among salmo trutta fario populations inferred from the analysis of coding and noncoding regions of the segments of cytochrome b gene.Regarding genetic variations salmonids, were studied brown trout that was found substantial genetic differentiation, as revealed by protein variation is found between native Mediterranean and Atlantic populations (Kerieg & Guyomard 1985;Guyomard 1989;Persa et al.1994).Also, Largiader et al., (1996), had reported there are common phenotype of the fluviatile ecotype of brown trout, salmo trutta f. fario.According to local fisherman, the striped trout represent the native form in the Doubs, whereas the fario phenotype originates from introductions of hatchery trout.In this research our aims to study among of homology between salmo trutta fario, salmo trutta caspius salmo salar and salmo trutta by cytochrome b gene, also the rate of relationships between salmo trutta fario and other salmonids for cytochrome b gene.

Sample collections, DNA extraction and gene amplification and sequence analysis:
Animals: Salmo trutta fario were obtained from the Rivers of Tonekabon -Iran, in the summer of 2011.All fish were approximately 1 kg in size.Species used in this study salmo trutta fario.DNA extracted from muscles samples was used to obtain sequence from the salmo trutta fario.Cytochrome b gene.cytochrome b gene sequences were amplified from salmo trutta fario.DNA was extracted from tissue muscles according to the method of Sambrook et al., (1998) Forward Primer 5' GACTTGAAAAACCACCGTTG 3' and Reverse Primer 5' CTCCGATCTCCGGATTACAAGAC 3',were based on conserved sequences from the promoter and terminator regions identified by the alignment of cytochrome b sequence data from several salmonid species.These amplification products were compared to the amplification products from a genomic DNA template using 1.5 percent agarose gel electrophoresis.

Sequencing:
For doing sequence we designed one side primer from end of the gene including, GTTTTCCTAAATTTCCAATTGC.By this primer amplified full length of sequence of cytochrome b gene in the salmo trutta fario.
Polymerase chain reaction (PCR; Saiki et al., 1988) amplification was performed on 200-500 ng of genornic DNA template with either Ultratherm (BioiCan Scientific) or Taq (Bethesda Research Laboratories-BRL) DNA Polymerase using the reagents and instructions provided by the manufacturer.Typically, the thermal profile of a PCR consisted of 2-4 min.incubation at 94" C, followed by 30 cycles of 30 s at 94°c, 30 s at 55°c, 60 s at 72°c, followed by a 4 min.incubation at 72°c.PCR amplification products were prepared for sequencing by purification with DNA Clean-Up kits (Promega).Where necessary, multiple amplification products were separated by electrophoresis in low-melting-point agarose using standard methods (Sambrook et al.1989).
Amplification products were sequenced directly using either the Thenosequenase sequencing kits (Amersham United States Biochemicals).
Sequencing, electrophoresis and autoradiography were performed according to the manufacturer's instructions.

Accessed in the GeneBank:
The cytochrome b gene was sequenced and deposited in GeneBank by Accession number (JN995186.1).

Mitochondrial sequence DNA analysis:
Overall, the mitochondrial DNA genomic has a relatively high substitution rate in salmonid fishes (Thomas and Beckenbach, 1989;McKay et al., 1996).The cytochrome b gene is part of mitochondrial DNA.
However, approximately, the complete sequence of the cytochrome b gene (1191 nt) was found to be identical between salmo trutta fario in Iran.Also observed among of variation between salmonids, including, salmo trutta caspius , salmo trutta and Salmo salar.Variation in exon sequences of the cytochrome b gene: The DNA sequence of cytochrome b gene from salmo trutta fario individuals was determined.To avoid confusion about geographic origin, only wild strains from known sampling locations were analyzed.Comparison of variation within species revealed that the higher degree of homozygosity with salmonid group.Although no fixed differences were observed between salmo trutta fario and salmo salar, because the degree of variation more than other salmonids likes salmo trutta and salmo trutta caspius.However results are shown there were almost variation between salmo salar, salmo trutta caspis and salmo trutta.The sequence of the same region of the cytochrome b gene was also obtained from salmo trutta fario, salmo trutta and salmo trutta caspius similar variation was detected within this gene: the (T to C, C to A, T to G and A to G), repeat sequences in the full length of the sequences.There were within sequences.The repeat single nucleotides, (A to G, C to A and etc.).In addition to variation in the number of repeat units in the cytochrome b gene had same with all of sequences exception salmo salar.(Fig. 1).DISCUSSION Phenotypic plasticity in salmo trutta fario and most salmonid species certainly limits its utility to resolve evolutionary and phylogenetic issues (Allendorf et al., 1987;Bernatchez et al., 1992;Bernatchez, 1995).In addition, we explained phenotypic and their distribution of striped between species of salmonids and rate of relationships with salmo trutta fario populations, in related to, in the Atlantic populations there are four black strips and variable number of small irregular black, spots white halos on the body sides.(Largiader et al. 1996).Salmo trutta fario, in hatchery trout has a bluish grey body colour and no black strips, However, they are larger than Atlantic salmon, salmo trutta caspius and salmo trutta, more regular in shape, and less intensively pigmented, moreover there are red spots are always observed in populations, however, the shape and size of salmo trutta fario is different with salmo trutta caspius and salmo trutta but the among of homology were high (99%).The salmo trutta fario and salmo trutta caspius are living in the Rivers of North of Iran that connected to Caspian Sea; perhaps these species with themselves has been conjugated, and regards salmo salar is living in the Atlantic Ocean and other Sea and Rivers connected to it, that so far to salmo trutta fario populations in Iran.However there is not any report regards complete sequence of cytochrome b gene in the salmo trutta fario and salmo trutta caspius , but regards salmo salar and salmo trutta have been reported in GeneBank.So, after sequencing of complete cytochrome b gene, we can exactly discuss about among of relationship between salmo trutta fario and other salmonids.Also, we should more research and using other method genetic techniques for finding ancestor of salmons specially relationships between salmo trutta fario, salmo trutta caspius, salmo trutta and salmo salar.Sequence analysis of salmo trutta fario and among of homology with other sequences of cytochrome b in salmonids: In this research, has examined variations in exonic sequence of a cytochrome b gene.Analysis of the sequence of cytochrome b gene that has one exon from first to end of the gene.There were some variation between salmo truutta fario and other salmonids that revealed a variable microsatellite locus within the sequence of full length.A direct, tandem repeat of a (CGT) sequence motif were observed.Also, there were tandem repeats single nucleotides including A to C, A to G and G to T, between salmo trutta fario, salmo trutta caspius and salmo trutta.Regards salmo salar the variation was more, so, the rate of variation on single nucleotides were low.We aim also research on the complete sequence of mitochondrial genomics and among relationships with mitochondrial salmo salar that reported in GeneBank.

CONCLUSION
The sequences of cytochrome b gene fragment we have identified in the Salmo trutta fario, that could be a potential genetic markers for Salmonids.Differences in trait association of the genetic markers may exist among different populations.More tests are needed in other populations of bony fishes to variety associated effects of genetic marker, as well as the effects of the other polymorphisms in the mitochondrial genome.However the mitochondrial genes traits more will be transformed from maternal, So, is better the studies on the other genes markers for example growth hormone genes, microsatellites and other marker genes related, that are associated with paternal traits.