Discrimination between GH-2 and GH-1 Genes in Rutilus Kutum Using Sequencing and Restriction Enzymes

Fish genome contains two copies of growth hormone (GH-1 and GH-2) as a result of duplication process during evolution. The aim of this study was to distinguish GH-1 from GH-2 genes in Rutilus kutum (Mahi Sefid) from the Caspian Sea using sequencing techniques and application of specific restriction enzymes for these loci. For this purpose, five fish were randomly selected and samples for DNA extraction were isolated from caudal fin clips. The DNA was extracted by salting-out method. Then, fragments of 333 and 410 bp from exon 4, intron 4 and exon 5 of GH-1 and GH-2 genes were amplified and, following purification, sequenced on the gel. GH-1 sequence of R. kutum was not different from that of common carp on the long allele showing 100% overlap with each other. On the short allele, however, they showed differences in GH-1 sequence of carp at four positions namely 89, 94, 105, and 164 bp with an overlap of 99%. The GH-1 overlapped with GH-2 sequences from both common carp and R. kutum as 99% and 86%, respectively. Comparison of the GH-1 and GH-2 sequences derived from R. kutum showed that they mainly differed in Intron 4. Other differences observed were mutations at one or more nucleotides. Despite the high similarity in the nucleotide sequences, the differences detected are applicable as specific genetic markers for the discrimination between GH-1 and GH-2. By identifying and sequencing GH-1 and GH-2 genes, the sequences obtained can be used to reconstruct the phylogenetic relations, study different allelic forms more precisely, and establish relationships between these loci with important economic traits in R. kutum from the Caspian Sea.