Coreius heterodon is one of the important economic fish species in the mainstem of the Yangtze River in China and its aquaculture has begun to rise in China in recent years. It is favored by consumers due to its high content of amino acids and unsaturated fatty acids, as well as its delicate and delicious meat. However, the presence of approximately 159 intermuscular bones (IBs) has strongly restricted its industrial development. Recently, runx2b has been identified as a key regulatory gene in the formation of IBs. In this study, the morphology and quantity of IBs in C. heterodon were characterized. Subsequently, the runx2b gene in C. heterodon was identified, and selection pressure analysis was performed across 20 different fish species, the results showed that two amino acid sites underwent positive selection, and one of the sites (site 17) was completely conserved as Val (V17) in fish species with IBs and as Ser (S17) in fish species without IBs. Furthermore, the process of IB ossification showed that IBs began to ossify at 11 days post hatching (dph) and developed from the posterior to the anterior region. The expression level of runx2b gene showed regular changes with the occurrence and ossification of IBs. Moreover, CRISPR/Cas9 method was applied to disrupt the function of runx2b , and fortunately, runx2b mutants consist of IB-free and IB-less C. heterodon individuals were obtained in F0 generation. This study demonstrates that the runx2b gene plays a crucial role in the formation of IBs in C. heterodon . The acquisition of C. heterodon runx2b mutants in F0 generation lays the important foundation to breed new strains without IBs in the future. • Coreius heterodon has a total of 159 intermuscular bones (IBs) in 6 different shapes. • The IBs-free and IB-less C. heterodon mutations were obtained through knocking out runx2b gene. • A key amino acid site (V17) in Runx2b is perfectly correlated with the presence of IBs.
Zhou et al. (Tue,) studied this question.