Analysis of β-amylase gene (Amyβ) variation reveals allele association with low enzyme activity and increased firmness in cooked sweetpotato (Ipomoea batatas) from East Africa

Abstract

β-amylase is a thermostable enzyme that hydrolyses starch during cooking of sweetpotato (Ipomoea batatas) storage roots, thereby influencing eating quality. Its activity is known to vary amongst genotypes but the genetic diversity of the beta-amylase gene (Amyβ) is not well studied. Amyβ has a highly conserved region between exon V and VI, forming part of the enzyme's active site. To determine the gene diversity, a 2.3 kb fragment, including the conserved region of the Amyβ gene was sequenced from 25 sweetpotato genotypes. The effect of sequence variation on gene expression, enzyme activity, and firmness in cooked roots was determined. Six genotypes carrying several SNPs within exon V, linked with an AT or ATGATA insertion in intron V were unique and clustered together. The genotypes also shared an A336E substitution in the amino acid sequence, eight residues upstream of a substrate-binding Thr344. The genotypes carrying this allele exhibited low gene expression and low enzyme activity. Enzyme activity was negatively correlated with firmness (R = -0.42) in cooked roots. This is the first report of such an allele, associated with low enzyme activity. These results suggest that genetic variation within the AmyB locus can be utilized to develop markers for firmness in sweetpotato breeding.

Keywords: Beta-amylase; Nucleotide variation; Optimal cooking time; Orange-fleshed; Sweetpotato; Texture.

Graphical abstract

Fig. 1

Map of the Amyβ genomic sequence based on Ipomoea batatas var ‘Kokei 14’ (D12882.1), showing exons (black) and introns (white). The map shows the conserved region (1204 bp) and the selected primer sites and for this study, covering a 2679 bp region.

Fig. 2

Multiple sequence alignment of a partial AmyB coding sequence (within exon V). D12882.1 is the sequence of Ipomoea batatas variety, ‘Kokei 14’. Coloured nucleotides in study genotypes indicate SNPs, asterisk (∗) below the alignment indicate conserved nucleotides and gaps indicate nonconserved nucleotides.

Fig. 3

The similarity of amino acid sequences inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The tree shows three distinct clades; G1-G3 (group 1, 2, and 3, respectively).

Fig. 4

Multiple sequence alignment of partial AmyB sequence (intron V). D12882.1 is the sequence of Ipomoea batatas variety, ‘Kokei 14’. Coloured nucleotides represent variation from reference (SNPs or insertion). Dashes (−) indicate a gap in the sequence. Asterisks (∗) below the alignment indicate conserved nucleotides and gaps indicate non-conserved nucleotides.

Fig. 5

Relative quantitative expression of the Amyβ gene in sweetpotato genotypes. Letters denote statistical significance. Genotypes sharing the same letter are not significantly different from each other (Tukey's HSD at p < 0.05).

Fig. 6

β-amylase activity in raw sweetpotato storage roots. Letters denote statistical significance. Genotypes sharing the same letter are not significantly different from each other (Tukey's HSD, at p < 0.05).