Multi-environment genome-wide association study reveals genetic loci associated with drought tolerance during wheat germination.

This study used multi-index evaluation and GWAS to identify QTLs and candidate genes for wheat germination drought tolerance, notably a 5D pleiotropic hotspot associated with multiple traits, offering breeding targets. Drought tolerance during wheat germination is critical for uniform seedling establishment in arid and semi-arid regions, ultimately affecting final yield. However, the genetic basis of drought tolerance at this stage remains less understood compared to later growth phases. In this study, a natural population of 389 wheat varieties was evaluated under three independent annual environments with drought stress simulated by 20% PEG-6000. Six germination-related traits-germination rate, shoot length, root length, root number, coleoptile length, and germination potential-were measured and their drought resistance coefficients (DRC) were calculated. To overcome the limitations of single-trait evaluation, principal component analysis combined with a membership function method was used to construct a comprehensive drought tolerance evaluation value (D-value). Rankings based on this D-value showed high consistency across environments, with pairwise correlation coefficients exceeding 0.82 (all P < 0.001). Genome-wide association study (GWAS) using 660 K SNP data identified 270 significantly associated SNPs at a threshold of P <1 × 10-4. Through stringent filtering-requiring detection in at least two environments or association with multiple traits-10 important quantitative trait loci (QTL) were defined, including eight stably expressed across multiple environments and four pleiotropic QTLs. Notably, a stable pleiotropic hotspot (qDT-G5D.1 and qDT-G5D.2) was identified on chromosome 5D, associated with root number, germination potential, and germination rate, highlighting its potential for the coordinated improvement of multiple drought tolerance traits. By integrating published transcriptome data, 12 putative candidate genes showing differential expression between drought-tolerant and sensitive varieties were screened within the important QTL intervals. Haplotype analysis revealed that 10 of these genes exhibited significant phenotypic differences among haplotypes. Overall, this study establishes a multi-index evaluation system for wheat germination-stage drought tolerance and identifies stable QTL, pleiotropic hotspots, and candidate genes with potential breeding value, providing genetic resources for marker-assisted breeding and further functional studies.