The relation between growth hormone (GH) gene and Cytochrome b gene in three salmon types

In the present study, growth hormone (GH) gene and Cytochrome b gene in Salmo trutta caspius and Salmo trutta fario were discussed, the rate of relationships between salmonids were analyzed by GH and Cytochrome b gene. The GH gene is a genetics marker in nuclear DNA that expressed paternal traits in salmons, furthermore, Cytochrome b gene also is genetics marker that expressed maternal DNA in mitochondrial genomics. With two genes we documented that there were high homology between sequences of GH gene and Cytochrome b gene, hence the salmonids types, specially salmo trutta caspius, salmo salar and salmo trutta fario probably had similar ancient in bony fishes.


INTRODUCTION
The salmo types including, salmo trutta caspius (S. t. caspius), salmo salar (S.Salar) and Salmo trutta fario (S. t. fario), is important for economic aquaculture industry.They live in the part of rivers and exhibit homing behavior.Therefore the difference of population related to migration of them (sometimes connected to the sea).There are studies related to physiological and genetic on the salmons, confirmed marker genetic differentiation (Fergusen, 1989, Guyomard, 1989).
The research on the marker of genetics most concentrated on the paternal and maternal traits in fishes.There are some genes for paternal traits specially growth hormone (GH) gene, that associated with various quantitative traits (reviewed in Peter and Marchant, 1995).However, GH gene is an important trait in other animals, like cattle that regulate somatic growth in muscle and the skeletal body (Harvey et al.,1995;Rocha et al., 1991;Hoj et al., 1993;Pilla et al., 1994;Schlee et al., 1994a, b;Lagziel et al., 1996), and pigs (Casas-Carrillo et al.,1994;Nielsen et al., 1995).
The full length of GH gene in salmonids sequenced and deposited in Genebank.In salmo trutta caspius was found 2048 bp.(Accession number, JN241634.1),containing, five exons and six introns in the length.(Rezaei and Akhshabi, 2011 a and b;Rezaei and Akhshabi, 2012).The result of sequence in Salmo Salar (Johansen et al., 1989), however there were not report regards sequence of GH gene in S. t. fario, but also we can conclude the variation is low between S. t. fario , S. Salar and S. t.Caspius, because the result of mitochondrial DNA that related to maternal traits had been high homology between salmonids.The mitochondrial genomic in salmonids including 13 protein coding genes, 22 transfer tRNA genes, and 2 ribosomal RNAs genes corresponding to the 12S and 16S transcripts, that was followed between the species of salmonids including, the O. mykiss (Zardoya et al., 1995), S. salar (Hurst et al., 1999), and other salmonids such as S. alpinus (Doiron et al., 1999), C. Lavaretus (Miya and Nishida, 2000) and S. fontinalis (Doiron et al., 2002).
Recently, in S. t.Caspius for the full length of cytochrome b gene in mitochondrial DNA was found and deposited by (Jamshidi and Kalbasi, 2009) in Genebank (Accession number, JN995186), this gene has one exon in full length, the result of alignment with another sequence of cytochrome b in S. t.Capius and S. Salar, there were high homology between sequences.In this study we have two aims, first, is their relationship between someone's or salmon types has been common ancestor?
Second, how much relationship between genetic markers engaged in paternal and maternal traits?

MATERIALS AND METHODS
Samples and DNA isolation: The samples are included, S. t.caspius and S. t. fario were obtained from the rivers of Tonekabon -Iran, the fishes both male and female had three years age old.
DNA isolation: Total genomic DNA from S.t.caspius and S.t.fario was isolated from powdered tissue has taken from muscle body following described by Sambrook et al., (1998).Briefly, the samples was extracted with an equal volume of phenol-chloroform-isoamyl alcohol (24:25: 1).Vortexed 10x is then centrifuged at 3000 x g for 5 min at room temperature.DNA was precipitated overnight at 4°C with then washed with 2 vol.100% ethanol.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 and PCR amplification:
There were not any report regards GH gene in S.t.Caspius in Genebank, hence we used other sequences specially S. Salar and S. t. fario for designing primers, so were designed three pair of primers from first to end of the GH gene, we used a DNAMAN program (USA) and also the BLAST NCBI Network system for designing primers (Table 1).The primers could amplify three different sizes of fragments including, 910, 312 and 819 bp.The primers has also able cross amplified, means a forward primer of first fragment can match with a reverse primer of the second or third fragment, hence, we could amplify that a full length of GH gene in S.t.Caspius.fario and other salmonids.The primers could amplify three fragments, including, 1495, 1500 and 1493 bp.These primers also could amplify by crossly, means, first forward primer with third reverse primer could amplify a fragment, also these results confirmed regards other forward and reverse primer in GH gene (Table 2).
However the nuclear DNA will express paternal traits and mithocondrial DNA will express maternal DNA in salmons but also together can have been similar aims for studies of phylogenetics analysis.In this study we sequenced both Cytochrome b and GH genes in s. t. fario and s. t. caspius, the results were compared between sequences of reported in Genebank -NCBI Network system, there were high homology between sequences of Cytochrome b and GH genes, however there is some variation in the shape of s. t. fario with s. t. caspius, and S. salar. in similarity the Atlantic populations there are four black stripes and variable number of small irregular black, spots white halos on the body sides.In S. t. fario, in hatchery trout has a bluish gray body color and no black strips, However, they are larger than Atlantic salmon, s. t. caspius but more regular in shape, and less intensively pigmented, moreover there are red spots are always observed in populations of s. t. fario.Nevertheless our studies showed the among of variation between salmons is very less(around 2-3 %) regarding Cytochrome b, GH gene, however we have to do more studies on mini and microsatellites, RFLP, RAPD and other genetics marker for getting better results.But the more reports indicated that GH gene is influence on the growth of the body and genetics marker that is part of nuclear DNA, expressed paternal traits.Furthermore, Cytochrome b and C oxidase expressed from mithocondrial DNA genomic that penetrated on the maternal traits, that's important for studies of phylogenetics and evolution of salmons.
F. Discarded the flow-through and placed the QIA quick column back in the same tube.G. Centrifuged the column for an additional 1 min at maximum speed.H. Placed QIA quick 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 GH gene and Cytochrome b gene: 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 Study variations at the DNA level contribute to the genetic characterization of Salmons.We used GH of gene and cytochrome b gene .According to the annotation GH genes, these are genes linked to economic traits and polymorphism genetics which are governed by many genes, Following to the sequences of the salmon GH gene and cytochrome b were published in the BLASTn on the National Centre for biotechnology information (NCBI) network service, was designed a fragment of almost 3kb.for S.t.caspius and also S.t.fario (Figure 1).Regarding cytochrome b gene a full length (1191 bp.) were amplified and are shown in Figure 2.

Fig. 1 :
Fig.1: PCR amplification of high quality salmo t. caspius using the primers of designed Samples were separated by electrophoresis in 1.5% gel electrophoresis.M. Size marker 500 bp.

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

Fig. 3 :
Fig. 3: Comparison of sequences of the Cytochrome b gene between S.t.caspius, s.salar and s. t. fario.The maximum identity is shown, there is a high homology between sequences.

Fig. 4 :
Fig. 4: Comparison of sequences of the GH gene between S.t.caspius and s.salar.The maximum identity is shown, there is a high homology between sequences.

Table 1 :
the primers for synthesis of GH gene in S. t.Caspius

Table 2 :
Primers were designed for amplified of GH gene in S. t. fario.