Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Six Ipomoea Species and the Identification of Anthocyanin-Related Members in Sweet Potatoes

Abstract

Sweet potatoes (Ipomoea batatas) are one of the important tuberous root crops cultivated worldwide, and thier storage roots are rich in antioxidants, such as anthocyanins. R2R3-MYB is a large gene family involved in various biological processes, including anthocyanin biosynthesis. However, few reports about the R2R3-MYB gene family of sweet potatoes have been released to date. In the present study, a total of 695 typical R2R3-MYB genes were identified in six Ipomoea species, including 131 R2R3-MYB genes in sweet potatoes. A maximum likelihood phylogenetic analysis divided these genes into 36 clades, referring to the classification of 126 R2R3-MYB proteins of Arabidopsis. Clade C25(S12) has no members in six Ipomoea species, whereas four clades (i.e., clade C21, C26, C30, and C36), including 102 members, had no members in Arabidopsis, and they were identified as Ipomoea-specific clades. The identified R2R3-MYB genes were unevenly distributed on all chromosomes in six Ipomoea species genomes, and the collinearity analysis among hexaploid I. batatas and another five diploid Ipomoea species suggested that the sweet potato genome might have undergone a larger chromosome rearrangement during the evolution process. Further analyses of gene duplication events showed that whole-genome duplication, transposed duplication, and dispersed duplication events were the primary forces driving the R2R3-MYB gene family expansion of Ipomoea plants, and these duplicated genes experienced strong purifying selection because of their Ka/Ks ratio, which is less than 1. Additionally, the genomic sequence length of 131 IbR2R3-MYBs varied from 923 bp to ~12.9 kb with a mean of ~2.6 kb, and most of them had more than three exons. The Motif 1, 2, 3, and 4 formed typical R2 and R3 domains and were identified in all IbR2R3-MYB proteins. Finally, based on multiple RNA-seq datasets, two IbR2R3-MYB genes (IbMYB1/g17138.t1 and IbMYB113/g17108.t1) were relatively highly expressed in pigmented leaves and tuberous root flesh and skin, respectively; thus, they were identified to regulate tissue-specific anthocyanin accumulation in sweet potato. This study provides a basis for the evolution and function of the R2R3-MYB gene family in sweet potatoes and five other Ipomoea species.

Keywords: Ipomoea species; R2R3-MYB gene family; anthocyanin biosynthesis; expression analysis; sweet potato.

Figure 1

Physicochemical properties of all identified R2R3-MYB proteins in six Ipomoea species. (a) Protein length; (b) Molecular weight (MW); (c) Isoelectric point (pI); (d) Instability index; (e) Aliphatic index; (f) Grand average of hydropathicity (GRAVY).

Figure 2

Phylogenetic tree of R2R3-MYB members in Arabidopsis and six Ipomoea species. All identified R2R3-MYB members from I. batatas (131), I. trifida (133), I. triloba (129), I. nil (127), I. purpurea (51), I. aquatica (124), and Arabidopsis (126) were used. The full-length amino acid sequences of R2R3-MYB proteins were aligned using MAFFT, and the tree was constructed by the maximum-likelihood (ML) method of IQ-tree using the JTT + F + R10 model. These R2R3-MYB proteins are clustered into 36 clades (designated as C1 to C36). The AtCDC5 was rooted as the outgroup, and 4 proteins did not fit well into the clade. The genes that belonged to the same organism were marked in the same shape and color. The dots on the branches represent bootstrap values based on 1000 replications, and bootstrap values >90% and ≥70% are shown as black and gray dots in the phylogenetic tree, respectively, while those <70% are not shown.

