Introgressive gene discovery of salt tolerance in Gossypium chromosome segment substitution lines with combined QTL mapping and RNA-seq

• QTLmapping for salt tolerance on 294 cotton CSSLs from CCRI36 × Hai1. • Crucial DEGs and signaling pathways identified by RNA-seq and physiological indicators. • Introgressive gene prediction by combining QTL intervals with WGCNAresults. • Verification of candidate genes related to cotton salt stress by VIGS experiment. • Deepening the understanding of cotton responding molecular to slat stress Soil salinization significantly limis crop yield and lowers produce quality. Wild plant species have developed various strategies to cope with soil salinity. However, the molecular responding mechanism to salt stress of domesticated crops is still open to discussion. Combining analysis of quantitative trait locus (QTL) mapping and transcriptome sequencing is an effective approach to identify candidate genes and study their regulation mechanisms of plant salt tolerance. In this study, 294 BC 5 F 3:5 chromosome segment substitution lines (CSSLs), which were constructed by introgressing chromosome segments of Gossypium barbadense Hai1 into G. hirsutum CCRI36 background, were utilized to evaluate relative germination rate (RGR) of seeds and relative survival rate (RSR) of seedlings under NaCl stress. Two salt-tolerant (ST) and salt-sensitive (SS) lines were screened from the CSSLs. With the basis of the previous SSR-based genotyping data, a total of 14 QTLs of RGR and RSR relating to salt tolerance were detected. Meanwhile, RNA-seq and physiological and biochemical indexes of ST and SS were detected. With the combination of weighted gene co-expression network analysis (WGCNA) and QTL intervals, we identified nine hub genes, four of which have nonsynonymous mutations in the protein sequences between G. hirsutum and G. barbadense . The unqiue common candidate gene located between qRGR-12-1 and qRSR-12-1 , namely GH_A12G0809 , were chosen to conduct functional validation vis VIGS, which confirmed its negative regulatory contribution to salt tolerance in cotton. Our results establish a foundation for elucidating the molecular mechanisms governing cotton's defense against salt stress.