Amino acid sequence alignment and phylogeny of the five Da-dio genes. (A) Alignment of the sequences of five Da-dio proteins this study, a D. japonica dioscorin homolog (dioscorin-5 precursor), and three carbonic anhydrases (CAs) from human (P00915), mouse (P13634) and Arabidopsis (CAB79100), respectively. Putative residues related to CA activity are indicated with a black arrow. Two cysteine residues that are implicated in activities of trypsin inhibitor (TI), dehydroascorbate (DHA) reductase, and monodehydroascorbate (MDA) reductase are indicated with an inverted triangles; Identical or conserved amino acids are shaded in black or red, respectively; (B) phylogenetic tree constructed for 23 dioscorin proteins from different yam species using MEGA version 6.0 (www. megasoftware.net). The five dioscorin proteins identified in this study are indicated with black triangles. The other dioscorin proteins are as follows: three D. alata dioscorins (Da-dioA1, AF242551; Da-dioA2, AF245019; Da-dioB1, AF243526), six D. japonica dioscorins (Dj-dioA1, AM849818; Dj-dioA2, AM849819; Dj-dioA3, AM849820; Dj-dioA4, AM849821; Dj-dioB1, AM849816; Dj-dioB2, AM849817), six D. pseudojaponica dioscorins (Dp-dioA1, GQ246171; Dp-dioA2, GQ246172; Dp-dioA3, GQ246173; Dp-dioA4, GQ246174; Dp-dioA5, GQ246175; Dp-dioB1, GQ246170), two D. batatas dioscorins (Db-DB3S, AB178473; Db-DB3L, AB178472), and one D. cayenensis dioscorin (Dc-dioA, X76187).