The stay-green phenotype is a valuable trait positively correlating with both forage quality and biomass production in perennial grasses. To date, with the exception of Festuca pratensis , no naturally occurring stay-green species have been reported among forage grasses. Leymus chinensis is an important forage species that presents two phenotypes: gray–green (GG) leaves and yellow–green (YG) leaves. In this study, we discovered that GG L. chinensis exhibited functional stay-green characteristics: both chlorophyll and photosynthesis were maintained at higher levels after 6 days of dark treatment, which is significantly different from that of the YG ecotype. Along with higher level of chlorophyll, GG also maintained high concentrations protein nutrient in darkness. In the case of YG, darkness significantly increased the expressions of NYC1 and NOL , which are responsible for initiating the chlorophyll degradation. However, the dark treatment did not alter the expression of NOL in the GG leaves, and the induction of NYC1 expression by darkness in GG was also significantly weaker than that observed in YG. This discrepancy could account for the slower rate of chlorophyll degradation observed in GG under dark conditions than in YG.

This research investigates the flavonoid biosynthesis pathways of two ecotypes of Leymus chinensis, distinguished by their gray-green (GG) and yellow-green (YG) leaf colors, to uncover the molecular bases of their adaptability to different environmental conditions. By integrating comprehensive transcriptomic and metabolomic analyses, we identified 338 metabolites, with 161 showing differential expression—124 upregulated and 37 downregulated. The transcriptomic data revealed substantial variation, with 50,065 genes differentially expressed between the ecotypes, suggesting complex genetic regulation of the flavonoid biosynthesis pathways involving 20 enzyme-coding genes. KEGG pathway enrichment analysis further highlighted the involvement of 26 genes in the synthesis of four distinct types of flavonoid metabolites, indicating the sophisticated modulation of these pathways. Our results demonstrate that the GG and YG ecotypes of Leymus chinensis exhibit distinct flavonoid profiles and gene expression patterns, with the GG ecotype showing a higher accumulation of quercetin and kaempferol (increased by 25% and 33%, respectively, compared to YG), suggesting enhanced antioxidant capacity. Conversely, the YG ecotype displayed a broader spectrum of flavonoid metabolites, possibly indicating an adaptive strategy favoring diverse ecological interactions. Our results show that the GG and YG ecotypes of Leymus chinensis exhibit distinct flavonoid profiles and gene expression patterns, suggesting divergent adaptive strategies to environmental stress. This study highlights the crucial role of flavonoid metabolites in plant adaptation strategies, enhancing our understanding of plant resilience and adaptability. The distinct metabolic profiles observed suggest that the GG ecotype may be better equipped to handle oxidative stress, while the YG ecotype could be predisposed to broader ecological interactions. This emphasizes the value of applying machine learning in predicting plant adaptability, providing a new perspective for the future exploration of how plants adapt to environmental challenges. Meanwhile, the information gleaned from this nuanced study offers a foundation for future investigations into the genetic and environmental factors involved in plant adaptation.

Intraorganismal genetic heterogeneity (IGH) exists when an individual organism harbors more than one genotype among its cells. In general, intercellular DNA diversity occurs at a very low frequency and cannot be directly detected by DNA sequencing from bulk tissue. In this study, based on Sanger and high-throughput sequencing, different species, different organs, different DNA segments and a single cell were employed to characterize nucleotide mutations in Leymus chinensis. The results demonstrated that 1) the nuclear DNA showed excessive genetic heterogeneity among cells of an individual leaf or seed but the chloroplast genes remained consistent; 2) a high density of SNPs was found in the variants of the unique DNA sequence, and the similar SNP profile shared between the leaf and seed suggested that nucleotide mutation followed a certain rule and was not random; and 3) the mutation rate decreased from the genomic DNA sequence to the corresponding protein sequence. Our results suggested that Leymus chinensis seemed to consist of a collection of cells with different genetic backgrounds.

Intraorganismal genetic heterogeneity (IGH) arises from the accumulation of somatic mutations during plant growth. Although leaves of Leymus chinensis exhibit pronounced IGH, its molecular basis remains unclear. Under strictly controlled growth conditions, this study compared a wild population (LC-W) with the cultivated cultivar Zhongke No. 2 (LC-ZK2) to search for DNA repair–related clues associated with leaf nuclear DNA diversity. Genomic DNA amplification and Sanger sequencing of three nuclear loci ( MCM7 , PsaE , and PsaL ) showed that, compared with LC-W, LC-ZK2 exhibited fewer polymorphic sites and lower haplotype diversity, indicating a more restricted leaf-scale sequence heterogeneity. De novo leaf transcriptome analysis identified 3,833 differentially expressed genes (DEGs; |log ₂ FC| ≥ 1, FDR < 0.05; log ₂ FC = log ₂ [LC-W/LC-ZK2]). GO and KEGG analyses indicated that DEGs were significantly enriched in DNA damage response and DNA repair pathways, with particularly prominent enrichment of base excision repair (BER) and homologous recombination (HR). The BER scaffold gene XRCC1 plays an important role in these pathways and was significantly upregulated in LC-ZK2 (~2.6-fold), suggesting transcriptional differences in repair-related genes between the two materials. Further Sanger sequencing of the XRCC1 BRCT domain indicated that LC-ZK2 possessed a more concentrated haplotype spectrum and exhibited distinct amino acid substitution combinations, providing candidate sites for subsequent functional validation. Overall, this study links differences in nuclear DNA diversity with repair-associated transcriptomic signatures and provides an interpretive framework for understanding leaf-scale heterogeneity divergence in L. chinensis .