Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation

Abstract

Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max) near-isogenic line (cgy-2-NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. To identify α-null-related transcriptional changes, the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). Seeds at 18 DAF served as the control. Comparison of the transcript profiles identified 3,543 differentially expressed genes (DEGs) between the two genotypes, with 2,193 genes downregulated and 1,350 genes upregulated. The largest numbers of DEGs were identified at 55 DAF. The DEGs identified at 25 DAF represented a unique pattern of GO category distributions. KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL. At 18DAF, 12 DEGs were involved in arginine and proline metabolism. The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes. The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality, and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality.

Fig 1

(A) Breeding process of the ‘cgy-2-NIL’. RPGR = recurrent parent genome recovery. Discontinuous lines link the last generation where the definitive near isogenic lines (NILs) were selected. Numbers alongside discontinuous lines show how many lines were obtained in each candidate line. (B) Graphical genotype for chromosome 20 in ‘cgy-2-NIL’ with the ‘DN47’ genetic background. (C) SDS-PAGE analysis of mature seed proteins of DN47 and four cgy-2-NIL lines: B-12088-3, B-12088-6, B-12088-8, and B-12088-12. (D) Immunoblot analysis of the seed extracts shown in (C) using antibodies specific for β-conglycinin subunits. The sizes of the protein markers (M) in kilodaltons are shown on the left of the image in (C). The arrows point to the α′, α, and β-subunits of β-conglycinin in Fig (C) and (D).

Fig 2

(A) Fully expanded flowers were marked individually with a tag at the 4^th, 5^th, 6^th, or 7^th nodes on cgy-2-NIL and DN47. (B) Pod samples were collected during seed development at 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, and 60 days after flowering (DAF). (C) Developmental changes in morphology, size, and weight of sampled seeds in cgy-2-NIL and DN47. (D) Comparison of transcript levels of the α-subunit gene between cgy-2-NIL and DN47. Differential expression of more than 50-fold was identified during the 25–55 days after flowering (DAF) development stages. Five stages of soybean seeds collected at 18, 25, 35, 50, and 55 DAF were finally selected to subjected to RNA-seq.

Fig 3

(A) Primer sequences used in quantitative real-time reverse transcription PCR (qRT-PCR) analysis for validation of the expressed genes in Illumina sequencing. (B) Comparison between the gene expression ratios obtained from RNA-seq data and that from qRT-PCR. The RNA-seq log2 value of the expression ratio (y-axis) was plotted against the developmental stages (x-axis). (C) qRT-PCR analysis of differentially expressed genes between cgy-2-NIL and DN47. The transcript abundance from the RNA-seq data is shown at the top of the panel for each gene. RPKM: reads per kilobase per million reads. DAF = days after flowering.

Fig 4. The number of differentially expressed genes (DEGs) between ‘cgy-2-NIL’ and ‘DN47’ at various developmental stages (18, 25, 35, 50, and 55 days after flowering (DAF)).

Numbers of up and downregulated genes are summarized.

Fig 5. Cluster dendrogram of differentially expressed genes in cgy-2 NIL and ‘DN47’ at five developmental stages (18, 25, 35, 50, and 55 days after flowering).

Fig 6. Venn diagram showing the overlap of identified differentially expressed genes (DEGs) between ‘cgy-2NIL’ and ‘DN47’ at 18, 25, 35, 50, and 55 days after flowering.