Figure 3

Chromosomal distribution and intraspecies synteny analysis of R2R3-MYBs in six Ipomoea species. (a) I. batatas; (b) I. trifida; (c) I. triloba; (d) I. nil; (e) I. aquatica; (f) I. purpurea. The circle represents 15 chromosomes of each Ipomoea species. The R2R3-MYBs are unevenly distributed on corresponding all chromosomes in six Ipomoea species’ genomes. The blue lines show syntenic gene pairs of R2R3-MYBs, and the red lines show syntenic gene pairs of R2R3-MYBs belonging to clade C24(S6). The detailed information is listed in Table S4.

Figure 4

Interspecies collinear analysis of R2R3-MYBs between I. batatas and Arabidopsis, and other five Ipomoea species. (a) Collinear analysis of IbR2R3-MYBs between AtR2R3-MYBs. (b) Collinear analysis of IbR2R3-MYBs between ItfR2R3-MYBs. (c) Collinear analysis of IbR2R3-MYBs between ItbR2R3-MYBs. (d) Collinear analysis of IbR2R3-MYBs between InR2R3-MYBs. (e) Collinear analysis of IbR2R3-MYBs between IaR2R3-MYBs. (f) Collinear analysis of IbR2R3-MYBs between IpR2R3-MYBs. The gray line links show the collinear block with I.batatas and six other plant species’ genomes. The blue lines show collinear gene pairs related to R2R3-MYBs, while the red lines show collinear gene pairs related to R2R3-MYBs belonging to clade C24(S6). The detailed information is listed in Table S5.

Figure 5

The number of R2R3-MYB gene pairs derived from different gene duplication events in the six Ipomoea species. (a) The phylogenetic relationship among the six Ipomoea species. The species tree was inferred from a concatenated alignment matrix of 9835 single-copy ortholog sequences across 6 Ipomoea species genomes. (b) The number of five models of duplicated gene pairs in each species. The gene duplication events include whole-genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD). The x-axis represents the number of duplicated gene pairs. The y-axis represents species.

Figure 6

Phylogenetic relationships, exon-intron structures, and conserved motifs of the R2R3-MYB genes in sweet potatoes. (a) The phylogenetic tree of the 131 IbR2R3-MYB proteins. The tree was constructed by the maximum-like (ML) method of IQ-tree with 1000 bootstrap replicates. (b) The gene structure of the 131 IbR2R3-MYB genes. The yellow boxes, green ellipses, and black lines represent exons, untranslated regions (UTR), and introns, respectively. The bar scale at the bottom shows the length of the gene. (c) The conserved motifs of the 131 IbR2R3-MYB proteins. The boxes with different colors represent Motif 1–10, and the black solid line represents non-conserved regions. While the dashed boxes represent the R2 and R3 domains, their sequences logo shown in Figure S1. The bar scale at the bottom shows the length of the amino acids.

Figure 7

Expression patterns analysis of differentially expressed genes (DEGs) of IbR2R3-MYB and anthocyanin biosynthesis genes (ABGs) in different pigmented tissues of sweet potatoes. (a) IbR2R3-MYBs and ABGs in white, yellow, and purple fleshes. (b,c) IbR2R3-MYBs and ABGs in red and yellow skins. (d,e) IbR2R3-MYBs and ABGs in green and purple leaves. The Log₂(FPKM + 1) values were row scaled and displayed according to the color code. The red and blue colors represent the highest and lowest expression levels, respectively. The hierarchical clustering and green boxes in each heatmap show similar expression patterns.

Figure 8

Pearson’s correlation analysis of differentially expressed genes (DEGs) of IbR2R3-MYB and anthocyanin biosynthesis genes (ABGs) in different pigmented tissues of sweet potatoes. (a) IbR2R3-MYBs and ABGs in white, yellow, and purple fleshes. (b,c) IbR2R3-MYBs and ABGs in red and yellow skins. (d,e) IbR2R3-MYBs and ABGs in green and purple leaves. The pie charts show the Pearson correlation coefficient values. The red and blue colors represent the positive and negative correlation, respectively. The asterisk shows the p-value, * p < 0.05, ** p < 0.01, and *** p < 0.001.