## 5 Conclusions  
To analyze the differences of genes related to glucosinolate biosynthesis in different plant parts of broccoli, we selected extreme individuals from an F\({}_{2}\) population of broccoli and mixed them to form low and high glucoraphanin content pools of leaf, stalk and floret, respectively. The combination of transcriptome and metabolome analysis revealed that some genes such as _B05g113720, Bo2g161100_ and _Bo7g09800, Bo4g018590, Bo5g021810_, and _Bo2g011730_ showed different expression trends between low and high glucoraphanin content pools, which increased the accumulation of glucoraphanin. These genes have different expression levels in the three plant parts. Notably, the accumulation of glucoraphanin upregulated the expression of plant hormone signal transduction-related genes, such as _TIFY_, _JAR1_, _IAA_, _GH3_ and _SAU_. In addition, the accumulation of glucoraphanin also increased the levels of flavonoid metabolites. Thus, our study deepens the understanding of glucosinolate biosynthesis at the molecular level, and also provides evidence for the crosstalk between the glucosinolates and flavonoids biosynthesis pathways. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13105837/s1](https://www.mdpi.com/article/10.3390/app13105837/s1)]([https://www.mdpi.com/article/10.3390/app13105837/s1](https://www.mdpi.com/article/10.3390/app13105837/s1)), Figure S1. The glucosinolates content of parents and their F2 population. (A) Comparison of glucosinolates content between B0401 and MS84003 at commercial stage. RAA, (RS)-4-(methylsulfonyl)butyl GSL (Glucoraphanin), GBC, Indol-3-methyl GSL (Glucobrassicin), ERU, 4-(Methylsulfonyl)butyl GSL (Glucorucrinch), ALY, (RS)-5-(Methylsulfonyl)pentyl GSL (Glucoolysissin), 4 ME, 4-Methocynidol-3-ylmethyl) GSL (4-methoxyglucobrassicin), NEO, 1-Methocynidol-3-ylmethyl GSL (Neoglocubrassicin). *** \(p\) < 0.01. Frequency histogram of RAA in leaf (B), stalk (C) at vegetative growth stage and floret (D) at commercial stage in F2 plants. FW, fresh weight; DW, dry weight. Figure S2. The number of transcripts annotated by KEGG, nonredundant (NR), Swiss-Prot, EuKaryotic Orthologous Groups (KOG), GO and Pfam databases. LTL, STL and FTL represent low RAA content pools in leaf, stalk and floret, respectively. LTH, STH and FTH represent high RAA content pools in leaf, stalk and floret, respectively. Figure S3. GO classification analysis of differential expression genes. (A) LTL vs. LTH, (B) STL vs. STH, (C)FTL vs. FTH. LTL, STL and FTL represent low RAA content pools in leaf, stalk and floret, respectively. LTH, STH and FTH represent high RAA content pools in leaf, stalk and floret, respectively. Figure S4. Phylogenetic analysis of DEGs involved in glucosinolate biosynthesis. Phylogenetic tree of _Bo5g113720_ (A), _Bo2g011730_ (B), _Bo2g161100_ and _Bo7g098000_ (C), _Bo4g018590_ (D), _Bo5g02181_ (E) and _Bo6g083740_ (F). Mega 7.0 was used to build the phylogenetic tree by the neighbor-joining method ([[https://www.megasoftware.net/](https://www.megasoftware.net/)]([https://www.megasoftware.net/](https://www.megasoftware.net/)), accessed on 25 August 2022). The scale bar indicates a branch length of 0.1. Red and blue boxes represent the gene itself and genes with functional annotations in _Brassica rap_ and _Arabidopsis thaliana_. Figure S5. PCA of differentially accumulated metabolites in LML vs. LMH (A), SML vs. SMH (B), and FML vs. FMH (C). LML, SML and FML represent low RAA content pools in leaf, stalk and floret, respectively. LMH, SMH and FMH represent high RAA content pools in leaf, stalk and floret, respectively. Figure S6. Coregulation among DAMs in all comparison groups. LML, SML and FML represent low RAA content pools in leaf, stalk and floret, respectively. LMH, SMH and FMH represent high RAA content pools in leaf, stalk and floret, respectively. Figure S7.