Identification and functional verification of salt tolerance hub genes in Salix matsudana based on QTL and transcriptome analysis

Salt tolerance hub genes play a vital role in the response to salt stress. Exploring the salt tolerance hub genes of Salix matsudana (Koidz.), an allotetraploid willow with certain salt tolerance, will be conducive to further research on the molecular mechanism of salt tolerance in trees. Here, 114 F1 populations of crossing salt-tolerant willow "9901" and salt-sensitive willow "Yanjiang" were used to perform the QTL of salt-tolerant traits and transcriptome analysis. 46 QTLs related to salt tolerance traits were obtained, of which 9 QTLs can regulate multiple traits. A total of 23795 differentially expressed genes (DEGs) were obtained by RNA-seq in different salt treatment periods (0 h/4 h/8 h/12 h). Weighted gene co-expression network analysis (WGCNA) and timeordered gene co-expression network (TO-GCN) analysis on these DEGs revealed candidate hub genes in regulating salt tolerance. Combining the QTL and transcriptome results, the number of salt tolerance hub genes was cut down and several hub genes were selected for further functional verification. qRT-PCR, VIGS and saltsensitive yeast complementation experiment showed that the silencing of one of salt tolerance hub gene, SmERF B1-2 significantly reduced salt tolerance in S. matsudana. And the expression of SmERF B1-2 in saltsensitive yeast strain AXT3K rescued its salt sensitive phenotype. In this study, the combination of QTL and transcriptome was shown to be effective in screening the salt-tolerant hub gene of S. matsudana. This salt-tolerant hub gene identified in this way provides new insight into the molecular mechanisms of salt tolerance in trees and a theoretical basis for molecular breeding of salt-tolerant trees.