Studies of Cytochrome b nucleotide sequence variation in the Salmo trutta fario *

The cytochrome b gene in the salmo trutta fario has been sequenced and characterized and deposited in GeneBank, Accession Number (JN995186), the gene of cytochrome b approximately 1.2 kb. and consists of one exon from first to end of the gene, as found for all of salmonids including, salmo salar, salmo trutta caspius, salmo trutta fario that reported in GeneBank. There were the first to end of gene repetitive regions C-G that was unique for cytochrome b gene. At this study DNA extracted from muscles of salmo trutta fario, after running PCR on the gel, the PCR products purify and sequenced. The fragments aligned with BLAST Network system, the results are shown there were high homology between solmonids, the rate of homology between salmo salar 93%, salmo trutta caspius almost 95% and salmo fario (was reported in GeneBank) 93%. However, the homology of sequences were very high, but the shape of salmo trutta fario and other salmonids different, salmo trutta fario has red to purple color dots on the skin but regards salmo salar and salmo trutta caspius have grey to black dots colors on the skin.


INTRODUCTION
Salmonids constitute one of the most manipulated fish in temperate countries.Moreover, Salmonid fishes, and specially the salmo trutta fario.Salmo trutta fario is a native salmonid from Eurasia to North Africa.There are natural distribution it exhibits an extreme phenotypic diversity and a considerable life history variation within geographical regions, including specialization for anadromous, fluviatile and lacustrine ecological modes of life (trutta, fario and lacustris, are the subspecies names currently assigned in the literature to fish possessing these specializations, respectively) (Behnke, 1972;Hamilton et al., 1989;Hindar et al., 1991).Present there are studies regard biological activities on the salmo trutta fario, including, marker genetics that contains microsatellites, alozymes which along with LDH5 and FBP1 (Poteaux 1995;Guyomard 1989) and mitochondrion DNA studies (Bernatchez et al., 1992).The using of DNA techniques and molecular researches for identification and characterization of fish for increasing production in them.(Gil,1992 andRasmussen andMorrissey, 2008).In related to, we studied regard mitochondrial DNA in salmo trutta fario.In general, 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.,199).Cytochrome b is a unique and essential required by cells to maintain aerobic metabolism, that proteins which contribute a function to the overall role of the mitochondrial encoded by mitochondrial DNA (mtDNA).In this study, we used PCR and direct sequencing techniques to compare Cytochrome b DNA sequence variation observed among morphologically distinct salmo trutta fario and other salmonid populations by analyzing segments of coding genes, the cytochrome b in related to fragments of the mitochondrial control region specially cytochrome b gene studied by Bernatchez et al. (1992).This allowed to us that discuss the congruence in phylogenetic relationships among salmo trutta fario populations inferred from the analysis of coding and noncoding regions of the cytochrome b. because there were reports 1293 bp.length in salmo salar and there was not any report regard full length of the cytochrome b gene in GeneBank.With align of sequences we observed high homology between sequences.

MATERIALS AND METHODS
Sample collections: The samples of salmo trutta fario were collected from Rivers of Tonekabon -Iran.These samples had three ages old that collected on July of 2011.The originated of samples from bloods and muscles of fishes.

DNA isolation:
Total cellular DNA was isolated from powdered tissue has taken from muscle body following described by Sambrook et al., (1998).Briefly, tissue was digested in a digestion buffer, containing; (100 mM NaCl, 10 mM Tris pH 8.0, 25 rnM EDTA pH 8.0, 0.5% sodium dodecyl sulphate (SDS), 0.1 mg/ml proteinase K) for 18 hours at 50°C.Samples were then extracted with an equal volume of phenol-chloroformisoamyl alcohol (24:25: 1).Vortexed 10 s then centrifuged at 3000 x g for 5 min at room temperature.DNA was precipitated overnight at 4°C with 1/2 vol 7.5 M ammonium acetate and 2 vol 100% ethanol.Then DNA solved in TE buffer (10 rnM Tris-CI, 1rnM EDTA) pH 8.0 and incubating at 37°C for 1 hour with 0.1% SDS.DNA samples were extracted again with an equal volume of phenol/ chlorofom / isoamyl alcohol and precipitated as described above.Upon the final resuspension in TE buffer pH 8.0.Samples were stored at-20°C until analysis.Then the DNA genomics amplified cytochrome b gene was separated by 1.5 % agarose gel electrophoresis.After electrophoresis, the DNA full length was visualized ethidium bromide and then was taken photos by gel DOC Bio RAD Company.

