Effect of GH gene polymorphisms on growth traits in Caspian white fish (Rutilus kutum) and common carp (Cyprinus carpio) by PCR-SSCP method

Master's Thesis

Fisheries major, Aquatic breeding and breeding

Growth hormone (GH) is the most important hormone that controls the growth of somatic cells and is effective in the synthesis of protein, fat and carbohydrates. The aim of this study was to identify polymorphisms of GH-1 gene in common carp and GH-2 gene in white fish using PCR-SSCP technique and its relationship with growth-related traits (condition factor, length and body weight). 150 pieces of carp in 4 age groups of 4, 6, 12 and 24 months and 150 pieces of white fish at the age of 3 months were randomly selected and the tail fin was used for DNA extraction. After extracting DNA by optimized salt method, two fragments of 373 and 410 base pairs were amplified and genotyped by SSCP method for GH-1 and GH-2 gene positions in common carp and white fish, respectively. In the studied samples, there are 8 different band patterns A, B, C, D, E, F, G and H in the carp population of better health fish with frequencies of 31/33, 10/67, 20/67, 22/67, 4, 2, 2/67 and 6% and 3 different band patterns A, B and C with frequencies of 24/67, 58/67 and 16/67 respectively. percentage was observed in the white fish population. Marker-trait analysis, there is no statistically significant relationship between different genotypes of GH-2 gene of white fish with weight, body length and condition factors. Also, there is a statistically significant relationship (P<0.05) between the GH-1 gene of carp in three age groups of 4 months, 6 months, and 12 months with the trait of weight, while no significant relationship was observed with the traits of length and condition factor. Duncan's multi-range test for the common carp population in three age groups of 4 months, 6 months and 12 months showed that fish with the D band pattern genotype had significantly (P<0.05) more weight than other genotypes. Also, Duncan's multi-range test for white fish showed that people with genotype C had higher CF (P<0.05) than people with genotype A. In terms of weight and length traits within the white fish population, none of the genotypes had significant differences with each other. The results of sequencing the replication fragment in white fish showed that in the C band pattern, nine SNPs were nucleotide changes from T to G at position 82 bp, A to C at position 113 bp, G to A at position 207 bp, G to A at position 254 bp, G to A at position 269 bp, G to A at position 296 bp, A to G at position 307 bp, C to A at position 308 pairs of games and G to A occurred in position 346 pairs of games. Also, a deletion type mutation was observed at the position of 366 base pairs in band pattern B. The observation of eight different band patterns in the locus studied in this research indicates a great diversity in the GH-1 gene locus in the common carp population. Therefore, considering the economic importance of common carp and white fish in the breeding industry and the existence of correlation between the observed polymorphisms and growth traits, it is possible to use this position as a marker in breeding programs in the studied populations. However, in order to obtain reliable results, it is necessary to repeat the test with more markers from these gene positions and larger sample sizes from these populations.

Key words: GH gene, common carp, white fish, PCR-SSCP

1-1 Introduction

The growing trend of the world population and the subsequent increase in needs Protein has made humans consume aquatic products, including fish. Also, the reduction of human aquatic resources has prompted him to take measures for the extensive breeding of aquatic animals in small and limited aquatic environments. Today, the demand for fish consumption is increasing in all parts of the world. For this reason, rapid progress in research programs will be needed, especially in the field of reproduction, breeding, nutrition, genetics and management of breeding systems. Global demand for consumption of fish and marine products is influenced by three factors: population growth, income and price. It is predicted that the global demand for fish and seafood consumption will reach more than 100 million tons in the next few years (FAO, 2008). In order to make more use of the production systems, we should look for solutions such as breeding, improving nutrition, or correct breeding management. Meanwhile, eugenics, which is carried out in order to change the genetic capacity for the desired economic traits, is of particular importance.Different traits of an animal can be divided into two categories, quantitative and qualitative traits, in terms of the number of controlling genes and the degree of influence of environmental factors. Qualitative traits are expressed by one or a small number of genes, and environmental factors are not very important in the occurrence of these traits, but quantitative traits are often controlled by a large number of genes, and environmental factors are effective in their occurrence. Most of the qualitative traits have high heritability, as a result, the selection and modification of these traits is relatively easy and often there is no need for indirect selection. But for few traits that are affected by environmental factors, the existing phenotype may not be indicative of the genotype of the traits. In order to solve this problem, advanced breeding techniques have been developed using the science of statistics. These methods have been useful in recent years and have caused significant progress (Harandi, 2017). Today, the technique of molecular genetics has created a revolution in the genetic analysis of domesticated species. Based on that, polymorphisms can be identified at the level of DNA [1] and used as molecular markers to study different people and organisms (Weller et al., 1990).

Caspian white fish[2] is one of the most valuable and economic species among bony fishes in the southern part of this sea, which due to its deliciousness, high nutritional value and excellent meat quality is more than It accounts for 50% of the total catch of these fishes, so that the amount of catch in 2016 was more than 17 thousand tons (Qasmi et al. 2016). In the three provinces adjacent to the Caspian Sea, the amount of white fish catch is 59.63% of the total fish catch of this sea in 2013 (Deputy of Fishing of Golestan, Mazandaran, Gilan provinces). There are two spring and autumn breeds of white fish in the Caspian Sea, and the spring breed makes up more than 98% of the Caspian sea white fish stocks. In recent years, the stocks of the autumn breed of white fish have decreased due to the loss of natural spawning grounds, overfishing and other influencing factors (Qasmi et al. 2016). The Caspian Sea is an important habitat for common wild carp, which is considered one of the economic fish and an important source of food, therefore it has a great contribution to strengthening the source of this fish in the sea (Yousefian, 2011). Although this species exists natively and naturally on all the shores of the Caspian Sea and enters the mouths of rivers for reproduction, in recent years due to excessive fishing and the destruction of breeding places, their generation has decreased so that it is considered among the species in need of protection in the region (Qalichpour et al. 2019). Parts of the world have drawn the attention of experts to the application of precise methods, including molecular methods, for the management of aquatic resources, and the identification of species and their breeding is of great importance in the exploitation of marine aquatic resources and aquaculture. Despite the importance of this valuable species, which is of high economic importance among the inhabitants of the southern basin of the Caspian Sea, few studies have been conducted in the field of its genetic and demographic structure (Qasmi et al. 2016). The importance of GH as an effective factor in increasing growth has been known for a long time. It was shown that the application of GH increases the growth rate in most animals, especially fish (Sansansen et al., 2009 and Zhang et al., 2014). GH was also shown to stimulate growth directly by increasing DNA, protein and lipolysis synthesis in muscle. Growth hormone indirectly controls the growth process by affecting the production and release of a mitogen called I-IGF [6], which is produced both by the liver and most of the surrounding tissues (Kalangi Miander et al., 2013).

Mutations in different regions of genes are always of interest to many specialists in eugenics. The association of polymorphisms of these genes with phenotypic traits such as growth has been extensively investigated in other animals and limited studies have been conducted in fish (Gross and Nielsen, 1999 and Tao and Bolding, 2003).