Table_2_Transcriptome analysis of the 2,4-dichlorophenoxyacetic acid (2,4-D)-tolerant cotton chromosome substitution line CS-B15sh and its susceptible parental lines G. hirsutum L. cv. Texas Marker-1 and G. barbadense L. cv. Pima 379.XLSX

The cotton chromosome substitution line, CS-B15sh, exhibits 41% lower injury from 2,4-D when applied at the field recommended rate of 1.12 kg ae ha−1 (1×) than does Texas Marker-1 (TM-1). CS-B15sh was developed in the genetic background of Gossypium hirsutum L. cv TM-1 and has chromosome introgression on the short arm of chromosome 15 from Gossypium barbadense L. cv. Pima 379. In a previous experiment, we observed reduced translocation of [14C]2,4-D outside the treated leaf tissue in CS-B15sh, which contrasted with an increased translocation of the herbicide in the tissues above and below the treated leaf in TM-1. Our results indicate a potential 2,4-D tolerance mechanism in CS-B15sh involving altered movement of 2,4-D. Here, we used RNA sequencing (RNA-seq) to determine the differential expression of genes between 2,4-D-challenged and control plants of the tolerant (CS-B15sh) and susceptible lines (TM-1 and Pima 379). Several components of the 2,4-D/auxin-response pathway—including ubiquitin E3 ligase, PB1|AUX/IAA, ARF transcription factors, and F-box proteins of the SCFTIR1/AFB complex—were upregulated with at least threefold higher expression in TM-1 compared with CS-B15sh, while both Pima 379 and TM-1 showed the same fold change expression for PB1|AUX/IAA mRNA. Some genes associated with herbicide metabolism, including flavin monooxygenase (Gohir.A01G174100) and FAD-linked oxidase (Gohir.D06G002600), exhibited at least a twofold increase in CS-B15sh than in TM-1 (the gene was not expressed in Pima 379), suggesting a potential relationship between the gene’s expression and 2,4-D tolerance. It is interesting to note that glutathione S-transferase was differentially expressed in both CS-B15sh and Pima 379 but not in TM-1, while cytochrome P450 and other genes involved in the oxidation–reduction process were significantly expressed only in CS-B15sh in response to 2,4-D. Gene set enrichment analysis on the union DEGs of the three cotton genotypes revealed the depletion of transcripts involved in photosynthesis and enrichment of transcripts involved in ABA response and signaling.

棉染色体替代系CS-B15sh在田间推荐用量1.12千克活性成分公顷−1（1倍）施用2,4-D时，相较于德克萨斯标记-1（TM-1），损伤降低了41%。CS-B15sh是在Gossypium hirsutum L. cv TM-1的遗传背景下开发的，并在染色体15的短臂上从Gossypium barbadense L. cv. Pima 379引入了染色体重组。在前一实验中，我们观察到CS-B15sh中[14C]2,4-D在处理叶片组织外的转移减少，这与TM-1中处理叶片上下组织的除草剂转移增加形成鲜明对比。我们的结果表明，CS-B15sh中存在一种潜在的2,4-D耐受机制，涉及2,4-D转移的改变化。在此，我们采用RNA测序（RNA-seq）技术，以确定耐受性（CS-B15sh）和易感性品种（TM-1和Pima 379）的2,4-D挑战植物与对照植物之间基因表达的差异。2,4-D/生长素响应途径的多个组分，包括泛素E3连接酶、PB1|AUX/IAA、ARF转录因子以及SCFTIR1/AFB复合物的F-box蛋白，在TM-1中的表达至少上调了三倍，比CS-B15sh高，而Pima 379和TM-1的PB1|AUX/IAA mRNA表达变化倍数相同。一些与除草剂代谢相关的基因，包括黄素单加氧酶（Gohir.A01G174100）和FAD连接的氧化酶（Gohir.D06G002600），在CS-B15sh中的表达至少增加了两倍，而在TM-1中则没有表达（该基因在Pima 379中未表达），这表明基因表达与2,4-D耐受性之间可能存在潜在关系。值得注意的是，谷胱甘肽S-转移酶在CS-B15sh和Pima 379中表达存在差异，但在TM-1中则没有，而细胞色素P450和其他参与氧化还原过程的基因仅在CS-B15sh中响应2,4-D显著表达。对三种棉花基因型的联合差异表达基因（DEGs）集进行富集分析，揭示了参与光合作用的转录本减少以及参与ABA响应和信号传导的转录本富集。