Genotyping and lipid profiling of 601 cultivated sunflower lines reveals novel genetic determinants of oil fatty acid content.

BACKGROUND: Sunflower is an important oilseed crop domesticated in North America approximately 4000 years ago. During the last century, oil content in sunflower was under strong selection. Further improvement of oil properties achieved by modulating its fatty acid composition is one of the main directions in modern oilseed crop breeding. RESULTS: We searched for the genetic basis of fatty acid content variation by genotyping 601 inbred sunflower lines and assessing their lipid and fatty acid composition. Our genome-wide association analysis based on the genotypes for 15,483 SNPs and the concentrations of 23 fatty acids, including minor fatty acids, revealed significant genetic associations for eleven of them. Identified genomic regions included the loci involved in rare fatty acids variation on chromosomes 3 and 14, explaining up to 34.5% of the total variation of docosanoic acid (22:0) in sunflower oil. CONCLUSIONS: This is the first large scale implementation of high-throughput lipidomic profiling to sunflower germplasm characterization. This study contributes to the genetic characterization of Russian sunflower collections, which made a substantial contribution to the development of sunflower as the oilseed crop worldwide, and provides new insights into the genetic control of oil composition that can be implemented in future studies.

QTL mapping of oleic acid content in modern VNIIMK sunflower (Helianthus annuus L.) lines by using GBS-based SNP map.

Oleic acid is a monounsaturated fatty acid increasing oil oxidative stability. High content of oleic acid is thus a valuable trait in oilseed crops. Sunflower (Helianthus annuus L.) normally accumulates linoleic acid as a major fatty acid, but a mutant expressing a high oleic phenotype form was previously obtained by chemical mutagenesis and mapped on the sunflower genome. Several studies suggest the presence of additional genes involved in the control of the high content of oleic acid, with their expression possibly depending on the genetic background. To test this hypothesis, we performed a QTL mapping of the high oleic acid trait within two independent F2 crosses involving lines with contrasting oleic acid content from the Pustovoit All-Russia Research Institute of Oil Crops (VNIIMK) collection. We applied genotyping-by-sequencing (GBS) to construct single nucleotide polymorphism-based genetic maps and performed QTL mapping using quantitative and qualitative encoding for oleic acid content. Our results support the finding that the oleic acid content in the assessed crosses is controlled by one major effect locus. However, different dominant/recessive effects of the major locus were reported for both crosses. Additionally, a possible translocation between chromosome 7 and 14 was reported in one assessed cross. We defined a set of single nucleotide polymorphism markers for each cross which could be used for marker-assisted selection.

Genetic mapping of loci involved in oil tocopherol composition control in Russian sunflower (Helianthus annuus L.) lines.

Tocopherols are antioxidants that preserve oil lipids against oxidation and serve as a natural source of vitamin E in the human diet. Compared with other major oilseeds like rapeseed and soybean, sunflower (Helianthus annuus L.) exhibits low phenotypic diversity of tocopherol composition, both in wild and cultivated accessions from germplasm collections. Two major mutations that alter tocopherol composition were identified in genetic collections, and several studies suggested additional loci controlling tocopherol composition, with their expression possibly depending on the genetic background. In the present study, we performed QTL mapping of tocopherol composition in two independent F2 crosses between lines with contrasting tocopherol composition from the Pustovoit All-Russia Research Institute of Oil Crops (VNIIMK) collection. We used genotyping-bysequencing (GBS) to construct single nucleotide polymorphism-based genetic maps, and performed QTL mapping using quantitative and qualitative encoding for phenotypic traits. Our results support the notion that the tocopherol composition in the assessed crosses is controlled by two loci. We additionally selected and validated two single nucleotide polymorphism markers for each cross which could be used for marker-assisted selection.

Genetic Characterization of Russian Rapeseed Collection and Association Mapping of Novel Loci Affecting Glucosinolate Content.

Rapeseed is the second most common oilseed crop worldwide. While the start of rapeseed breeding in Russia dates back to the middle of the 20th century, its widespread cultivation began only recently. In contrast to the world's rapeseed genetic variation, the genetic composition of Russian rapeseed lines remained unexplored. We have addressed this question by performing genome-wide genotyping of 90 advanced rapeseed accessions provided by the All-Russian Research Institute of Oil Crops (VNIIMK). Genome-wide genetic analysis demonstrated a clear difference between Russian rapeseed varieties and the rapeseed varieties from the rest of the world, including the European ones, indicating that rapeseed breeding in Russia proceeded in its own independent direction. Hence, genetic determinants of agronomical traits might also be different in Russian rapeseed lines. To assess it, we collected the glucosinolate content data for the same 90 genotyped accessions obtained during three years and performed an association mapping of this trait. We indeed found that the loci significantly associated with glucosinolate content variation in the Russian rapeseed collection differ from those previously reported for the non-Russian rapeseed lines.

Ultra-performance liquid chromatography-mass spectrometry for precise fatty acid profiling of oilseed crops.

Oilseed crops are one of the most important sources of vegetable oils for food and industry. Nutritional and technical properties of vegetable oil are primarily determined by its fatty acid (FA) composition. The content and composition of FAs in plants are commonly determined using gas chromatography-mass spectrometry (GS-MS) or gas chromatography-flame ionization detection (GC-FID) techniques. In the present work, we applied ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) technique to FA profiling of sunflower and rapeseed seeds and compared this method with the GC-FID technique. GC-FID detected 11 FAs in sunflower and 13 FAs in rapeseed, while UPLC-MS appeared to be more sensitive, detecting about 2.5 times higher numbers of FAs in both plants. In addition to even-chain FAs, UPLC-MS was able to detect odd-chain FAs. The longest FA detected using GC-FID was an FA with 24 carbon atoms, whereas UPLC-MS could reveal the presence of longer FAs with the tails of up to 28 carbon atoms. Based on our results, we may conclude that UPLC-MS has great potential to be used for the assessment of FA profiles of oil crops.

UPLC⁻MS Triglyceride Profiling in Sunflower and Rapeseed Seeds.

Sunflower and rapeseed are among the most important sources of vegetable oil for food and industry. The main components of vegetable oil are triglycerides (TAGs) (about 97%). Ultra- performance liquid chromatography coupled with mass spectrometry (UPLC⁻MS) profiling of TAGs in sunflower and rapeseed has been performed and the TAG profiles obtained for these species have been compared. It has been identified that 34 TAGs are shared by sunflower and rapeseed. It was demonstrated that TAGs 52:2, 52:5, 52:6, 54:3; 54:4, 54:7, 56:3, 56:4, and 56:5 had the highest variability levels between sunflower and rapeseed with the higher presence in rapeseed. TAGs 50:2, 52:3, 52:4, 54:5, and 54:6 also showed high variability, but were the most abundant in sunflower. Moreover, the differences in TAG composition between the winter-type and spring-type rapeseed have been revealed, which may be associated with freezing tolerance. It was shown that winter-type rapeseed seeds contain TAGs with a lower degree of saturation, while in spring-type rapeseed highly saturated lipids are the most abundant. These findings may give new insights into the cold resistance mechanisms in plants the understanding of which is especially important in terms of global climate changes.

Active Nuclear Import of Membrane Proteins Revisited.

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast.