*2.2. Phylogenetic and Comparative Analyses of PODs in Cassava*

The homology and similarity of the *POD* genes in cassava were determined by performing multiple sequence alignments. Then, the radiation phylogenetic tree of the 91 *POD* genes was constructed using the neighbor-joining (NJ) method with a bootstrap value of 1000 using MEGA 5.1 (University College Dublin, Dublin, Ireland). The phylogenetic analyses indicated that *MePOD* genes can be divided into six subgroups on the basis of the observed genetic distance and bootstrap support (Figure 1). The large subgroups A and D consist of 23 and 24 MePOD members, respectively, whereas the small subgroups C and F contain 9 and 8 MePOD members, respectively. Subgroups B and E are composed of 15 and 12 MePODs members, respectively. These results show that a diversified POD family exists in cassava. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 3 of 18 94 constructed using the neighbor-joining (NJ) method with a bootstrap value of 1000 using MEGA 5.1 95 (University College Dublin, Dublin, Ireland). The phylogenetic analyses indicated that *MePOD* genes 96 can be divided into six subgroups on the basis of the observed genetic distance and bootstrap support 97 (Figure 1). The large subgroups A and D consist of 23 and 24 MePOD members, respectively, whereas 98 the small subgroups C and F contain 9 and 8 MePOD members, respectively. Subgroups B and E are 99 composed of 15 and 12 MePODs members, respectively. These results show that a diversified POD 100 family exists in cassava.

102 **Figure 1.** Phylogenetic analyses of PODs from cassava. A total of 91 PODs from cassava were used to 103 create the neighbor-joining (NJ) tree with 1000 bootstraps. **Figure 1.** Phylogenetic analyses of PODs from cassava. A total of 91 PODs from cassava were used to create the neighbor-joining (NJ) tree with 1000 bootstraps.

#### 104 *2.3. Conserved Motif and Gene Structure Analysis of POD Families in Cassava 2.3. Conserved Motif and Gene Structure Analysis of POD Families in Cassava*

105 The structural features of MePODs were investigated by identifying 10 conserved motifs using 106 the MEME database in accordance with the phylogenetic relationship. Then, the conserved motifs 107 were submitted in their entirety to the InterProScan database for annotation. Eight domains (domains 108 1, 2, 3, 4, 5, 6, 7, and 9) were noted as POD protein motifs, which are an essential feature of the The structural features of MePODs were investigated by identifying 10 conserved motifs using the MEME database in accordance with the phylogenetic relationship. Then, the conserved motifs were submitted in their entirety to the InterProScan database for annotation. Eight domains (domains 1, 2, 3, 4, 5, 6, 7, and 9) were noted as POD protein motifs, which are an essential feature of the peroxidase

109 peroxidase family. On the basis of the motif analyses, 83 MePODs were assigned to one of five 110 subgroups (A–E). Each of these 83 MePODs contains at least nine POD motifs, except for MePOD57 111 (in subgroup D) and MePOD69 (in subgroup B), which have seven and five motifs, respectively. The

family. On the basis of the motif analyses, 83 MePODs were assigned to one of five subgroups (A–E). Each of these 83 MePODs contains at least nine POD motifs, except for MePOD57 (in subgroup D) and MePOD69 (in subgroup B), which have seven and five motifs, respectively. The presence of these motifs suggests that the identified proteins are characteristic of the POD family (Figure 2). Subgroup F is distinct from the others: its members contain domains 2, 4, 5, 7, and 8. These results indicate that the proteins assigned to the same subfamilies share similar POD motif characteristics, further supporting their phylogenetic classification as PODs in cassava. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 18 113 (Figure 2). Subgroup F is distinct from the others: its members contain domains 2, 4, 5, 7, and 8. These 114 results indicate that the proteins assigned to the same subfamilies share similar POD motif 115 characteristics, further supporting their phylogenetic classification as PODs in cassava.

117 **Figure 2.** The motif analyses of POD family members in cassava according to their evolutionary 118 relationship. The POD motifs were identified by the MEME database. The 10 different colors of the 119 boxes on the right represent diverse conserved motifs, while the gray lines indicate non-conserved **Figure 2.** The motif analyses of POD family members in cassava according to their evolutionary relationship. The POD motifs were identified by the MEME database. The 10 different colors of the boxes on the right represent diverse conserved motifs, while the gray lines indicate non-conserved sequences.

120 sequences. 121 Next, the exon–intron structures of cassava *POD* genes were analyzed. Subgroup F is exon-rich, Next, the exon–intron structures of cassava *POD* genes were analyzed. Subgroup F is exon-rich, with five to nine exons, whereas other subfamilies have fewer (between one and four) exons, except for

 with five to nine exons, whereas other subfamilies have fewer (between one and four) exons, except for *MePOD02* (in subgroup B), which has five exons (Figure 3). High proportions of *POD* genes contain four exons in subgroups A, B, C, and D, with four-exon *POD* genes forming 84%, 67%, 57%, and 76% of the genes in these subgroups, respectively, whereas only 50% of the subgroup E genes

127 providing further evidence of their phylogenetic relationship.

*MePOD02* (in subgroup B), which has five exons (Figure 3). High proportions of *POD* genes contain four exons in subgroups A, B, C, and D, with four-exon *POD* genes forming 84%, 67%, 57%, and 76% of the genes in these subgroups, respectively, whereas only 50% of the subgroup E genes have four exons. Generally, *POD* genes in the same subgroup show similar exon–intron features, providing further evidence of their phylogenetic relationship. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 5 of 18

129 **Figure 3.** The exon–intron organization analyses of cassava PODs on the basis of the phylogenetic 130 relationship. The exon–intron distribution was established using the GSDS database. The yellow 131 boxes and the black lines represent exons and introns, respectively. **Figure 3.** The exon–intron organization analyses of cassava PODs on the basis of the phylogenetic relationship. The exon–intron distribution was established using the GSDS database. The yellow boxes and the black lines represent exons and introns, respectively.

 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

132 *2.4. Analyses of Chromosomal Distribution and Duplication Events of the Cassava* POD *Genes*
