Efficiently whole-genomic mutagenesis approach by ARTP in blunt snout bream (Megalobrama amblycephala)

Abstract

The atmospheric and room temperature plasma (ARTP) mutagenesis technique was first used in the present study to produce whole-genomic mutants in blunt snout bream (Megalobrama amblycephala). The optimal ARTP mutation parameters for semen were established according to sperm motility and abnormal embryonic rate. A total of 2026 mutated offsprings were produced with normal and abnormal individuals with a ratio of approximately 1:1. Among them, 384 normal mutant fish (~20%), after three months of cultivation, demonstrated improved performance with either fast growth or hypoxia tolerance, and 89 mutant individuals with both hypoxia tolerance and fast growth (H&F). The average bodyweight of the H&F fish increased by 26.48% compared with the control group. The average oxygen tension threshold for loss of equilibrium (LOEcrit) of the H&F fish was 0.45 mg/L, significantly (p < 0.05) lower than 0.86 mg/L in the control group. Meanwhile, the red blood cell count, hemoglobin concentration, and Na+/K+-ATPase enzyme activity of the H&F group increased significantly (p < 0.05), whereas, the gill tissue cell apoptosis rate decreased considerably (p < 0.05), suggesting that they are hypoxia tolerant. According to the genome resequencing analysis, 3,195,434 SNPs and 927,930 InDels were found in the H&F group, which were significantly higher than the 9127 SNPs and 2908 InDels in the control group. The average mutation rate in the H&F fishes reached 2.98 × 10−3 at the genome level, approximately 350 times that of the control group. Importantly, 3651 special non-synonymous SNP mutations were found in the H&F fish, which are located in 1223 genes. Among them, eight hypoxia- and growth-related genes with non-synonymous mutations at one or more sites were differentially expressed (DE) in H&F fish. Taken together, our results establish a novel whole-genomic mutagenesis approach using ARTP in blunt snout bream, which should be useful for future genetic breeding.