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Knockout of the  MSTN  gene is linked to the enlarged tongue, and it causes suckling difficulty in animals. The suckling difficulty has a severe effect on animal mortality. Thus, special care was required to ensure their survivability. Here, it is critical to promptly ascertain the genotype of all pigs after birth. The main objective of the present study was to develop the restriction enzyme-mediated PCR-RFLP assay for  MSTN  mutant pig genotyping. To accomplish this, conserved oligonucleotide primer and restriction site were deduced according to the mutated sequence of the  MSTN  mutant pigs. PCR amplification yielded a 176 bp band for all homozygous  MSTN  mutant ( MSTN–/– ), heterozygous  MSTN  mutant ( MSTN+/– ) and wild-type (WT) pigs. However,  MSTN +/–  samples produced two fragments with 176 and 87 bp, and WT samples produced one fragment with 87 bp after being digested by  BstNI .  MSTN–/–  samples were not digested by  BstNI  and yielded a 176 bp band. Thus, we were able to determine the genotype of all pigs using  BstNI  restriction enzyme-mediated PCR-RFLP method. Overall, the present study reported a simple and fast PCR-RFLP genotyping method for  MSTN  mutant pig breeding. The present study may contribute to the establishment of commercial breeding systems and the production of double muscle pigs.

Application of PCR-RFLP for quick identification of MSTN mutants in MSTN mutant pig breeding