![bioedit code heterozygote bioedit code heterozygote](https://www.genome.gov/sites/default/files/tg/en/illustration/heterozygous.jpg)
Practically, the use of molecular markers should be followed by simple and easy molecular analysis techniques, such as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method (Hashim and Al-Shuhaib, 2019). Thus, the exploration of molecular markers is consistently employed in both partial and total genome approaches and molecular tools used (Singh et al., 2014). The use of molecular markers resolves the problems encountered in traditional breeding, which has limitations in terms of recording and selection. Thus, the MSTN gene has the potential for use as a molecular marker (Singh et al., 2014). The MSTN gene has been reported to affect the characteristics of cattle significantly, including the double-muscled phenotype and other economic traits (Casas and Kehrli, 2016). Furthermore, numerous studies that discovered the association between the MSTN gene and the economic features of cattle have been conducted intensively (Bennett et al., 2019), primarily focusing on the use of the MSTN gene for genome-based selection tools (Mrode et al., 2019) as well as marker-assisted selection (Sánchez-Ramos et al., 2012). The MSTN gene has been explored and characterized, including its targeted editing by the genetic manipulation technique (Qian et al., 2015, Zhao et al., 2016). Hence, the MSTN gene and its correlation with the double-muscled phenotype have been massively researched not only in cattle breeds (Cassar-Malek and Picard, 2016, Grisolia et al., 2009) but also in horses (Dall'Olio et al., 2010), goats (Khichar et al., 2016), sheep (Wadood, 2019), and geese (Smołucha et al., 2019). However, several mutations were also found in other MSTN gene regions (Grisolia et al., 2009, Aiello et al., 2018).
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The most successful finding on the double-muscled phenotype investigation is an 11-bp deletion in the MSTN gene in Belgian blue (BB), Asturiana, and Maine-Anjou cattle (Grobet et al., 1997, McPherron and Lee, 1997, Kambadur et al., 1997). The MSTN gene consists of three exons and two introns and encodes a 375-amino acid precursor protein (Jeanplong et al., 2001).
![bioedit code heterozygote bioedit code heterozygote](https://www.researchgate.net/profile/Mabel-Taracena/publication/322895455/figure/fig6/AS:589572312408065@1517576401404/Multiple-amino-acid-sequence-alignment-of-R-prolixus-mitoferrin-predicted-amino-acid.png)
Mutation of the MSTN gene prompts negative muscle growth regulation, which mediates muscle hypertrophy (McPherron and Lee, 1997, Miretti et al., 2013). Myostatin (GDF-8) is a member of the transforming growth factor-β superfamily that primarily regulates the development of skeletal muscle. In this case, by applying the PCR-RFLP technique using the restriction enzyme NmuCI ( Tsp45I) in indel 11-bp, the genotypes that were successfully observed were +/+, +/del.11, and del.11/del.11. Moreover, in this study, an 11-bp deletion in exon 3 of the MSTN gene in BB cattle was found. However, the four SNPs could not differentiate normal and double-muscled phenotypes although they are polymorphic.
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The MSTN gene in the coding region was detected in four SNPs found in PO cattle and its crossbreed. SNPs and indel 11-bp variation in the coding region of the MSTN gene were found using the sequencing method, followed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. A total of 86 blood samples were collected from 28 individual BB, 43 individual PO, and 15 individual BB × PO crossbred cattle. This study aimed to investigate single-nucleotide polymorphisms (SNPs) and 11-bp deletions in the coding region of the MSTN gene and their relationship with the double-muscled phenotype in BB × PO crossbred cattle. Determining double muscle based on the myostatin ( MSTN) gene in Belgian blue (BB), Peranakan Ongole (PO) and BB × PO crossbred cattle is very important for crossbreeding programs.