Phylogenetic and evolutionary investigation of MSTN gene sequence of Turkmen camel in comparison with other species

Introduction: The evolution of single and two humped camels dating back to 40 to 45 million years ago, when the Camelida family was evolved during the Eocene in North America. Genus Camelini and Genus Lamini, which includes species of Guanaco, Lama, Alpaca, and Vicugna, were evolved from the Camelida family, about 11 to 25 million years ago. Old world camel can be divided into wild and domestic one and two humped camels, in which wild type of one humped camel has been distanced centuries ago (Burger 2016). Among 150,000 of Iranian one-humped camel, there are about 2,000 one-humped (Turkmen) camels, which are the country’s major species (Ansari Renani et al 2010), whom mainly rearing in Golestan province. Single-humped camels are used mainly for the purpose of producing meat, milk, transport, hair, and sports (Abdel-Aziem et al. 2015; Ramadan and Inoue-Murayama 2017). Several studies in different species have shown that the genetic polymorphisms of the Myostatin gene can increase the lean meat (Mosher et al. 2007). The identified polymorphisms of the gene in the camel included mutations in non-coding regions and synonymous mutations in coding regions (Shah et al. 2006; Sajadi Zirjani et al. 2016). Myostatin or Growth differentiation factor 8 (GDF8) is a member of beta-transforming family (TGF-β) that limits muscle mass (Wang and Mcpherron 2012). The mutation in the Myostatin gene increases muscle mass through the growth and proliferation of its cells (Mosher et al. 2007). The Myostatin gene with its three exons and two introns, has been sequenced in wild and domestic two-humped and domestic Arabian camel. The aim of this study was to investigate the sequence of the Myostatin gene (Gene ID: 105099167) of Camellus Bactrianus, Ferus, Dromedarius (Arabian) and some other species with Iranian Turkmen Dromedarius camel. Therefore, the evolutionary relationships among different species in terms of the sequence of this gene can be determined using phylogenetic study. For this reason, the whole genome sequence of Turkmen camel was performed and Myostatin gene sequences of different species were obtained from data banks.
Material and methods: The blood sample was obtained using syringe from the abdominal vein of a pure Iranian Turkmen camel, then stored in an EDTA containing plastic tube. After extraction, one microliter of the product was used to measure concentration using Qubit^TM Fluorometer (Invitrogen, Cat. No. Q32857). After evaluation, the sample was sent for whole genome sequencing using the Illumina HiSeq 2000 platform. After trimming the results, reads were assembled and the reads containing the Myostatin gene were extracted from the whole data set. To compare the different species of camels, the Myostatin gene sequence of Arabic camels, wild Bactrian camel, domesticated Bactrian camel, and Alpaca were obtained from NCBI. Using the CLC Genomics Workbench 12 software, alignment was performed to compare and obtain the sequences of the Myostatin gene in old and new world camels. Alignment for comparison of Myostatin gene sequence parameters of different camel species were conducted using CLC Genomics Workbench 12 software. Then, the number of gaps, differences, distances, identity percent, and identity of Myostatin gene sequence nucleotides was considered in different camel species. To study the degree and the relative time of divergence of the camel species, a phylogeny tree using neighbor-joining method was prepared. Also, annotation information of Myostatin was used to extract exons and introns of the gene. Then, the number of gaps, differences, distances, identity percent, and identity of Myostatin protein sequences was considered in different camel species. The phylogenetic and evolutionary situation of the Turkmen camel compared to many other species.
Results and discussion: Concentration of extraction product was 304 ng/μl. A total of 589,326,158 clean reads of about 88 billion nucleotides in paired end mode with the length of 150 base pairs were obtained. The Myostatin gene sequence was extracted from the assembled whole data set. Alignment results of Myostatin gene sequence in different camel species showed the greatest gap, differences and distance, and the least similarity between the alpaca species and other camel species were obtained. Also, the most similarity obtained between wild and domestic two-humped camels. The phylogenetic tree obtained from the gene sequence alignment revealed that Alpaca is far apart from other camel species. Among old world camels, the two-humped camels are more distinct from the Arabian one-humped camel. The length of the exons in different species was equal, but not the sequence, and exon alignment showed evolutionary conservation of exon length and ultimately the protein length. Considering identity and differences of exons and gene sequence revealed that Alpaca is more distant from other species. Also, one-humped and two-humped camels were similar to each other. The results are consistent with the results of Shah et al. (2006), which did not observe any polymorphism by sequencing of exon 1 of this gene. In the study of Yi et al. (2017), domestic and wild two-humped camel were located in different branches of the phylogenetic tree. In this study, the Iranian Turkmen camel was located in a separate branch between the two-headed and one-humped Arab camels. Myostatin gene alignment was conducted between different species and the phylogenetic tree showed that camels, horses, sheep, goats and cows belong to different groups. In the study of Hedayat-Evrigh et al. (2016), it was found that one-humped and two-humped camels were far away from cattle and horses. Homology between sheep and goat, as well as przewalski horse and the horse were complete. The lowest homology of 92.82 was found between humans and buffaloes. The greatest gaps of 3236 nucleotide were seen between sheep and donkeys.
Conclusion: Although there are differences between the nucleotide sequence of the introns and the exons of Camelida family, there was no difference in amino acid sequence of the gene, which indicates the strong protection of this gene during the evolutionary period. It can be said that numerous mutations in the gene have been eliminated during natural selection and no evolutionary opportunity has been provided for the mutations. The study of the homology and difference between the 16 species revealed that the homology of the protein sequence is very high in different species.