## 3 Results
### DEGs Responding to Low-Temperature Stress in Sandrice  
A total of 18 samples generated 127.18 G of raw data, resulting in 121.60 G of clean data after the removal of the low-quality reads. The genome mapping rates for each sample ranged from 96.4% to 98.57%, while the unique mapping rates for clean reads varied between 90.53% and 95.28%. The distribution of Q30 bases ranged from 92.63% to 93.17%, and the average GC content was 43.83% (Table S4). In the PCA plot, the 18 samples were divided into control groups and low-temperature treatment groups alongside PC1, while the _Central_ lineage (DL and DK), and the _Eastern_ lineage (AEX) were alongside PC2 (Figure 3A). These findings indicate that the low-temperature treatment significantly  
Figure 2: The flavonoid-targeting metabolites identified in the above-ground tissue of sandrice. (**A**) The hierarchical cluster heatmap analysis of metabolites in six groups; (**B–F**) 5 flavonoid metabolites with high content; (**G**) Venn diagram of metabolite significance analysis in three ecotypes. \({}^{\star}\), VIP \(\geq\) 1 and \(p\)\(\leq\) 0.05; \({}^{\star\star}\), VIP \(\geq\) 1 and \(p\)\(\leq\) 0.01.  
affected all three ecotypes of sandrice at the transcriptome level. Furthermore, it was noticed that the transcriptomic samples of DK and DL clustered together under both the control and low-temperature conditions, suggesting that these two ecotypes may exhibit similar transcriptional responses to cold stress. In the DL, AEX, and DK groups, 2097, 2475, and 2329 up-regulated DEGs were identified, respectively. Additionally, 2823, 3150, and 2771 DEGs were found to be downregulated in the DL, AEX, and DK groups, respectively (Figure 3B). In all three ecotypes, the number of down-regulated DEGs exceeded that of the up-regulated DEGs. The Venn diagram illustrates that among the 2676 genes shared by the three ecotypes (Figure 3C), 1125 were up-regulated (Figure 3D), and 1478 were down-regulated (Figure 3E). Additionally, there were 1039, 1334, and 794 unique DEGs identified in DL, AEX, and DK, respectively (Figure 3C). The KEGG enrichment bubble plot (Figure 4A) for the top 20 metabolic pathways of the common up-regulated 1125 DEGs showed that the transcription of flavonoid biosynthesis was highly sensitive to low-temperature stress in sandrice. This result was consistent with the KEGG enrichment analysis of the up-regulated DEGs in the different comparison groups (Figure S2). In addition, the KEGG enrichment bubble plot (Figure 4B) of the common down-regulated 1478 DEGs included pathways, such as starch and sucrose metabolism (ko00500), glyoxylate and dicarboxylate metabolism (ko00630), pentose and glucuronate interconversions (ko00040), photosynthesis (ko00195), and photosynthesis-antenna proteins (ko00196). These findings suggested that sandrice responds to low-temperature stress by inhibiting most of the metabolic processes, such as photosynthesis, and sugar metabolism. Based on the sequence alignments of candidate genes, a total of 22 kinds of genes putatively involved in flavonoid synthesis were identified in the transcriptome of sandrice (Figure 5A). These included fifteen _COMTs_, ten _FNSs_, ten _4 CLs_, eight _CHSs_, five _CCOAOMTS_, and other low copy genes, accounting for a total of eighty-five genes (Figure 5B). Also identified as potentially regulating flavonoid biosynthesis were 85 _bHLHs_ (Figure 5C) and 78 _MYBs_ (Figure 5D) TFs. Among these, there were 19 common DEGs related to flavonoid  
Figure 3: Overall description of DEGs in sandrice in response to low-temperature stress. (**A**) The PCA plot; (**B**) the bar plot of DEGs; (**C**) Venn diagram of all DEGs in the three ecotypes; (**D**) Venn diagram of up-regulated DEGs in the three ecotypes; (**E**) Venn diagram of down-regulated DEGs in the three ecotypes. synthesis (Figure 5E), 20 common DEGs of _bHLHs_ (Figure 5F), and 13 common DEGs of _MYBs_ (Figure 5G).