Cotton Genomics and Genetics 2025, Vol.16, No.6, 290-299 http://cropscipublisher.com/index.php/cgg 294 5 Case Study: Spatial Gene Expression inGossypium hirsutum 5.1 Sampling design: dissecting elongation zones and applying spatial barcoding methods To truly understand the "elongation map" of cotton fibers on land, researchers often have to start from the most minute details. Recent studies have utilized spatial barcode technology in combination with high-resolution sampling to conduct analyses on different parts of fibers. Not all fibers are the same. The tip, middle and base each have different active genes. By dissecting these regions at specific developmental stages, researchers were able to map out the local transcriptome distribution. Furthermore, genetic materials such as chromosome fragment replacement lines (CSSL) and gene infiltration populations have also played significant roles in the research (Qi et al., 2024). They can reveal the differences under different genetic backgrounds, making it possible to compare gene expression in various regions. This design not only showcases spatial differences but also helps clarify the genetic basis behind fiber elongation. 5.2 Findings: expression gradients of GhEXPA1, GhRDL1, andGhMYB25-like along fiber tips Research has found that some key genes controlling fiber elongation, such as GhEXPA1 (expansion protein), GhRDL1 (cell wall modifier related), and GhMYB25-like protein (MYB transcription factor), are not evenly distributed but form clear expression gradients along the fiber axis. Their activity is highest in the tip and early elongation regions. This "local overheating" pattern suggests that they are closely related to the relaxation of the cell wall and the rapid expansion of the cell. When upstream regulatory factors (such as GhHDZ76) are disrupted, the expression levels of these genes decrease significantly and the fibers become shorter accordingly (Figure 2) (Wu et al., 2024). This kind of phenomenon makes people realize that elongation is not the task of a single gene, but a process maintained by multiple layers of regulation. Further co-expression network analysis revealed that GhEXPA1, GhRDL1 and GhMYB25-like proteins would also form interactive networks with other factors (such as GhHOX3), jointly participating in the coordination of fiber growth. Figure 2 The fiber length measurements of GhHDZ76 knockout lines in T3 andT4 generations. (a) Comparison of mature fiber length between GhHDZ76 knockout lines and WT. Bar =1 cm. (b) Measurement of fiber length in GhHDZ76 knockout lines and WT in T3 generation. (c) Measurement of fiber length in GhHDZ76 knockout lines and WT in T4 generation. Data represent the mean ± SD. WT: WT; CR2, CR3, CR11: GhHDZ76knockout line by CRISPR/Cas9 (Adopted from Wu et al., 2024)
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