Development of KASP Molecular Markers and Candidate Gene Mining for Heat Tolerance-Related Traits in Gossypium hirsutum

<b>Background</b>: High-temperature stress is one of the major abiotic stresses limiting cotton production. Identifying genetic loci and genes for heat tolerance is crucial for breeding heat-tolerant varieties. <b>Methods</b>: Given the complexity of heat tolerance phenotypes in cotton, this study, which focused on resource materials, identified an A/C SNP mutation at position 5486185 on chromosome D06 within the heat tolerance interval through genome-wide association studies (GWAS) of natural <i>Gossypium hirsutum</i> populations. <b>Results</b>: A total of 308 resource materials were identified and evaluated for their heat tolerance phenotypes over two years of field research. Kompetitive allele-specific PCR (KASP) molecular markers were developed on the basis of the D06-5486185 SNP to characterize the heat tolerance phenotypes of these 308 resource materials. Genotyping for heat tolerance-related traits and agronomic traits was also performed. Materials with the C/C haplotype at position D06-5486185 presented increased heat tolerance (higher pollen viability (PV), leaf area (LA), chlorophyll (Chl) and number of bolls on the third fruit branch (FB3) and a lower number of dry buds (DBs) and drop rate (DR)) without negatively impacting key yield traits. This locus is located in the intergenic region of two adjacent bZIP transcription factor genes (<i>GH_D06G0408</i> and <i>GH_D06G0409</i>). Expression analysis revealed that the expression levels of these two genes were significantly greater in heat-tolerant accessions (C/C type) than in sensitive accessions and that their expression levels were significantly correlated with multiple heat-tolerant phenotypes. <b>Conclusions</b>: In summary, this study developed a Kompetitive Allele Specific PCR (KASP) marker associated with heat tolerance in <i>G. hirsutum</i> and identified two key heat tolerance candidate genes. These results provide an efficient marker selection tool and important genetic resources for the molecular breeding of heat-tolerant <i>G. hirsutum</i>, laying an important foundation for further establishing a molecular marker-assisted breeding system for heat tolerance in <i>G. hirsutum</i>.