Genome-Wide Association Study and RNA-Seq Analysis Uncover Candidate Genes Controlling Growth Traits in Red Tilapia (Oreochromis spp.) Under Hyperosmotic Stress

Abstract

Growth traits are the most important economic traits in red tilapia (Oreochromis spp.) production, and are the main targets for its genetic improvement. Increasing salinity levels in the environment are affecting the growth, development, and molecular processes of aquatic animals. Red tilapia tolerates saline water to some degree. However, few credible genetic markers or potential genes are available for choosing fast-growth traits in salt-tolerant red tilapia. This work used genome-wide association study (GWAS) and RNA-sequencing (RNA-seq) to discover genes related to four growth traits in red tilapia cultured in saline water. Through genotyping, it was determined that 22 chromosomes have 12,776,921 high-quality single-nucleotide polymorphisms (SNPs). One significant SNP and eight suggestive SNPs were obtained, explaining 0.0019% to 0.3873% of phenotypic variance. A significant SNP peak associated with red tilapia growth traits was located on chr7 (chr7-47464467), and plxnb2 was identified as the candidate gene in this region. A total of 501 differentially expressed genes (DEGs) were found in the muscle of fast-growing individuals compared to those of slow-growing ones, according to a transcriptome analysis. Combining the findings of the GWAS and RNA-seq analysis, 11 candidate genes were identified, namely galnt9, esrrg, map7, mtfr2, kcnj8, fhit, dnm1, cald1, plxnb2, nuak1, and bpgm. These genes were involved in 'other types of O-glycan biosynthesis', 'glycine, serine and threonine metabolism', 'glycolysis/gluconeogenesis', 'mucin-type O-glycan biosynthesis' and 'purine metabolism signaling' pathways. We have developed molecular markers to genetically breed red tilapia that grow quickly in salty water. Our study lays the foundation for the future marker-assisted selection of growth traits in salt-tolerant red tilapia.

Keywords: GWAS; RNA-seq; candidate gene; growth traits; red tilapia.

Figure 1

The comparison of growth traits in red tilapia between the fast-growing and slow-growing groups. Note: FGG: fast-growing group; SGG: slow-growing group; **: p < 0.001; *: p < 0.05; Each grey dot represented an individual.

Figure 2

The density and distribution of the high-quality SNPs genotyped on each chromosome of red tilapia. Notes: red denotes high density, yellow denotes moderate density, and green denotes low density.

Figure 3

Population structure analysis. (A): The phylogenetic tree analysis of genotype in the red tilapia. (B): The PCA scatter plot revealed the spread of red tilapia populations; each point represented a sample, with red and blue points indicating the slow-growing and fast-growing groups, respectively. (C): The K-value for CV error estimation ranges from 2 to 12. (D): The population genetic structure analysis of the red tilapia (K = 10). Each column indicated an individual; The lengths of the different colored segments indicated the proportion of that individual’s genome that is attributable to a particular ancestor.

Figure 4

Manhattan plot (A1–D1) and QQ plot (A2–D2) for GWAS, using the total body length (A), body height (B), body mass (C), and caudal peduncle height (D) of red tilapia. The red solid line indicated the significance threshold line; the blue dashed line denoted the suggestive threshold line.

Figure 5

The fine localization of the candidate region of chr 7. (A) The locus zoom plot revealed fine localization near significant SNPs in chr 7. Red dots denoted SNP signals above the significance threshold, other color dots were below the threshold. (B) Haplotype block counting of QTL areas with significant SNP spikes on chr7. The black depth of the square increased with the value of r2. (C) The distribution of growth traits of different haplotypes; *: p < 0.05.

Figure 6

Transcriptome analyses based on fast-growing and slow-growing groups of red tilapia. (A) Volcano plot of DEGs in the fast-growing and slow-growing groups for muscle in red tilapia. (B) Histogram of DEGs in the female (SF vs. FF) and male (SM vs. FM) comparison group for muscle in red tilapia. (C) Venn maps of DEGs in the female (SF vs. FF) and male (SM vs. FM) comparison group for muscle in red tilapia. (D) The top 30 GO terms of 501 overlapping DEGs for muscle in red tilapia. (E) The top 10 KEGG pathways of 501 overlapping DEGs for muscle in red tilapia. (F) The qRT-PCR validation for 10 DEGs in red tilapia; **: p < 0.001; *: p < 0.05;.

Figure 7

Eleven candidate genes for combined GWAS and RNA-seq analyses. (A) Hierarchical cluster analysis of the candidate genes in SF vs. FF comparison group. (B) Hierarchical cluster analysis of the candidate genes in SM vs. FM comparison group. (C) The top 20 GO terms of the 11 candidate genes. (D) The KEGG pathways enriched with 11 candidate genes.