*2.4. Analyses of Chromosomal Distribution and Duplication Events of the Cassava* POD *Genes*

The locations of the cassava *POD* genes were determined by analyzing their chromosomal distribution (Figure 4). The 91 MePODs were mapped to chr1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, and 18, and scaffold01119. The 24 *POD* genes in subgroup D were distributed among chr1, 2, 3, 5, 6, 8, 11, 12, 13, 15, 16, and 17, making subgroup D the most widely distributed subgroup. Subgroup F contains eight *POD* genes, which are located on chr1, 2, 4, 8, 11, 16, and 18; thus, of the six subgroups identified, subgroup F is dispersed among the fewest chromosomes. The 23 members of subgroup A are found on chr1, 2, 7, 8, 9, 10, 15, 17, and 18, among which chromosomes 7, 9, and 10 only contain subgroup A genes. Generally, the cassava *POD* genes are widely distributed among chromosomes. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 6 of 18 137 F contains eight *POD* genes, which are located on chr1, 2, 4, 8, 11, 16, and 18; thus, of the six subgroups 138 identified, subgroup F is dispersed among the fewest chromosomes. The 23 members of subgroup A 139 are found on chr1, 2, 7, 8, 9, 10, 15, 17, and 18, among which chromosomes 7, 9, and 10 only contain 140 subgroup A genes. Generally, the cassava *POD* genes are widely distributed among chromosomes.

142 **Figure 4.** Chromosome distribution analyses of *POD* gene subgroups in cassava. The chromosomal 143 information of 91 MePODs was collected from the Phytozome 12.0 cassava database, and the genes 144 were then mapped to 17 chromosomes and one scaffold. MapInspect software (Wageningen 145 University, Wageningen, Netherlands) was used to draw this figure. **Figure 4.** Chromosome distribution analyses of *POD* gene subgroups in cassava. The chromosomal information of 91 MePODs was collected from the Phytozome 12.0 cassava database, and the genes were then mapped to 17 chromosomes and one scaffold. MapInspect software (Wageningen University, Wageningen, Netherlands) was used to draw this figure.

146 To further investigate the expansion of *POD* genes in cassava, we aligned the total nucleotide 147 sequences of the 91 *MePOD* genes to identify duplication events. We identified 15 events involving 148 16 paralogs (*MePOD2/MePOD33*, *MePOD29/36/79*, *MePOD30/32/39*, *MePOD34/44/56/84*, 149 *MePOD42/MePOD49*, *MePOD60/MePOD62*), suggesting that tandem duplication played a significant 150 role in POD family expansion in the cassava genome (Figure 5). To further investigate the expansion of *POD* genes in cassava, we aligned the total nucleotide sequences of the 91 *MePOD* genes to identify duplication events. We identified 15 events involving 16 paralogs (*MePOD2*/*MePOD33*, *MePOD29*/*36*/*79*, *MePOD30*/*32*/*39*, *MePOD34*/*44*/*56*/*84*, *MePOD42*/*MePOD49*, *MePOD60*/*MePOD62*), suggesting that tandem duplication played a significant role in POD family expansion in the cassava genome (Figure 5).

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152 **Figure 5.** Analyses of *POD* gene duplication in cassava. The program Circos (Canada's Michael Smith 153 Genome Sciences Center, Vancouver, Canada) was used to draw different chromosomes in a circular 154 distribution. The brown connection lines represent tandem duplication events of *POD* genes in **Figure 5.** Analyses of *POD* gene duplication in cassava. The program Circos (Canada's Michael Smith Genome Sciences Center, Vancouver, Canada) was used to draw different chromosomes in a circular distribution. The brown connection lines represent tandem duplication events of *POD* genes in cassava.

155 cassava. 156 Next, we calculated nonsynonymous (Ka) and synonymous (Ks) ratios to understand the modes 157 of evolutionary selection for the duplicated *MePOD* genes. We found that the Ka/Ks ratios of the 158 paralogous genes are between 0.05 and 0.29, indicating that these genes underwent purifying Next, we calculated nonsynonymous (Ka) and synonymous (Ks) ratios to understand the modes of evolutionary selection for the duplicated *MePOD* genes. We found that the Ka/Ks ratios of the paralogous genes are between 0.05 and 0.29, indicating that these genes underwent purifying selection during evolution (Table S2).

#### 159 selection during evolution (Table S2). *2.5. Expression Profiles of POD Genes in Di*ff*erent Tissues of Two Cassava Genotypes*

170 role in the development and function of different cassava tissues.

160 *2.5. Expression Profiles of POD Genes in Different Tissues of Two Cassava Genotypes* 161 The expression levels of *MePOD* genes in different tissues were investigated by performing 162 RNA-Seq analysis on the storage roots, stems, and leaves of a cultivated variety (Arg7) and wild 163 subspecies (W14). The resulting expression data covered 59 and 56 *MePOD* genes in the 164 transcriptome dataset of Arg7 and W14, respectively (Figure 6A; Table S3). Of these genes in Arg7, 165 15 (25%), 9 (15%), and 8 (14%) *MePODs* had high transcriptional levels (log2-based > 4) in stems, 166 leaves, and storage roots, respectively. The number of *MePODs* with high expression (log2-based > 4) 167 in the stems, leaves, and storage roots of W14 was 9 (16%), 7 (13%), and 10 (18%), respectively. 168 Notably, *MePOD5* in subgroup E and *MePOD89* in subgroup F were strongly expressed (log2-based 169 fold change > 4) in the three diverse tissues of Arg7 and W14. These *POD* genes may play a molecular The expression levels of *MePOD* genes in different tissues were investigated by performing RNA-Seq analysis on the storage roots, stems, and leaves of a cultivated variety (Arg7) and wild subspecies (W14). The resulting expression data covered 59 and 56 *MePOD* genes in the transcriptome dataset of Arg7 and W14, respectively (Figure 6A; Table S3). Of these genes in Arg7, 15 (25%), 9 (15%), and 8 (14%) *MePODs* had high transcriptional levels (log2-based > 4) in stems, leaves, and storage roots, respectively. The number of *MePODs* with high expression (log2-based > 4) in the stems, leaves, and storage roots of W14 was 9 (16%), 7 (13%), and 10 (18%), respectively. Notably, *MePOD5* in subgroup E and *MePOD89* in subgroup F were strongly expressed (log2-based fold change > 4) in the three diverse tissues of Arg7 and W14. These *POD* genes may play a molecular role in the development and function of different cassava tissues.

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172 **Figure 6.** Transcriptomic analysis of cassava *POD* genes. (**A**) Expression of MePODs in the stem (S), 173 leaf (L), and storage root (SR) of W14 and Arg7. The log2-based FPKM value was applied to build the 174 heat map using Mev4.9.0 software (CCCB, Boston, USA). (**B**) Expression of MePODs in the leaf (L) **Figure 6.** Transcriptomic analysis of cassava *POD* genes. (**A**) Expression of MePODs in the stem (S), leaf (L), and storage root (SR) of W14 and Arg7. The log2-based FPKM value was applied to build the heat map using Mev4.9.0 software (CCCB, Boston, USA). (**B**) Expression of MePODs in the leaf (L) and root (R) of Arg7, SC124, and W14 after drought treatment relative to under normal conditions. Log2-based fold changes (L/control; R/control) were applied to build the heat map using Mev4.9.0 software. (**C**) Expression of MePODs in the storage root at 6, 12, and 48 h relative to 0 h after harvest. Log2-based fold changes were applied to build the heat map using Mev4.9.0 software.
