Soybean (*Glycine max*) is a crucial global crop, meeting significant oilseed and protein demands. To meet projected increases in demand, global soybean yield must double by 2050. Soybeans are short-day plants originating in temperate China, limiting their cultivation to specific latitudes due to photoperiod sensitivity affecting maturity and plant architecture. While long juvenile traits have facilitated expansion to lower latitudes (e.g., Brazil), high-latitude regions (over 40°N) offer substantial potential for increased production but pose challenges. In 2021, China's northeastern provinces accounted for nearly half the country's soybean production, yet global high-latitude production is under 20%, indicating significant untapped potential. High-latitude long-day photoperiods lengthen maturity and cause excessive stem growth, conflicting with short frost-free seasons. Although several flowering time genes have been identified, the genetic basis of plant height adaptation to high latitudes remains poorly understood. Plant height is determined by internode number and length, and is sensitive to light conditions. Long-day photoperiods at high latitudes, coupled with low blue light (LBL) under high-density planting, induce ESE syndrome. This weakens stems, causing lodging and increased susceptibility to pests and diseases, significantly hindering yield improvements through high-density planting. GmCRY1s, blue light receptors, repress LBL-induced ESE by stabilizing STF1/2 bZIP transcription factors. Modulating the GmCRY1-mediated pathway enhances shade tolerance and yield. This study focuses on identifying and characterizing a key gene regulating plant height in soybeans adapted to high latitudes, aiming to provide insights for breeding improved cultivars.

Previous research has explored the genetic basis of flowering time in soybeans adapted to high-latitude environments, identifying several key genes such as the early flowering loci (E1-E4), *TofS*, and homologs of PSEUDO-RESPONSE-REGULATOR3 (*Tof1l* and *Tof12*). These genes have been successfully utilized in breeding programs to develop cultivars suitable for northern China. However, much less is known about the genetic factors that control plant height in these environments, a crucial trait for preventing lodging and maximizing yield in high-density planting systems. Studies have shown that exaggerated stem elongation (ESE) is induced by long-day photoperiods and low blue light conditions, leading to lodging and reduced yields. The blue light receptor GmCRY1s has been identified as a key regulator of this process, acting to repress ESE by stabilizing the bZIP transcription factors STF1 and STF2. Manipulation of the GmCRY1-mediated signaling pathway has shown promise in improving shade tolerance and overall yield in soybean cultivars.

This study employed a multi-faceted approach to identify and characterize genes affecting plant height in soybean. Initially, a genome-wide association study (GWAS) was conducted on a panel of 2214 soybean accessions, utilizing 3.47 million SNPs (minor allele frequency >5%). Concurrently, a transcriptome-wide association study (TWAS) was performed on a subset of 488 accessions to identify genes associated with plant height variations. Both GWAS and TWAS utilized the FarmCPU model to identify significant associations, with Bonferroni correction applied to adjust for multiple testing. A mixed linear model was also used in TWAS to validate findings and account for potential false negatives due to the FarmCPU model's treatment of associated markers as covariates. The study identified *Glyma.13G276700*, located on chromosome 13, as a key candidate gene (*PH13*) through both GWAS and TWAS analyses. The subsequent analyses included: 1. **Haplotype Analysis:** The coding DNA sequence (CDS) of *PH13* was examined in 1254 accessions to identify different haplotypes. PCR and resequencing were used to detect an insertion of a *Tyl*/Copia-like retrotransposon in one of the haplotypes (PH13H3), resulting in a truncated protein. 2. **Functional Validation:** CRISPR/Cas9 technology was used to generate loss-of-function mutants (*ph13*) and overexpression lines in different genetic backgrounds (W82 and TL1). Phenotypic analysis evaluated plant height, internode length, cell length, flowering time, and node number in these lines. Near-isogenic lines (NILs) were created to compare the effects of different PH13 haplotypes on plant height under field conditions. 3. **Protein Interaction Studies:** Yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) assays were conducted to investigate the interaction between PH13 protein and GmCOP1a/b, which are involved in protein degradation. 4. **Gene Expression Analysis:** RT-qPCR was used to analyze the expression patterns of *PH13*, *GmCOP1s*, and *STF1/2* in different genotypes and under different light conditions. 5. **Paralog Gene Analysis:** A paralogous gene, *PHP*, was identified, and CRISPR/Cas9 was used to create *php* and *phd* (double mutant) lines to study their combined effect on plant height. 6. **Field Trials:** Field experiments were conducted at different latitudes (Beijing, Changchun, and Harbin) and planting densities to evaluate the effects of *ph13*, *php*, and *phd* mutations on agronomic traits, including yield and lodging resistance, comparing them to wild type plants and elite cultivars. Maize-soybean intercropping trials were also conducted to evaluate the impact of the mutations under these conditions.

