Meta-Analysis of QTL Mapping and GWAS Reveal Candidate Genes for Heat Tolerance in Small Yellow Croaker, Larimichthys polyactis

Abstract

High temperatures present considerable challenges to global fish growth and production, yet the genetic basis of heat tolerance remains underexplored. This study combines quantitative trait locus (QTL) mapping and genome-wide association studies (GWAS) to examine the genetics of heat tolerance in Larimichthys polyactis. As a result, a genetic linkage map was constructed with 3237 bin markers spanning 24 linkage groups and totaling 1900.84 centimorgans, using genotyping-by-sequencing of a full-sib family comprising 120 progeny and their two parents. Based on this genetic linkage map, QTL mapping identified four QTLs associated with heat tolerance, which encompassed 18 single nucleotide polymorphisms and harbored 648 genes within the QTL intervals. The GWAS further disclosed 76 candidate genes related to heat tolerance, 56 of which overlapped with the QTL results. Enrichment analysis indicated that these genes are involved in immune response, development, lipid metabolism, and endocrine regulation. qPCR validation of 14 prioritized genes, which were simultaneously enriched in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways, confirmed significant upregulation of smpd5, polr3d, rab11fip2, and gfpt1, along with downregulation of gpat4 and grk5 after 6 h of heat stress. These findings demonstrate their responsiveness to elevated high temperatures. This meta-analysis of QTL mapping and GWAS has successfully identified functional genes related to heat tolerance, enhancing understanding of the genetic architecture underlying this critical trait in L. polyactis. It also provides a molecular breeding tool to improve genetic traits associated with heat tolerance in cultured L. polyactis.

Keywords: GWAS; Larimichthys polyactis; QTL; genetic linkage map; heat tolerance.

Figure 1

Temperature changes at each time point during the heat stress experiment. Temperature increased from 15 °C to 32 °C by 1 °C every 4 h. Subsequently, if no mortality occurred for four consecutive hours at this temperature, an additional 1 °C increase was implemented, and this process continued until all fish had succumbed.

Figure 2

Survival curve of Larimichthys polyactis during heat stress. Cumulative mortality reached nearly 50% after 18 h of heat stress. The heat stress experiment lasted for 36 h, with the first fish succumbing to stress after only 190 min. The average survival duration was calculated to be 19.72 h, with peak mortality occurring between the 12th and 30th hours of the heat stress exposure.

Figure 3

Length and marker distribution of 24 linkage groups in the sex-averaged bin map. The ordinate indicates the genetic distance.

Figure 4

QTL analysis for heat tolerance traits in the full-sib family using SNP data of L. polyactis. (A) Survival duration under high-temperature stress. Three major genome-wide significant QTL intervals were identified with LOD values of 5.85, 6.01, and 5.00, with percentages of explained variance of 10.13%, 13.08%, and 6.98%, respectively. (B) Survival status under high-temperature stress. One major genome-wide significant QTL interval was identified with a LOD value of 5.02 and a percentage of explained variance of 17.52%. The horizontal axis indicates linkage group numbers. The vertical axis represents LOD values. The red line indicates the determined LOD (=4.95).

Figure 5

GWAS analysis of resistance to high temperature in L. polyactis: (A) Manhattan plot for GWAS of survival duration; (B) Q-Q plot of survival duration; (C) Manhattan plot for GWAS of survival status; (D) Q-Q plot of survival status. The red and blue dashed lines indicate a genome-level suggestive significance threshold p value of 7.77 × 10⁻⁹. The red lines in B and D represent the expected distribution of −log₁₀ (p) values under the null hypothesis, while deviations from this line (blue) reflect the p-value from which the regression of the phenotype on the SNP deviates from what would be expected by chance.

Figure 6

GO enrichment analysis of overlap genes identified by QTL and GWAS for heat tolerance trait in L. polyactis. Genes were annotated in three categories: biological process, cellular component, and molecular function. Top 20 terms of GO enrichment analysis. The Y-axis represents the name of each GO term, and the X-axis represents the number of genes in each term.

Figure 7

KEGG enrichment analysis of gene overlap identified by QTL and GWAS for heat tolerance trait in L. polyactis. All 21 enriched pathways are shown, and the most enriched pathways include the Adherens junction, melanogenesis, Wnt signaling pathway, insulin signaling pathway, Endocytosis, and metabolic pathways. Among these pathways, the two pathways of Adherens junction and melanogenesis were significantly enriched. The pathways were ranked according to their statistical significance.

Figure 8

Expression of candidate genes subjected to heat stress. The fish in the test group were subjected to a temperature of 32 °C for 6 h, and the fish in the control group were cultured in the normal water temperature of 20 °C. After heat stress for 6 h, six candidate genes were significantly differently expressed; smpd5, polr3d, rab11fip2, and gfpt1 were significantly upregulated, while gpat4 and grk5 were significantly downregulated. * indicates a significant difference (p ≤ 0.05); ** indicates a highly significant difference (p ≤ 0.01).