Improving Soybean Seed Sucrose Content using TILLING by Sequencing Analyses of The Soybean Sucrose Synthase Gene Family

Abstract

Soybean seed quality is influenced by its soluble sugar composition, with high sucrose content being desirable for nutritional and industrial applications. In contrast, excessive raffinose and stachyose levels are considered undesirable due to their adverse effects on gastrointestinal function in humans and monogastric animals. Therefore, developing soybean mutant lines with elevated sucrose content and optimal raffinose and stachyose content is desirable. In this study, we characterized twelve sucrose synthase genes through a comprehensive phylogenetic tree analysis, synteny analysis, gene structure evaluation, and variations in conserved domains. Additionally, we conducted a TILLING by Sequencing approach to identify EMS mutations in the characterized Sucrose synthase genes. Numerous mutations have been identified in soybean sucrose synthases that resulted in high sucrose content, including the sucrose synthases mutants SL446 (R582W) and F1115 (G249E) on Glyma.02G240400 with a sucrose content of 9.5% and 9.1%, respectively. The obtained soybean mutants with enhanced sugar content can be useful in breeding programs to improve soybean nutritional quality without potential developmental trade-offs.

Keywords: EMS mutagenesis; TILLING; glycine max; raffinose; soybean; stachyose; sucrose; sucrose synthase gene family.

Figure 1

Phylogenetic gene tree for sucrose synthase gene family from nine plant species including Arabidopsis thaliana, Phaseolus vulgaris, Medicago truncatula, Zea mays, Triticum aestivum, Beta vulgaris, Selaginella moellendorffii, Physcomitrium patens and Sorghum bicolor. The protein sequences were subjected to a MUSCLE multiple alignment and a phylogenetic gene tree was constructed by the maximum likelihood (ML) method using Mega 11.

Figure 2

Phylogenetic relationships and gene structures of soybean sucrose synthase. The protein sequences of each gene family were aligned using MUSCLE and the phylogenetic tree was constructed using MEGA 11. The structures of 12 soybean sucrose synthase genes were illustrated with yellow boxes representing exons (coding DNA sequence, CDS), black lines illustrating introns, and blue boxes indicating 5’-UTR and 3’-UTR regions. The size of gene structures can be measured by the base pair (bp) scale at the bottom. The gene structure was drawn using the Gene Structure Display Server (Hu et al., 2015).

Figure 3

Expression heatMap of the soybean sucrose synthase genes in Williams 82 (RPKM) retrieved from publicly available RNA-seq data from the soybase database (Brown et al., 2021). The color key represents the relative transcript abundance from low (red) to high (green). RNA-seq data is not available at Soybase for Glyma.17G045800, Glyma.03G216300, and Glyma.19G212800.

Figure 4

(A) Chromosomal locations and duplications of 12 soybean sucrose synthase. The chromosome size and the gene locations were drawn based on soybean genome annotation a2.v1 on SoyBase (Brown et al., 2021). The scale is on the left and it’s in megabase (Mb). Each duplicated pairs of sucrose synthase genes are linked by a purple line, respectively. (B) Percent Identity Matrix of the Sucrose Synthase Genes, created by Clustal2.1 using the multiple sequence alignment of the soybean sucrose synthase protein sequences performed at Mview (Multiple Sequence Alignment (MSA)) at EMBL-EBI (https://www.ebi.ac.uk/).

Figure 5

Structural analysis and protein homology modeling of mutants of sucrose synthase genes, including (A) Glyma.02G240400 sucrose synthase gene mutants, (B) Glyma.09G167000 sucrose synthase gene mutants, (C) Glyma.09G073600 sucrose synthase gene mutants, (D) Glyma.19G212800 sucrose synthase gene mutants.

Figure 6

The plant (A) height and (B) number of pods of the sucrose synthase mutants. One-way ANOVA analysis and Student’s t-test were performed using JMP.