Genome-wide association analysis and transgenic characterization for amylose content regulating gene in tuber of Dioscorea zingiberensis

Abstract

Background: Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation.

Results: To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele.

Conclusions: Overall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes.

Keywords: Starch biosynthesis; Food quality; Food security; Genome wide association study; Yams.

Fig. 1

A simplified schematic view of the potential routes of amylose biosynthesis in D. zingiberensis. The diagram includes two compartments: the cytosol and the amymoplast. The stepwise reactions of sucrose synthase (SuSy), UGP-glucose pyrophosphorilase (UGPase) and plastidial phosphoglucomutase (pPGM) take place in the cytosol to convert sucrose to glucose-6-phosphate, which enters the amyloplast to be used for amylose biosynthesis. The enzymes involved in amylose biosynthesis in yam tubers include the precursor ADP-glucose pyrophosphorylase (AGPase) and granule-bound starch synthase (GBSS). Abbreviations are defined as follows Fructokinase (FRK); Glucose 1-phosphate (G1P); Glucose 6-phosphate/phosphate transporter (GPT/G6PPT); Fructose 6-phosphate (F6P); Cytosolic phosphoglucomutase (cPGM); cytosolic phosphoglucose isomerase (cPGI); inorganic phosphate (Pi); inorganic pyrophosphate (PPi); 3-phosphoglyceric acid (3-PGA); AGPase large subunit (AGPLS); AGPase small subunit (AGPSS).

Fig. 2

Distribution of amylose content data of D. zingiberensis accessions at Luohe (a) and Hainan (b)

Fig. 3

The results of the population structure (a) and principal component (b) analyses of the 150 D. zingiberensis accessions

Fig. 4

Genome-wide association mapping for amylose content in D. zingiberensis. Manhattan plot for amylose content (a). Quantile-quantile plot for amylose content (b)

Fig. 5

Characterization of D. zingiberensis AGPase (Dzin_AGPase) gene structure showing the location of the SNP Chr6_75609202 in the first exon. A single nucleotide polymorphism (SNP) was detected within this exon, leading to a non-synonymous alteration in the resulting protein sequence (a). Comparative amylose quantification for accessions exhibiting A and G alleles (b). Relative expression of the both versions of the gene via qRT-PCR experiment (c)

Fig. 6

Generation of Arabidopsis thaliana transgenic plants for the overexpression of the DZin_AGPase gene (a). The construct of plasmid contains the Dzin_AGPase gene, the CaMV35S promoter, and NOS terminator. The NPT II was employed as a selective marker. RB, right border; LB, left border; NOS-P, nopaline synthase promoter; NOS-T, nopaline synthase terminator. Relative expression (b) and amylose content quantification (c) from the T3 generation transgenic plants. Four lines for each allele were selected. WT-VC is vector control (transformed with empty vector). Mean comparison significance of the amylose content was depicted with the letter A, B, and C