Intermuscular bones (IBs) are small spicule-like bones locating in the muscle fillet of teleost. They are found to be ubiquitous in most of freshwater fishes, especially in farmed carps. Therefore, the creation of IB-free mutants has become a hot issue in fish genetic breeding. 

Gibel carp (Carassius gibelio) is an important aquaculture species in China, it contains approximately 80 IBs. A research group led by Prof. GUI Jianfang from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences (CAS) revealed that the gibel carp is amphitriploid (AAABBB) by comparative genome analysis, which means that it contains two triploid sets of chromosomes, and developed an efficient approach to rapidly generate mutants by gene editing in amphitriploid gibel carp and amphidiploid goldfish (C. auratus) (AABB). 

In a study published in Aquaculture, based on these breakthroughs, Prof. GUI’s group has developed a precision breeding biotechnology for polyploid fish and created IB-free mutants in amphitriploid gibel carp, in collaboration with Prof. GAO Zexia from Huazhong Agricultural University, who has identified runx2b as a decisive player of IBs in zebrafish. 

The researchers first identified two homeologs of runx2b (Cgrunx2b-A and Cgrunx2b-B) and found that each of them possessed three almost identical alleles in gibel carp.  

They then traced the IB ossification process in gibel carp. Alizarin red S staining showed that gibel carp IB began to ossify in the tail area at 14 approximately days post hatching and developed from the posterior to the anterior regions. By comparing the differential expression of Cgrunx2b-A and Cgrunx2b-B, the researchers found that they displayed similar expression pattern and differential expression level during IB development. 

Using CRISPR/Cas9, the researchers have edited CgRunx2b-A and CgRunx2b-B singly or simultaneously, and found that single loss of CgRunx2b-A or CgRunx2b-B function was unable to affect normal IB development, but simultaneous disruption of CgRunx2b-A and CgRunx2b-B led to complete loss of IBs. Through the above research, they obtained 291 gibel carp mutants with completely lost IBs. 

These findings indicated that CgRunx2b-A and CgRunx2b-B cooperatively regulate IB development. Importantly, the researchers created IB-free mutants, which laid the foundation for breeding IB-free strains of gibel carp.  

In the next step, the detailed biological and economic traits, including growth and development, swimming performance, bone density, disease resistance, food competitiveness and muscle nutrient content, need to be compared between wild-type (WT) and IB-free gibel carp strain under rigorous raising and other experiment conditions.