*4.2. Analysis of Physicochemical Parameters of IabHLH Proteins*

The physicochemical characteristics of the IabHLH proteins were analyzed using the ExPASy program (http://web.expasy.org/protparam/. accessed on 22 November 2022), including the number of amino acids, molecular weight (kDa), theoretical isoelectric point (pI), instability index (Ii), aliphatic index (Ai), and grand average of hydropathicity index (GRAVY). Subcellular localization prediction of IabHLHs was carried out using BUSCA (http://busca.biocomp.unibo.it/. accessed on 13 January 2023).

#### *4.3. Analysis of Phylogenetic Relationships of IabHLHs and AtbHLHs*

The AtbHLH protein sequences without conserved bHLH domain were excluded [43,60,67,68]. The ClustalW2.0 program with default parameters was used to perform multiple sequence alignment of IabHLHs and AtbHLHs, and then the phylogenetic tree was constructed using the neighbor-joining (NJ) method within MEGA-X (bootstrap value: 1000) [69]. The phylogenetic tree was visualized using the online tool Evolviewv2 [70] (https://evolgenius.info/. accessed on 13 January 2023). The TBtools (version 1.108) software was used to visualize the sequence logo of the IabHLH domain.

The SWISS-MODEL (https://www.swissmodel.expasy.org/. accessed on 14 January 2023) homology modeling was performed to predict protein tertiary structures. The PyMOL (version 4.6) was used to visualize the protein tertiary structural models [71].

#### *4.4. Analysis of Conserved Motif, Gene Structure, and Cis-Acting Elements*

The conserved motif within IabHLH proteins was analyzed using the online program MEME (https://meme-suite.org/meme/tools/meme. accessed on 10 December 2022). The GFF annotation file of *I. aquatica* was used to obtain the intron-exon distributions of the *IabHLHs*.

The promoter sequences of the *IabHLH* genes (the upstream 2000 bp sequences from CDS of *IabHLH* genes) were extracted using TBtools based on the full-length DNA sequences of the *I. aquatica* genomes. The PlantCare online database (http://bioinformatics. psb.ugent.be/webtools/PlantCare/html/, accessed on 1 January 2023) was used to predict the cis-acting elements within promoter regions of *IabHLHs*. The results were visualized using TBtools.

#### *4.5. Analysis of Chromosomal Distribution, Collinearity, and Gene Duplication Events*

The chromosomal distribution of *IabHLHs* and the gene density within the chromosome of *I. aquatica* were visualized using TBtools. The collinearity relationships of *I. aquatica*, *A. thaliana*, *Ipomoea batatas*, and *Oryza sativa* were analyzed using Multiple Collinearity Scan Toolkit (MCScanX), The results were visualized using the "Advanced Circos" function within TBtools [72].

Gene duplication events of *IabHLHs* were analyzed using the dupGen\_finder pipeline, which includes WGD (whole-genome duplication), TD (tandem duplication), PD (proximal duplication), TRD (transposed duplication), and DSD (dispersed duplication). The

MYN method within KaKs\_Calculator 2.0 (https://GitHub.com/qiao-xin/scripts\_for\_gb. accessed on 23 December 2022) was used to calculate the value of Ka, Ks, and Ka/Ks.

#### *4.6. Plant Materials and Sampling*

The purple-stemmed *I. aquatica* and green-stemmed *I. aquatica* were cultivated by cutting in Hoagland nutrient solution at 28 ◦C with a light cycle of 16 h (light)/8 h (dark). The uniformly colored stems of two varieties were harvested after four weeks of culture and immediately frozen in liquid nitrogen for a moment, which was then stored at −80 ◦C until use.

#### *4.7. Transcriptome Analysis of IabHLH Genes*

The transcript abundance of IabHLH genes was quantified by calculating the value of FPKM (fragments per kilobase per million mapped reads) obtained from the RNA-seq data (BioProject: PRJNA814206). The genes with expression levels that met the criteria (|log2FC| > 1, FDR < 0.05, and *p*-value < 0.05) were considered differentially expressed genes (DEGs).

#### *4.8. qRT-PCR Analysis*

Total RNA was extracted from the stems of two varieties using the RNAprep Pure extraction kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The RNA quality was assessed using the microspectrophotometer K5600C (KAIAO, Beijing, China) and agarose gel electrophoresis. The cDNA was synthesized using the FastKing cDNA kit (Tiangen, Beijing, China). The gene-specific primers were designed using Primer Premier 6.0 (Table S7). The reaction mixture of qRT-PCR includes 10 µL 2xSYBR green mix (ChamQ Universal SYBR qPCR Master Mix), 1 µL cDNA, 1 µL primers (0.5 µL forward primer and 0.5 µL reverse primer), and 8 µL PCR-grade water. The qRT-PCR was conducted on the QuantStudio Real-Time Fluorescence PCR System (ThermoFisher, MA, USA). The conditions of two-step qRT-PCR are as follows: 95 ◦C 3 min followed by 40 cycles of 95 ◦C 10 s and 55 ◦C 30 s. The *GAPDH* gene of *I. aquatica* was used as the internal reference gene. The relative expression of the target gene was calculated using the 2−∆∆Ct method. Data were plotted using GraphPad Prism 8.

#### **5. Conclusions**

In this study, a total of 157 *bHLH* genes were identified in *I. aquatica* at the whole genome level, which were subdivided into 23 subgroups. The *bHLHs* within the identical subgroup had similar motifs and gene structures. The DSD and WGD duplication events made significant contributions to the expansion of the *IabHLH* gene family. All duplicated gene pairs were subjected to the purifying selection. A total of 13 DEGs were identified between the purple-stemmed and green-stemmed varieties. The expression profiling revealed that the *IabHLH027* belonging to subgroup XII was dramatically upregulated in the purple-stemmed *I. aquatica* relative to the green-stemmed *I. aquatica,* suggesting a potential positive role in regulating anthocyanin accumulation in the *I. aquatica* stems. Our study provides important gene candidates for further exploring the regulatory network of anthocyanin biosynthesis of *I. aquatica*.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24065652/s1.

**Author Contributions:** Conceptualization, W.B.; methodology, W.B. and Z.L.; software, Z.L. and X.F.; validation, Z.L., X.F. and H.X.; formal analysis, Z.L., X.F., H.X., Y.Z., Z.S. and G.Z.; investigation, Z.L., X.F., H.X., Y.Z., Z.S. and G.Z.; writing—original draft preparation, Z.L. and X.F.; writing—review and editing, W.B.; funding acquisition, W.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hainan Provincial Natural Science Foundation of China (2019RC059 and 320MS008) and the Initial Funds for the High-level Talents of Hainan University (KYQD(ZR)1935).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
