Generation of high oleic acid sunflower lines using gamma radiation mutagenesis and high-throughput fatty acid profiling

Sunflower (Helianthus annuus L.) is the second most important oil seed crop in Europe. The seeds are used as confection seeds and, more importantly, to generate an edible vegetable oil, which in normal varieties is rich in the polyunsaturated fatty acid linoleic acid. Linoleic acid is biosynthesized from oleic acid through activity of the oleate desaturase FATTY ACID DESATURASE 2 (FAD2), which in seeds is encoded by FAD2-1, a gene that’s present in single copy in sunflowers. Defective FAD2-1 expression enriches oleic acid, yielding the high oleic (HO) acid trait, which is of great interest in oil seed crops, since HO oil bears benefits for both food and non-food applications. Chemical mutagenesis has previously been used to generate sunflower mutants with reduced FAD2-1 expression and here it was aimed to produce further genetic material in which FAD2-1 activity is lost and the HO trait is stably expressed. For this purpose, a sunflower mutant population was created using gamma irradiation and screened for fad2-1 mutants with a newly developed HPLC-based fatty-acid profiling system that’s suitable for high-throughput analyses. With this approach fad2-1 knock-out mutants could be isolated, which stably hyper-accumulate oleic acid in concentrations of 85-90% of the total fatty acid pool. The genetic nature of these new sunflower lines was characterized and will facilitate marker development, for the rapid introgression of the trait into elite sunflower breeding material

BSTA: a targeted approach combines bulked segregant analysis with next- generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower

BackgroundSunflower belongs to the largest plant family on earth, the genomically poorly explored Compositae. Downy mildew Plasmopara halstedii (Farlow) Berlese & de Toni is one of the major diseases of cultivated sunflower (Helianthus annuus L.). In the search for new sources of downy mildew resistance, the locus Pl(ARG)on linkage group 1 (LG1) originating from H. argophyllus is promising since it confers resistance against all known races of the pathogen. However, the mapping resolution in the Pl(ARG) region is hampered by significantly suppressed recombination and by limited availability of polymorphic markers. Here we examined a strategy developed for the enrichment of molecular markers linked to this specific genomic region. We combined bulked segregant analysis (BSA) with next-generation sequencing (NGS) and de novo assembly of the sunflower transcriptome for single nucleotide polymorphism (SNP) discovery in a sequence resource combining reads originating from two sunflower species, H. annuus and H. argophyllus.ResultsA computational pipeline developed for SNP calling and pattern detection identified 219 candidate genes. For a proof of concept, 42 resistance gene-like sequences were subjected to experimental SNP validation. Using a high-resolution mapping population, 12 SNP markers were mapped to LG1. We successfully verified candidate sequences either co-segregating with or closely flanking Pl(ARG).ConclusionsThis study is the first successful example to improve bulked segregant analysis with de novo transcriptome assembly using next generation sequencing. The BSTA pipeline we developed provides a useful guide for similar studies in other non-model organisms. Our results demonstrate this method is an efficient way to enrich molecular markers and to identify candidate genes in a specific mapping interval

BSTA: a targeted approach combines bulked segregant analysis with next- generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower

BACKGROUND: Sunflower belongs to the largest plant family on earth, the genomically poorly explored Compositae. Downy mildew Plasmopara halstedii (Farlow) Berlese & de Toni is one of the major diseases of cultivated sunflower (Helianthus annuus L.). In the search for new sources of downy mildew resistance, the locus Pl(ARG) on linkage group 1 (LG1) originating from H. argophyllus is promising since it confers resistance against all known races of the pathogen. However, the mapping resolution in the Pl(ARG) region is hampered by significantly suppressed recombination and by limited availability of polymorphic markers. Here we examined a strategy developed for the enrichment of molecular markers linked to this specific genomic region. We combined bulked segregant analysis (BSA) with next-generation sequencing (NGS) and de novo assembly of the sunflower transcriptome for single nucleotide polymorphism (SNP) discovery in a sequence resource combining reads originating from two sunflower species, H. annuus and H. argophyllus. RESULTS: A computational pipeline developed for SNP calling and pattern detection identified 219 candidate genes. For a proof of concept, 42 resistance gene-like sequences were subjected to experimental SNP validation. Using a high-resolution mapping population, 12 SNP markers were mapped to LG1. We successfully verified candidate sequences either co-segregating with or closely flanking Pl(ARG). CONCLUSIONS: This study is the first successful example to improve bulked segregant analysis with de novo transcriptome assembly using next generation sequencing. The BSTA pipeline we developed provides a useful guide for similar studies in other non-model organisms. Our results demonstrate this method is an efficient way to enrich molecular markers and to identify candidate genes in a specific mapping interval