Molecular characterization of two high-palmitic-acid mutant loci induced by X-ray irradiation in soybean

Abstract

Palmitic acid is the most abundant (approx. 11% of total fatty acids) saturated fatty acid in conventional soybean seed oil. Increasing the saturated acid content of soybean oil improves its oxidative stability and plasticity. We have developed three soybean mutants with high palmitic acid content by X-ray irradiation. In this study, we successfully identified the mutated sites of two of these high-palmitic-acid mutants, J10 and M22. PCR-based mutant analysis revealed that J10 has a 206,203-bp-long deletion that includes the GmKASIIA gene and 16 other predicted genes, and M22 has a 26-bp-long deletion in the sixth intron of GmKASIIB. The small deletion in M22 causes mis-splicing of GmKASIIB transcripts, which should result in nonfunctional products. In addition, we designed co-dominant marker sets for these mutant alleles and confirmed the association of genotypes and palmitic acid contents in F(2) seeds of J10 X M22. This information will be useful in breeding programs to develop novel soybean cultivars with improved palmitic acid content. However, in the third mutant, KK7, we found no polymorphism in either GmKASIIA or GmKASIIB, which suggests that several unknown genes in addition to GmKASIIA and GmKASIIB may be involved in elevating the palmitic acid content of soybean seed oil.

Keywords: Glycine max; GmKASIIA; GmKASIIB; X-ray irradiation; palmitic acid.

Fig. 1

Southern blot and PCR analysis of GmKASIIA and GmKASIIB genes in the normal soybean cultivar Bay and three high-palmitic-acid mutants. (A) Hind III-digested DNA fragments (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22) were hybridized with a GmKASIIA cRNA probe. (B) GmKASIIA and GmKASIIB DNA fragments were amplified with gene-specific primer sets (lane 1, Bay; lane 2, KK7; lane 3, J10; lane 4, M22).

Fig. 2

PCR-based analysis of the deleted region in the J10 mutant. (A) Location of the GmKASIIA gene and primer sets on soybean chromosome 17. The large arrow indicates the position of GmKASIIA, and the small arrows indicate the primer positions. Amplification result for J10 (even-numbered lanes) and Bay (odd-numbered lanes) with (B) 5′-upstream primer sets and (C) 3′-downstream primer sets.

Fig. 3

Gene structures and splicing products from a normal Bay and a mutant M22 GmKASIIB gene. The 26-bp-long deleted region is indicated by the gray triangle. The normally spliced transcript is shown by solid lines and two different mis-spliced transcripts are shown by short- and long-dashed lines.

Fig. 4

Development of PCR-based markers for the J10 and M22 mutations. (A) Locations of two primer sets for detecting the mutation in J10. N.D. means not detected. (B) Location of a primer set for detecting the mutation in M22. (C) Detection of the J10 mutation: Bay (lane 1), heterozygous (lane 2) and J10 (lane 3) DNA templates. (D) Detection of the M22 mutation: Bay (lane 1), heterozygous (lane 2) and M22 (lane 3) DNA templates.

Fig. 5

Comparison of palmitic acid content and marker segregation on F₂ seed individuals obtained from the cross between J10 and M22. The palmitic acid contents are arranged in the order of increasing palmitic acid content. The patterns of individual bars indicate nine genotypes as illustrated in the figure.

Fig. 6

Phylogenic tree of βKAS family proteins in soybean. The AtKASI, AtKASII, AtKASIII and AtmtKAS were used as outgroups. The reliability of each branch was tested by bootstrap analysis with 1,000 replications. The asterisks indicated only partial amino acid sequences were registered in Glyma1 at Phytozome v.6.0.