Designing of primers:
Primers designed to specifically amplify the cytochrome b gene based on conserved sequences from regions identified by the alignment of all the available sequences data from several salmonid species.These primers can amplified from first to end of cytochrome b gene including 1191 bp.These primers including: Forward Primer 5' GACTTGAAAAACCACCGTTG 3' Reverse Primer 5' CTCCGATCTCCGGATTACAAGAC 3' The PCR programs: The PCR reaction used 10 microgram PCR reactions contained: 1 µl template DNA, 2 µl forward primer (100 ng/µl), 2 µl reverse primer (100 ng/µl), 2 µl dNTP mix (2.5mM each), 5 µl 10X ChromTaq Assay buffer, 0.5 µl ChromTaq enzyme (3U/µl), Water 37.5 µl, in a total volume, 50 µl.94° of 5 min, 35 cycles of 94°C 30 Sec., 55°C 30 Sec., and 72°C 1 min.Two to ten µl of each PCR reaction were run on 1.5% agarose gels in TAE buffer containing ethidium bromide.One µl 500bp, DNA ladder (Gibco-BRL) was used as a size standard.Then the PCR products after purification by the Chromous kit purification were sent to the Choromous Geni Company-India for doing sequence.

Sequencing of the cytochrome b gene in salmo trutta fario:
For sequencing of cytochrome b gene we designed one set of primer, that process of the sequencing including: Sequencing was performed along with the Forward and reverse primers in ABI 3730XL high throughput sequencer machine.Forward and reverse sequences were assembled and edited.

RESULTS
The ability of designed primers sets to detect of salmo salar, salmo trutta and salmo trutta.The amplified full length of gene cytochrome b had 1191 bp.Therefore the results are shown; there is high homology between sequences.In figure 3. A 99% DNA sequence similarity was observed, but there were five nucleotides differences from first of the two sequences and end of two sequences between sequence of salmo trutta fario and salmo trutta caspius.But regards salmo salar there was 93 percent homology between sequences.However there is some different the shape of salmons, salmo trutta caspius and salmo salar with salmo trutta fario but the analysis are shown homology was 93 percent, especially with salmo salar.Moreover these results innovated that salmo trutta fario in Iran is originated from salmo salar that the ancestor of salmo trutta fario had migrated from Atlantic Ocean to White Sea and then to Caspian Sea, because we collected samples has been connected from Rivers to Caspian Sea..