This study's key findings include: 1. **Identification of PH13:** A novel plant height gene (*PH13*) was identified using GWAS and TWAS, significantly associated with plant height variation. 2. **Haplotype Variation and Retrotransposon Insertion:** Three main haplotypes of *PH13* were identified, with haplotype 3 (PH13H3) containing a *Tyl*/Copia-like retrotransposon insertion leading to a truncated PH13 protein lacking a portion of the WD40 domain. 3. **Functional Role of PH13:** CRISPR/Cas9-generated *ph13* mutants displayed significantly reduced plant height due to shorter internodes and slightly earlier flowering. Overexpression lines showed increased plant height, confirming PH13's role in promoting stem elongation. The PH13H3 allele, while shorter, retains some function. 4. **Artificial Selection of PH13H3:** The PH13H3 allele was found to be strongly selected during soybean improvement, particularly at high latitudes, highlighting its role in adapting soybeans to these environments. 5. **Interaction with GmCOP1s and STF1/2:** PH13 interacts with GmCOP1a and GmCOP1b, two E3 ubiquitin ligases. The truncated PH13H3 protein shows reduced interaction with these ligases, leading to an accumulation of STF1/2 proteins, repressors of stem elongation. 6. **Cooperative Function with PHP:** PH13 and its paralog, PHP, act cooperatively in regulating plant height. Double mutants (*phd*) showed the most significant reduction in height and an increased accumulation of STF1/2. 7. **Improved Shade Tolerance and Yield:** The *phd* double mutants showed significantly improved shade tolerance, a trait important for high-density planting, and they also exhibited increased yields under high-density conditions in high latitudes. The *phd* mutants were also highly resistant to lodging. 8. **Adaptation to High Latitudes:** The *phd* mutants exhibited reduced stem elongation, lower lodging rates, earlier maturity, and higher yields in field trials conducted at different latitudes and under intercropping conditions. These improvements enhance the adaptation of mid-latitude cultivars to high-latitude environments for high-density planting and intercropping.

This research successfully identified PH13, a key regulator of plant height in soybean, and elucidated its mechanism of action in relation to shade avoidance. The discovery of the retrotransposon insertion in PH13H3 provides a clear explanation for the shorter stature of many high-latitude cultivars. The observation that PH13H3 has undergone strong artificial selection further underscores its importance in improving soybean adaptation to high-latitude environments. The study's findings on the interaction between PH13, GmCOP1s, and STF1/2 shed light on a conserved mechanism for regulating plant height in response to light conditions. The development of *phd* double mutants, exhibiting enhanced shade tolerance and lodging resistance, provides a valuable tool for breeding high-yielding soybean cultivars suitable for high-density planting and intercropping in high-latitude regions. This approach offers a promising avenue for increasing soybean production in areas currently underutilized, contributing to global food security.

This study identified PH13, a major plant height regulator in soybean, whose function is modulated by a retrotransposon insertion. This insertion, present in the widely used PH13H3 allele, leads to decreased plant height through reduced interaction with GmCOP1s and subsequent STF1/2 accumulation. The creation of *phd* double mutants demonstrates a successful strategy for improving shade tolerance, lodging resistance, and ultimately yield in high-latitude soybean production. Future research could focus on incorporating *phd* mutations into elite cultivars to further improve yield and adaptability in high-density planting and intercropping systems.

The study primarily focused on the PH13 gene and its paralog PHP, and further research is needed to investigate the potential involvement of other genes in the regulation of plant height and shade avoidance. While the *phd* mutants showed improved performance in high-density planting and intercropping systems, the long-term effects of these mutations on other agronomic traits need to be evaluated in diverse environments. Moreover, the study primarily focused on Chinese soybean varieties. Further investigations are needed to validate the findings in other soybean germplasm across diverse genetic backgrounds.