EccDNA Analysis Provides Novel Insights Into the Molecular Mechanism of Firmness of Fish Fillet

Abstract

Extrachromosomal circular DNAs (eccDNAs) play a significant role in regulating various biological processes, including abnormal muscle development. The molecular functions and impact of eccDNAs in the muscle development of fish are poorly understood. To investigate the potential roles of eccDNAs in the muscle development of fish, we analyzed and compared the expression profile of muscle eccDNAs of crisp grass carp, fed a faba bean meal-based diet, and ordinary grass carp, fed a practical diet. Using the Circle-seq strategy, we found the eccDNA abundance in crisp grass carp (211,920 eccDNAs) was significantly higher than that in ordinary grass carp (25,857 eccDNAs), suggesting that the faba bean diet likely independently influences eccDNA production. Compared to ordinary grass carp, crisp grass carp exhibited 10,565 upregulated and 129 downregulated eccDNAs, indicating eccDNAs were possibly associated with the muscle development of grass carp. GO and KEGG enrichment analyses indicated that the upregulated eccDNAs were related to muscle fiber development, cellular structure, and cell junctions. Based on our results, we speculated that the overexpression of genes involved in muscle fiber, calcium metabolism, and collagen driven by eccDNAs likely contributes to the observed increase in muscle fiber density, calcium levels, and collagen content in crisp grass carp, thereby enhancing muscle hardness. Notably, eccDNAs were identified as potential innate immunostimulants capable of eliciting immune responses in fish. In summary, our findings demonstrate that eccDNAs are aberrantly expressed in the muscles of fish fed a faba bean diet, offering novel insights into the molecular mechanisms underlying muscle hardening in fish.

Keywords: extrachromosomal circular DNA; fillet quality improvement; muscle texture.

FIGURE 1

Electrophoresis results in the amplification of COX3 (A) and NDL6 (B) genes.

FIGURE 2

Electrophoresis results in the purification of eccDNAs after RCA reaction (A)and enzyme digestion verification (B).

FIGURE 3

The length distribution of eccDNAs in two grass carp groups.

FIGURE 4

The distribution of eccDNAs in different chromosomes.

FIGURE 5

The GC content of eccDNA in crispy grass carp (A ~ C) and common grass carp (D ~ F).

FIGURE 6

GO enrichment analysis of differentially expressed eccDNA‐associated genes. (A ~ C) referred to the GO enrichment analysis of up‐regulated eccDNAs in crisp grass carp, (A) Biological process, (B) Cellular component, (C) Molecular function; (D ~ F) referred to the GO enrichment analysis of down‐regulated eccDNAs in crisp grass carp, (D) Biological process, (E) Cellular component, (F) Molecular function.

FIGURE 7

Pathway analysis of eccDNA‐associated genes. (A, B) referred to the up‐regulated pathways of eccDNAs; (C, D) referred to the down‐regulated pathways of eccDNAs.