Identification of key genes related to growth of largemouth bass (Micropterus salmoides) based on comprehensive transcriptome analysis

Abstract

Introduction: Largemouth bass is an economically important farmed freshwater fish species that has delicious meat, no intermuscular thorns, and rapid growth rates. However, the molecular regulatory mechanisms underlying the different growth and developmental stages of this fish have not been reported.

Methods: In this study, we performed histological and transcriptomic analyses on the brain and dorsal muscles of largemouth bass at different growth periods. The brain and muscle tissue were dehydrated, embedded, sliced and stained with hematoxylin-eosin. Images were captured under a microscope and acquired using a microphotographic system. Differential expression between groups was analyzed using DESeq2. GO functional analysis and KEGG pathway analysis were then performed for differentially expressed genes. RT-qPCR validates the reliability of transcriptome sequencing data.

Result: Smaller fish had more new muscle fiber numbers and wider intermuscular spaces compared to big specimens. Axons and nerve fibers were more pronounced in the telencephalons of big fish than in small fish. A total of 19,225 differentially expressed genes (DEGs) were detected in the muscle tissue, among which 7,724 were upregulated and 11,501 were downregulated, while a total of 5,373 DEGs were detected in the brain, among which 2,923 were upregulated and 2,450 were downregulated. GO and KEGG enrichment analyses indicated that nucleic acid binding, cytoskeletal motor activity, DNA binding, circadian rhythm, glycolysis/gluconeogenesis, and osteoclast differentiation were related to brain development while binding, cytoskeletal protein binding, biological processes, c-type lectin receptors, mitogen-activated protein kinase (MAPK) signaling pathways, and osteoclast differentiation were related to muscle growth. Stat3, pparg, akt1, mapk3, and mapk1 genes were mainly involved in the growth and development of largemouth bass.

Conclusion: These results provide novel perspectives for deepening our understanding of the mechanisms underlying the growth and development and performing genetic selection in largemouth bass.

Keywords: DEGs; different growth periods; histological analysis; largemouth bass; transcriptome.

FIGURE 1

Telencephalon and muscle transverse slices of largemouth bass. (A) small fish telencephalon slice; (B) medium fish telencephalon slice; (C) big fish telencephalon slice; (D) small fish muscle transverse slice; (E) medium fish muscle transverse slice; (F) big fish muscle transverse slice. (PC) Pyramid Cell; (AX) Axon; (NF) Nerve Fibers; (IS) Interosseous space; (NMF) new muscle fiber.

FIGURE 2

Evaluation of sample transcriptomic correlation. BBr, big largemouth bass’s brain; MBr, medium largemouth bass’s brain; SBr, small largemouth bass’s brain; BMu, big largemouth bass’s muscle; MMu, medium largemouth bass’s muscle; SMu, small largemouth bass’s muscle. (A) Pearson correlation coefficients for comparisons among all samples; (B) The sample relationship cluster analysis; (C) The PCA distribution of 18 samples. In the heat map, each row represents one gene, and each column represents one sample. Different color areas represent different clustering information, and the color from red to blue represents the expression intensity of differentially expressed genes from high to low.

FIGURE 3

Identification of differentially expressed genes (DEGs) in different groups. (A) the pairwise comparison of the numbers of DEGs in different periods of tissues. (B) Venn diagram showing the overlapping number of DEGs in the pairwise comparisons. (C) Venn diagram showing the overlapping number of DEGs in the pairwise comparisons. All DEGs were determined based on statistical significance according to an FDR<0.05.

FIGURE 4

Top 20 terms in the GO term enrichment of significant DEGs in brain and muscle. (A) BBr-vs-MBr; (B) MBr-vs-SBr; (C) BBr-vs-SBr; (D) Mu-vs-MMu; (E) MMu-vs-SMu; (F) BMu-vs-SMu; X-axis indicates GO terms while the Y-axis indicates the rich factor. Rich factor refers to the ratio of DEGs relative to all genes annotated in the GO term. The higher the rich factor, the greater the intensity. The input number represents the number of DEGs enriched in the specific GO term. p-value indicates the enrichment significance of the GO term, and a lower p-value represents greater intensity.

FIGURE 5

Top 15 pathways in the KEGG enrichment of significant DEGs in brain and muscle. (A) KEGG enrichment is the pairwise comparison of the numbers of DEGs in different periods of brain tissues; (B) KEGG enrichment is the pairwise comparison of the numbers of DEGs in different periods of muscle tissues. The Y-axis represents the KEGG pathway while the X-axis represents the rich factor. Rich factor indicates the ratio of DEGs relative to all genes enriched in the specific pathway. The higher the rich factor, the greater the intensity. The input number represents the number of DEGs enriched in the specific pathway. p-value represents the enrichment significance of the signaling pathway, and a lower p-value represents greater intensity.

FIGURE 6

PPI networks of DEGs in brain (A) and muscle (B) growth-related pathways.

FIGURE 7

Schematic representation of DEGs in growth-related pathways. Red and blue colors indicate upregulation and downregulation of DEGs, respectively.

FIGURE 8

Validation of RNA-seq expression values by qPCR.