Molecular genetic characterization of a high oleic/no hydroxy fatty acid seed oil mutant of Lesquerella fendleri (brassicaceae)

Authors: SALYWON, A - Desert Botanical Garden; Dierig, David; Tomasi, Pernell; Dahlquist, Gail

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/18/2010
Publication Date: 9/18/2010
Citation: Salywon, A.M., Dierig, D.A., Tomasi, P., Dahlquist, G.H. 2010. Molecular genetic characterization of a high oleic/no hydroxy fatty acid seed oil mutant of Lesquerella fendleri (brassicaceae). Meeting Abstract. AAIC 22nd Annual meeting, Fort Collins, CO. September 18-22, 2010. pp 35.

Interpretive Summary: Lesquerella fendleri (Gray) Wats. (Brassicaceae) is being developed as a new crop for its seed oil that is typically comprised of ca. 55 percent of the hydroxy fatty acid (HFA) lesquerolic acid. Most breeding efforts for lesquerella have been geared towards improving harvest index, seed yield or production practices, with little attention paid to modifying seed oil composition. However, with the recent application of the half-seed method to our breeding population, we have identified greater variability in the fatty acid (FA) composition in the seed oil than had previously been characterized, including a mutant with high oleic and no HFA. The objectives of this study was to characterize the underlying molecular genetic trait of the high oleic/no HFA mutation in order to inform breeding practices of lesquerella for altered HFA levels. Fatty acid profiles were obtained for forty S1 and thirty-six S2 half-seeds from the mutant line. The oleate 12-hydroxylase gene (LFAH12) was investigated using Southern blot analysis, PCR, RT-PCR and chromosomal walking to determine the gene number, DNA sequence and expression patterns for a control and mutant. HFA inheritance and Southern blot analyses data indicate the LFAH12 gene is present in as one copy. Further, the mutant phenotype segregates in 1 to 3 ratio revealing that it is a recessive trait. Chromosomal walking uncovered a large indel in the 3’ end of the LFAH12 mutant and a corresponding premature stop codon, resulting in gene truncated by 29 amino acid residues. RT-PCR analysis of the LFAH12 mutant in developing seeds indicates that the gene is expressed, although at lower levels than the control - results that may be expected from nonsense-mediated mRNA decay. No other noticeable phenotypic differences were observed in the mutant. The isolation and molecular genetic characterization of the LFAH12 mutant benefits selection for altered HFA levels in lesquerella by providing a null parent for selecting high HFA alleles by crossing or for transgenic HFA programs.

Technical Abstract: Lesquerella fendleri (Gray) Wats. (Brassicaceae) is being developed as a new crop for its seed oil that is typically comprised of ca. 55 percent of the hydroxy fatty acid (HFA) lesquerolic acid. Most breeding efforts for lesquerella have been geared towards improving harvest index, seed yield or production practices, with little attention paid to modifying seed oil composition. However, with the recent application of the half-seed method to our breeding population, we have identified greater variability in the fatty acid (FA) composition in the seed oil than had previously been characterized, including a mutant with high oleic and no HFA. The objectives of this study was to characterize the underlying molecular genetic trait of the high oleic/no HFA mutation in order to inform breeding practices of lesquerella for altered HFA levels. Fatty acid profiles were obtained for forty S1 and thirty-six S2 half-seeds from the mutant line. The oleate 12-hydroxylase gene (LFAH12) was investigated using Southern blot analysis, PCR, RT-PCR and chromosomal walking to determine the gene number, DNA sequence and expression patterns for a control and mutant. HFA inheritance and Southern blot analyses data indicate the LFAH12 gene is present in as one copy. Further, the mutant phenotype segregates in 1 to 3 ratio revealing that it is a recessive trait. Chromosomal walking uncovered a large indel in the 3’ end of the LFAH12 mutant and a corresponding premature stop codon, resulting in gene truncated by 29 amino acid residues. RT-PCR analysis of the LFAH12 mutant in developing seeds indicates that the gene is expressed, although at lower levels than the control - results that may be expected from nonsense-mediated mRNA decay. No other noticeable phenotypic differences were observed in the mutant. The isolation and molecular genetic characterization of the LFAH12 mutant benefits selection for altered HFA levels in lesquerella by providing a null parent for selecting high HFA alleles by crossing or for transgenic HFA programs.