Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica

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Abstract

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.
Original languageEnglish
Pages (from-to)471-481
JournalPlant Biotechnology Journal
Volume13
Issue number4
DOIs
Publication statusPublished - 2015

Fingerprint

Crambe Plant
Crambe abyssinica
mutagenesis
Oils
mutation
oils
Mutation
polyunsaturated fatty acids
Genes
Crambe (Brassicaceae)
genes
nonsense mutation
Unsaturated Fatty Acids
missense mutation
fatty acid composition
erucic acid
oil crops
industrial applications
hexaploidy
polyploidy

Keywords

  • induced point mutations
  • crop improvement
  • reverse genetics
  • oleic-acid
  • functional genomics
  • fad2 gene
  • discovery
  • arabidopsis
  • populations
  • cloning

Cite this

@article{7af7b5f9b53442a2bc96d381c26373f3,
title = "Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica",
abstract = "Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25{\%} higher content of C18:1 and 25{\%} lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.",
keywords = "induced point mutations, crop improvement, reverse genetics, oleic-acid, functional genomics, fad2 gene, discovery, arabidopsis, populations, cloning",
author = "J. Cheng and E.M.J. Salentijn and {Huang Bangquan} and C. Denneboom and A.C. Dechesne and F.A. Krens and R.G.F. Visser and {van Loo}, E.N.",
year = "2015",
doi = "10.1111/pbi.12269",
language = "English",
volume = "13",
pages = "471--481",
journal = "Plant Biotechnology Journal",
issn = "1467-7644",
publisher = "Wiley",
number = "4",

}

TY - JOUR

T1 - Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica

AU - Cheng, J.

AU - Salentijn, E.M.J.

AU - Huang Bangquan, null

AU - Denneboom, C.

AU - Dechesne, A.C.

AU - Krens, F.A.

AU - Visser, R.G.F.

AU - van Loo, E.N.

PY - 2015

Y1 - 2015

N2 - Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.

AB - Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.

KW - induced point mutations

KW - crop improvement

KW - reverse genetics

KW - oleic-acid

KW - functional genomics

KW - fad2 gene

KW - discovery

KW - arabidopsis

KW - populations

KW - cloning

U2 - 10.1111/pbi.12269

DO - 10.1111/pbi.12269

M3 - Article

VL - 13

SP - 471

EP - 481

JO - Plant Biotechnology Journal

JF - Plant Biotechnology Journal

SN - 1467-7644

IS - 4

ER -