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<p>Drought-responsive transcriptional programmes in wheat are strongly shaped by genetic background, complicating the prioritisation of reproducible candidate genes for crop improvement. In this study, publicly available RNA-seq data from fourteen genetically diverse wheat accessions were reanalysed using a genotype-aware paired modelling framework to identify drought-responsive signals that remain stable across genetic backgrounds. In the original discovery experiment, four biological plants per accession and treatment were pooled before sequencing; therefore, the present analysis was designed to prioritise cross-accession stability-supported candidates rather than to estimate replicate-level differential responses within individual accessions. A strict conserved drought-responsive core of 7,228 genes was identified, including 2,897 genes exhibiting large-effect, directionally consistent responses across all fourteen accessions. Intersection of this strict core with RefSeq v2.1-mapped genes annotated with GO:0003700 (DNA-binding transcription factor activity) identified 286 candidate transcription factor genes. To avoid prioritisation of partially stable candidates solely because of large effect sizes, the previous score-based ranking was replaced by a transparent stability-tiered framework, with primary candidates restricted to genes showing complete stability across all fourteen accessions. Independent transcriptomic reproducibility was assessed using GSE136683 with a blocked DESeq2 model accounting for plant-type background. Of the 286 candidate transcription factor genes, 166 met the independent reproduction criteria, and 164 showed concordant regulatory direction between discovery and independent datasets (98.80%). Effect sizes were strongly correlated across datasets (Pearson <i>r</i> = 0.924877; Spearman ρ = 0.867831), and a sensitivity analysis restricted to WT and BSMV00 backgrounds yielded similarly high agreement. GO Biological Process enrichment using the 76,427 genes tested in the discovery analysis as the primary background identified nine significant terms, including response to gibberellin, response to sucrose, adventitious root development, plant organ formation and response to lipid, in addition to expected transcription-related terms. No significant enrichment was detected when the comparison was restricted to the initial candidate transcription factor background, indicating that these functions represent transcriptome-relative features of the reproduced candidate set rather than selective enrichment within the transcription factor subset. Overall, this study provides a genotype-aware and stability-tiered transcriptome reanalysis framework for prioritising reproducible drought-responsive candidate transcription factor genes in wheat while explicitly acknowledging the limitations associated with pooled sequencing and cross-dataset comparison.</p>

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Genotype-aware transcriptome reanalysis prioritises conserved drought-responsive candidate transcription factor genes across diverse wheat accessions

  • 期刊论文

  • 发表日期：

    2026年7月1日

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  • Musa Kar

摘要

Drought-responsive transcriptional programmes in wheat are strongly shaped by genetic background, complicating the prioritisation of reproducible candidate genes for crop improvement. In this study, publicly available RNA-seq data from fourteen genetically diverse wheat accessions were reanalysed using a genotype-aware paired modelling framework to identify drought-responsive signals that remain stable across genetic backgrounds. In the original discovery experiment, four biological plants per accession and treatment were pooled before sequencing; therefore, the present analysis was designed to prioritise cross-accession stability-supported candidates rather than to estimate replicate-level differential responses within individual accessions. A strict conserved drought-responsive core of 7,228 genes was identified, including 2,897 genes exhibiting large-effect, directionally consistent responses across all fourteen accessions. Intersection of this strict core with RefSeq v2.1-mapped genes annotated with GO:0003700 (DNA-binding transcription factor activity) identified 286 candidate transcription factor genes. To avoid prioritisation of partially stable candidates solely because of large effect sizes, the previous score-based ranking was replaced by a transparent stability-tiered framework, with primary candidates restricted to genes showing complete stability across all fourteen accessions. Independent transcriptomic reproducibility was assessed using GSE136683 with a blocked DESeq2 model accounting for plant-type background. Of the 286 candidate transcription factor genes, 166 met the independent reproduction criteria, and 164 showed concordant regulatory direction between discovery and independent datasets (98.80%). Effect sizes were strongly correlated across datasets (Pearson r = 0.924877; Spearman ρ = 0.867831), and a sensitivity analysis restricted to WT and BSMV00 backgrounds yielded similarly high agreement. GO Biological Process enrichment using the 76,427 genes tested in the discovery analysis as the primary background identified nine significant terms, including response to gibberellin, response to sucrose, adventitious root development, plant organ formation and response to lipid, in addition to expected transcription-related terms. No significant enrichment was detected when the comparison was restricted to the initial candidate transcription factor background, indicating that these functions represent transcriptome-relative features of the reproduced candidate set rather than selective enrichment within the transcription factor subset. Overall, this study provides a genotype-aware and stability-tiered transcriptome reanalysis framework for prioritising reproducible drought-responsive candidate transcription factor genes in wheat while explicitly acknowledging the limitations associated with pooled sequencing and cross-dataset comparison.