Integrated Bioinformatic, Molecular, Phenotypic, and Anatomical Analysis for Laser-Induced Mutation in Sugarcane (Saccharum spp.)

Abstract Background Although laser irradiation is an emerging physical mutagen for crop improvement, its effects on sugarcane (Saccharum hybrid) at the phenotypic, anatomical, molecular, and bioinformatic levels have not been comprehensively integrated. Methods Sugarcane buds (variety G.95 − 21) were pre-illuminated using He-Ne laser (630 nm) with six power levels (5–100 mW) and five exposure times (0.5–10 min), generating 20 treatments. Forty-eight clones were evaluated for five agronomic traits (stalk height, stalk diameter, stalk number/stool, stalk weight, Brix) across two seasons (2020/2021–2021/2022). Seven selected clones underwent SCoT marker analysis (7 primers, 83 fragments). Statistical analyses included principal component analysis (PCA), broad-sense heritability (H²), genetic advance (GA), two-way ANOVA with Tukey's HSD, marker-trait association (MTA), quantitative anatomy (9 parameters), and comprehensive bioinformatic annotation (GO, KEGG, InDel characterization, protein modeling). Results Treatment T1 (5 mW/0.5 min) exhibited superior performance, with increases in stalk height (272.9 cm, + 53% vs. control), stalk weight (1.43 kg, + 113%), and Brix (23.5%, + 88%). Broad-sense heritability was high for both stalk diameter (H² = 0.79 ± 0.09) and Brix (H² = 0.74 ± 0.11). PCA explained 78.6% of the total variance (PC1 = 51.2%, PC2 = 27.4%), clearly separating the elite mutants from the control. SCoT markers revealed 68 polymorphic fragments (81.9% polymorphism). A 2,173 bp deletion in the dehydrin gene (DHN1) was identified exclusively in all laser-treated clones (SCoT21-InDel marker), and was absent in the control. This deletion likely occurred via microhomology-mediated end joining (5 bp direct repeat AGCTT). The predicted SoDHN1 protein (237 aa, 25.8 kDa) is a YSK₂-type intrinsically disordered dehydrin (85% disordered residues) with three K-segments, one S-segment, and one Y-segment. Marker-trait association analysis revealed that SCoT-21 (dehydrin) explained 58% of stalk weight variance (R² = 0.58, β = -0.76, P = 0.001). Quantitative anatomy revealed T1 increased lamina thickness (+ 8.33%), metaxylem vessel diameter (+ 11.89%), and bulliform cell thickness (+ 20.67%), with strong correlations to agronomic traits (r = 0.71–0.94). Phylogenetic analysis placed SoDHN1 within Poaceae YSK₂-type clade (98% bootstrap with sorghum). The SCoT21-InDel marker was validated using specifically designed primers (287 bp intact / 124 bp deleted) and showed a low natural frequency (6.7% in the germplasm). Conclusion Low-power He-Ne laser irradiation (5 mW/0.5 min) is an effective mutagen for generating elite sugarcane mutants with enhanced yield and quality. The SCoT21-InDel marker provides a simple, PCR-based tool for marker-assisted selection. Deletion of DHN1 eliminates the fitness cost associated with constitutive stress-protein expression under well-watered conditions, reallocating resources to growth and sugar accumulation. However, caution is warranted for rain-fed environments, where dehydrin-mediated drought tolerance may be essential. Further multi-location trials of T1 and T15 are recommended.