Identification and Candidate Gene Evaluation of a Large Fast Neutron-Induced Deletion Associated with a High-Oil Phenotype in Soybean Seeds

Abstract

Since the dawn of agriculture, crops have been genetically altered for desirable characteristics. This has included the selection of natural and induced mutants. Increasing the production of plant oils such as soybean (Glycine max) oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybeans, however, usually results in reduced seed protein. A soybean fast neutron population was screened for oil content, and three high oil mutants with minimal reductions in protein levels were found. Three backcross F2 populations derived from these mutants exhibited segregation for seed oil content. DNA was pooled from the high-oil and normal-oil plants within each population and assessed by comparative genomic hybridization. A deletion encompassing 20 gene models on chromosome 14 was found to co-segregate with the high-oil trait in two of the three populations. Eighteen genes in the deleted region have known functions that appear unrelated to oil biosynthesis and accumulation pathways, while one of the unknown genes (Glyma.14G101900) may contribute to the regulation of lipid droplet formation. This high-oil trait can facilitate the breeding of high-oil soybeans without protein reduction, resulting in higher meal protein levels.

Keywords: comparative genomics hybridization; fast neutron mutagenesis; renewable oil; triacylglyceride.

Figure 1

Scatter plots of the protein (x-axes) and oil (y-axes) content of F2:3 seeds. Each data point represents the NIRS values for each respective F2:3 family (e.g., corresponding to the rows in Table 5 for the A1, A2, and A3 populations). Seeds were analyzed for percent oil and protein content on a dry-weight basis via bulk-seed NIRS with n = 3 technical replications for each F2:3 seed lot.

Figure 2

A large copy number variation (CNV) event detected in chromosome 14 of the A1 and A3 populations (both derived from separate crosses between 1R22 × WT) exhibited strong inverse correlations of oil and protein content. The x-axis indicates the location of each microarray feature along chromosome 14, according to the Williams 82 genome version 2 assembly (Wm82.a2.v1). The y-axis shows the log2 ratio of the CGH intensity from the high-oil versus the normal-oil bulk for each microarray feature. The blue dots show the CGH comparison between A1 high-oil versus A1 normal-oil. The orange dots show the CGH of high-oil versus normal-oil for A2, and the grey dots show the CGH of high-oil versus normal-oil for A3. These data indicate that a large deletion is enriched in the high-oil plants of the A1 and A3 populations, while the A2 population does not show this enrichment. This deletion event was detected on Chr14, from bp 9,994,086 to 10,301,954, a span of approximately 308 kb.

Figure 3

Predicted topology of Glyma.14G101900. It has one transmembrane region, designated by the blue 1. Extra means extra-cellular, and intra means inside the plasma membrane.

Figure 4

Protein network for Glyma.14G101900 clustered in three groups. Glyma.14G101900 is referred to as I1M988 (red color) in the STRING database. Green lines represent all relationships to Glyma14G101900, whereas other colors represent multiple unique connections between genes in the associated network families.

Figure 5

Some views of Glyma.14G101900 PDB model predicted by Phyre2 and illustrated by Chimera. (A) Ribbon view, (B) mesh surface view, (C) mesh surface with ball and stick view, and (D) hydrophobicity surface (hydrophobicity surface preset from dodger blue for the most hydrophilic to white to orange-red for the most hydrophobic).

Figure 6

Some views of Glyma.14G102100 PDB model predicted by Phyre2 and illustrated by Chimera. (A) Ribbon view, (B) mesh surface with ball and stick view, (C) surface view, and (D) hydrophobicity surface (hydrophobicity surface preset from dodger blue for the most hydrophilic to white to orange-red for the most hydrophobic).