Rapid and high-throughput identification of thermal-responsive genes based on cDNA-library in upland cotton

Severe high temperature (HT) climate significantly impacts cotton quality and yield. Consequently, it is essential to mine thermal-responsive genes and explore the underlying mechanisms of HT response in cotton. In this study, we employed a high-throughput cDNA-library method in conjunction with the ALRS system to screen thermotolerant genes in Upland cotton. As a result, a total of 16,120, 13,216 and 172 effective survival genes were filtered after HT stress exposure (42 °C, 220 rpm) for 48 h, 60 h and 72 h, respectively. Functional annotation and enrichment analysis revealed that 170 common genes were involved in regulatory processes associated with HT stress, and the relevant transcriptome data indicated that the majority of these genes responded to temperature fluctuations. Twenty-one genes were randomly selected for verification, and it was found that these genes could enhance yeast resistance to HT stress. Additionally, we selected mutants of homologous Arabidopsis genes for four candidate genes to validate plant thermotolerance during flowering; the thermotolerances of SALK_201915 and SALK_120540.1 were significantly worse. The results demonstrate that numerous candidate genes identified from the cDNA-library contribute to the highly complex molecular network that governs the response and resistance to HT stress in Upland cotton. The high-throughput heat-screening method utilized in this study was optimized for mining thermotolerant genes including improvement in yeast library construction, screening system, gradient reverse pressure, and sequencing library construction. We hope that this new method can be applied in future studies on stress in cotton and other species.