| Grouper aquaculture is an important industry in China's marine aquaculture. In recent years, inter-specific hybridization has become a common method for grouper breeding, with new hybrid species such as the Hulong and Yunlong Grouper accounting for more than 70% of domestic grouper production. Although inter-specific hybrid selection has significantly promoted the development of the grouper industry, it has also brought challenges to species identification and germplasm preservation. Additiothe enally, scape of hybrid fish can pollute the gene pool of species. Hybrid fish possess morphological characteristics from both parents, making morphological identification difficult. Therefore, there is an urgent need for precise identification and authentication of hybrid groupers and their parents to ensure the healthy and sustainable development of the grouper industry. This study compared the morphological differences between F1 hybrids and their parents and used mitochondrial gene COI and nuclear gene RYR3 as dual molecular markers to trace the parents of five common hybrid species:Yunlong grouper, Hulong grouper, Jinhu grouper, Shanhu grouper, Longshu grouper. The results showed: (1) The body shape of hybrid is similar to that of the maternal parent, such as Yunlong grouper, Hulong grouper, Jinhu grouper, Shanhu grouper are all oval-shaped, Longshu grouper has a pointed head and a humpback. The color and pattern of hybrid groupers combine the characteristics of both parents, among which Jinhu grouper and Shanhu grouper have high similarity with their parents' color and pattern, making it difficult to distinguish hybrid offspring from parents by color alone; Yunlong grouper, Hulong grouper, and Longshu grouper exhibit new color patterns by blending parental colors; (2) The genetic distance between the five hybrid species and their maternal parents based on COI gene is 0.000-0.002, all less than 0.020; in their phylogenetic tree, hybrid species are clustered with their maternal parents. It is evident that COI gene can accurately identify the maternal parent of hybrid species; (3) Given that nuclear genes carry genetic information from both parents, calculating the genetic distance between parental RYR3 genes of hybrids (range 0.006-0.023) and constructing a phylogenetic tree can effectively distinguish each purebred grouper, indicating that RYR3 is suitable as a molecular marker to assist COI gene in identifying the parents of hybrid groupers; (4) Based on these results, a method for identifying the parents of hybrid groupers using COI and RYR3 dual molecular markers was established. Initially, whether a sample is a hybrid is determined by the heterozygosity of its RYR3 gene sequence; subsequent sequence comparison confirms its parental information; if it is a hybrid, then the parents are identified using COI results; (5) This method was applied to identify the parents of 16 hybrid grouper samples, and the identification results were consistent with their recorded parental information, demonstrating that the hybrid parent identifying method is feasible and effective. These findings indicate that the COI and RYR3 dual molecular marker technology can accurately identify the parents of hybrid groupers, providing important references for the conservation of stingray germplasm resources and genetic breeding. |
