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Development of low-linolenic acid Brassica oleracea lines through seed mutagenesis and molecular characterization of mutants

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Abstract

Designing the fatty acid composition of Brassica napus L. seed oil for specific applications would extend the value of this crop. A mutation in Fatty Acid Desaturase 3 (FAD3), which encodes the desaturase responsible for catalyzing the formation of α-linolenic acid (ALA; 18:3cisΔ9,12,15), in a diploid Brassica species would potentially result in useful germplasm for creating an amphidiploid displaying low ALA content in the seed oil. For this, seeds of B. oleracea (CC), one of the progenitor species of B. napus, were treated with ethyl-methane-sulfonate to induce mutations in genes encoding enzymes involved in fatty acid biosynthesis. Seeds from 1,430 M2 plants were analyzed, from which M3 seed families with 5.7–6.9 % ALA were obtained. Progeny testing and selection for low ALA content were carried out in M3–M7 generations, from which mutant lines with <2.0 % ALA were obtained. Molecular analysis revealed that the mutation was due to a single nucleotide substitution from G to A in exon 3 of FAD3, which corresponds to an amino acid residue substitution from glutamic acid to lysine. No obvious differences in the expression of the FAD3 gene were detected between wild type and mutant lines; however, evaluation of the performance of recombinant Δ-15 desaturase from mutant lines in yeast indicated reduced production of ALA. The novelty of this mutation can be inferred from the position of the point mutation in the C-genome FAD3 gene when compared to the position of mutations reported previously by other researchers. This B. oleracea mutant line has the potential to be used for the development of low-ALA B. napus and B. carinata oilseed crops.

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Acknowledgments

HR and RJW are grateful for the support provided by AVAC Ltd, the Canada Foundation for Innovation and the Research Capacity Program of Alberta Enterprise and Advanced Education. RJW is also grateful for the support provided by the Alberta Innovates Bio Solutions, Canada Research Chairs Program and the Natural Sciences and Engineering Research Council of Canada. The authors also thank Dr. Mohan Thiagarajah for suggestions on EMS treatments, Dr. Nidhi Sharma for collecting developing siliques, Ms. An Vo for FA analysis of the mutagenized populations, and other laboratory staff for the technical assistance provided.

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Authors and Affiliations

  1. Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada

    Habibur Rahman, Stacy D. Singer & Randall J. Weselake

Authors
  1. Habibur Rahman
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  2. Stacy D. Singer
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  3. Randall J. Weselake
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Correspondence to Habibur Rahman.

Additional information

Communicated by C. Quiros.

Electronic supplementary material

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122_2013_2076_MOESM1_ESM.doc

Supplementary material 1 Fig. S1 Sequence alignment of wt BoFAD3-1 from B. oleracea var. alboglabra and BnaC.FAD3.b (ortholog) from B. napus as reported by Yang et al. (2012). ‘Asterisks’ indicates identical nucleotide identities (DOC 1909 kb)

122_2013_2076_MOESM2_ESM.doc

Supplementary material 2 Fig. S2 Sequence alignment of wt BoFAD3-2 from B. oleracea var. alboglabra and BnaC.FAD3.a (ortholog) from B. napus as reported by Yang et al. (2012). ‘Asterisks’ indicates identical nucleotide identities (DOC 1935 kb)

122_2013_2076_MOESM3_ESM.doc

Supplementary material 3 Fig. S3 Comparison of BoFAD3-1 (top) and BoFAD3-2 (bottom) amino acid sequences from wt Brassica oleracea var. alboglabra. ‘Asterisk’ indicates identical amino acid identities; ‘colon’ denotes conserved substitutions; ‘dot’ designates semi-conserved substitutions (DOC 612 kb)

122_2013_2076_MOESM4_ESM.doc

Supplementary material 4 Fig. S4 Semi-quantitative RT-PCR of BoFAD3-1 and BoFAD3-2 expression in wt and low-linolenic mutant B. oleracea var. alboglabra lines. NTC, no template control; M, molecular weight ladder (DOC 910 kb)

122_2013_2076_MOESM5_ESM.doc

Supplementary material 5 Fig. S5 Analysis of desaturase activity of wt and mutant BoFAD3-1 in yeast by GC–MS. FAMEs of total lipids of Saccharomyces cerevisiae grown under inducing conditions in the presence (a) or absence (b) of 150 µM exogenously supplied C18:2. Chromatograms shown are representative of yeast bearing either wt BoFAD3-1 (wt), mutant BoFAD3-1 (mut) or empty vector (EV). Common peaks were identified as C14:0 (peak 1), C15:0 (peak 2), C15:1 (peak 3), C16:0 (peak 4), C16:1 (peak 5), C18:0 (peak 6) and C18:1 (peak 7). While C18:2 (peak 8) was produced in all lines, the level differed dramatically depending on whether the culture had been supplemented with exogenous C18:2. The standard, C17:0 (STD) is also denoted in each case. The additional C18:3 generated by the wt and mutant BoFAD3-1 enzymes in the C18:2 supplemented cultures (a) are indicated by arrows (DOC 474 kb)

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Rahman, H., Singer, S.D. & Weselake, R.J. Development of low-linolenic acid Brassica oleracea lines through seed mutagenesis and molecular characterization of mutants. Theor Appl Genet 126, 1587–1598 (2013). https://doi.org/10.1007/s00122-013-2076-y

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