Sequencing of cytochrome b gene:
For doing sequence from PCR products, we designed gene specific forward and reverse primers for synthesis of fragment of cytochrome b gene.Moreover was sequenced one fragment (figure 4), from first to end of the gene.That including: The choice of a distant homology between outgroup of salmonids which could explain the apparent homology between salmo salar and salmo trutta at these nucleotide position, also was studied cytochrome b gene affected the rate of penetration and retention of ancestral sequence could have determined the fixation of genotypepic grouping specially cytochrome b gene in early times of differentiation between salmo salar and salmo trutta populations from a common ancestor (Avise et al., 1993;Avise, Neigel & Arnold 1984) .It could also be evidence of a variation from the constant rate of molecular evolution (Gillespie 1984(Gillespie , 1986) ) or of selective processes (MacRae & Anderson 1988).Also, regarding cytochrome b in the salmo trutta and salmo salar the levels of variation detected among coding regions and sequence divergence estimates among variant that the cytochrome b gene segment represent the least variable region between sequences, however there were deletion or different nucleotides in the regions of segments, for example, in the 5' and 3' end segments of the control regions, the transition: transversion ratio was nearly 3:1(176) and deletions were found (Bernatchez et al., 1992;Brown et al., 1982 and1993).The cytochrome b gene in salmonids have different length, in the salmo salar has, Accession Number, U12143.1 and EF584212, 1140 bp.(Philips et al., 1998).salmo trutta caspius, 1069 bp.Accession Number, FJ655773.1 (Jamshidi and Kalbasi, 2009) and salmo trutta fario, 310 bp.Accession Number, DQ394286.1,(De finizio et al., 2010) however the sequence of cytochrome b gene in the salmo trutta caspius was partial sequence.In salmo trutta fario were designed a pair of primers for amplify of full length, polymorphism was assessed at 9 samples and was observed on the gel a fragment of almost 1200 bp. then were purified PCR products and designed primers for the sequencing.These primers is expected to amplify one fragment 1 to 1191.Related to encompassing nucleotides of DNA in cytochrome b gene, the nucleotide-nucleotide BLAST search recovered numerous significant matches (>99% similarity) for cytochrome b gene sequences from other unionids.Empirical nucleotide base frequencies were biased towards thymine and adenine (27.7% A; 17.5% C; 29.9% G; 24.9% T).In related to, encompassing the fragments, positions of richness G-C, these characters are indicate unique at salmo trutta fario which almost like nucleotides situation in the salmo salar.This resulted also in higher frequency of the nucleotides GC, and GT, compared to the lower frequency of the nucleotides AT.These are fragments aligned with other sequences, especially salmo salar (accession no.U12143.1),salmo trutta caspius (accession number, FJ655773), these results are shown, there is high homology, 100% with salmo salar and almost 95% with salmo trutta caspius.Regards the salmo trutta caspius and salmo salar the results are shown however the variation between sequences was low but for typically salmo trutta caspius have grey to black dots color on the skin, but on the salmo trutta fario there is red to purple color on the skin.In other hand, the rate of growth in the salmo trutta caspius, salmo salar and salmo trutta more than salmo trutta fario, also there was high variation between sequences of the growth hormone gene in the salmo trutta caspius, salmo salar and salmo trutta with salmo trutta fario, (Rezaei et al., 2011, a, b) because the growth hormone gene relationship with paternal traits for increasing of growth body in the salmonids, (Gross et al.,1999;Nilsson et al., 2001) but regard of cytochrome b gene there were high homology between sequences.
PCR Purification: Amplified PCR product was purified using QIAquick PCR Purification Kit Protocol: A. Added 5 volumes of Buffer PB to 1 volume of the PCR sample and mixed.B. Placed a QIAquick spin column in a provided 2 ml collection tube.C. Centrifuged at 8000 rpm for 30-60 s.D. Discarded flow-through.Placed the QIAquick column back into the same tube.E. Washed with 0.75 ml Buffer PE to the QIAquick column and centrifuged for 30-60 s.F. Discarded the flowthrough and placed the QIAquick column back in the same tube.G. Centrifuged the column for an additional 1 min at maximum speed.H. Placed QIAquick column in a clean 1.5 ml microcentrifuge tube.K. To elute PCR product, added 40 μl of B H2O to the center of the QIAquick membrane and centrifuged the column for 1 min.Sequencing of Amplified Cytochrome b gene:

Fig. 2 :
Fig. 2: PCR amplification of high quality salmo trutta fario using the primers of designed Samples were separated by electrophoresis in 1.5% gel electrophoresis.M. Size marker 1000bp.

Figure 3 :
Figure 3: Comparison of sequences of the Cytochrome b gene between salmo trutta fario with other salmons.The maximum identity are shown, there are high homology between sequences.Regards salmo trutta caspius was 99% , salmo trutta 93% and salmo salar 93%.

Fig. 4 :
Fig. 4: The DNA sequencing of cytochrome b gene in the salmo trutta fario, from 1 to 1191 nucleotides.The major fragments have one exon from first to end of the gene.The fragment also contains GGGCC richness regions.