Local Duplication of TIR-NBS-LRR Gene Marks Clubroot Resistance in Brassica napus cv. Tosca.

Clubroot, caused by Plasmodiophora brassicae infection, is a disease of growing importance in cruciferous crops, including oilseed rape (Brassica napus). The affected plants exhibit prominent galling of the roots that impairs their capacity for water and nutrient uptake, which leads to growth retardation, wilting, premature ripening, or death. Due to the scarcity of effective means of protection against the pathogen, breeding of resistant varieties remains a crucial component of disease control measures. The key aspect of the breeding process is the identification of genetic factors associated with variable response to the pathogen exposure. Although numerous clubroot resistance loci have been described in Brassica crops, continuous updates on the sources of resistance are necessary. Many of the resistance genes are pathotype-specific, moreover, resistance breakdowns have been reported. In this study, we characterize the clubroot resistance locus in the winter oilseed rape cultivar "Tosca." In a series of greenhouse experiments, we evaluate the disease severity of P. brassicae-challenged "Tosca"-derived population of doubled haploids, which we genotype with Brassica 60 K array and a selection of SSR/SCAR markers. We then construct a genetic map and narrow down the resistance locus to the 0.4 cM fragment on the A03 chromosome, corresponding to the region previously described as Crr3. Using Oxford Nanopore long-read genome resequencing and RNA-seq we review the composition of the locus and describe a duplication of TIR-NBS-LRR gene. Further, we explore the transcriptomic differences of the local genes between the clubroot resistant and susceptible, inoculated and control DH lines. We conclude that the duplicated TNL gene is a promising candidate for the resistance factor. This study provides valuable resources for clubroot resistance breeding programs and lays a foundation for further functional studies on clubroot resistance.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene.

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low ("00") cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new "00" varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5'-C↓TAG-3' sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Marker assisted selection of new high oleic and low linolenic winter oilseed rape (Brassica napus L.) inbred lines revealing good agricultural value.

Development of oilseed rape (Brassica napus L.) breeding lines producing oil characterized by high oleic and low linolenic acid content is an important goal of rapeseed breeding programs worldwide. Such kind of oil is ideal for deep frying and can also be used as a raw material for biodiesel production. By performing chemical mutagenesis using ethyl methanesulfonate, we obtained mutant winter rapeseed breeding lines that can produce oil with a high content of oleic acid (C18:1, more than 75%) and a low content of linolenic acid (C18:3, less than 3%). However, the mutant lines revealed low agricultural value as they were characterized by low seed yield, low wintering, and high content of glucosinolates in seed meal. The aim of this work was to improve the mutant lines and develop high-oleic and low-linolenic recombinants exhibiting both good oil quality and high agronomic value. The plant materials used in this study included high-oleic and low-linolenic mutant breeding lines and high-yielding domestic canola-type breeding lines of good agricultural value with high oleic acid content and extremely low glucosinolates content. Field trials were conducted in four environments, in a randomized complete block design. Phenotyping was performed for wintering, yield of seed and oil, and seed quality traits. Genotype × environment interaction was investigated with respect to the content of C18:1 and C18:3 acids in seed oil. Genotyping was done for the selection of homozygous high oleic and low linolenic lines using allele-specific CAPS markers and SNaPshot assay, respectively. Finally, new high oleic and low linolenic winter rapeseed recombinant lines were obtained for use as a starting material for the development of new varieties that may be of high value on the oil crop market.

The effect of drying on the native tocopherol and phytosterol content of Sinapis alba L. seeds.

BACKGROUND: Postharvest processing, including drying, has a significant effect on the processability of oil crop seeds. High drying air temperature may significantly affect the levels of bioactive compounds in plant raw materials. We decided to investigate the subject given the lack of data on the drying of mustard seeds. The aim was to determine the effect of drying temperature on free fatty acid, phytosterol and tocopherol levels in the oil obtained from white mustard seeds. Seeds were dried in a thin layer at 40, 60, 80, 100, 120 and 140 °C. Changes in phytosterol levels were assessed using gas chromatography-mass spectrometry and changes in tocopherol levels by high-performance liquid chromatography. RESULTS: The analysis showed that, upon completion of air drying at 40-100 °C, losses of sterols did not exceed 5%, while upon completion of drying at 120 °C and 140 °C these losses amounted to 17-50%. Our investigation also showed that during drying of white mustard seed the total tocopherol levels increased, and the higher the drying temperature, the greater the increment. In seeds air-dried at 120 °C and 140 °C, the increase in tocopherol amounted to 7-9%. CONCLUSION: We also showed that the temperature of the drying agent significantly affects the tocopherol and phytosterol levels. It was found that a maximum temperature of 100 °C provides optimal drying conditions for mustard seeds. Principal component analysis identified two subgroups of oils obtained from seeds dried at 120 °C and 140 °C, which differed considerably from the other samples in terms of their bioactive component contents. © 2019 Society of Chemical Industry.

Genetic and Molecular Regulation of Seed Storage Proteins (SSPs) to Improve Protein Nutritional Value of Oilseed Rape (Brassica napus L.) Seeds.

The world-wide demand for additional protein sources for human nutrition and animal feed keeps rising due to rapidly growing world population. Oilseed rape is a second important oil producing crop and the by-product of the oil production is a protein rich meal. The protein in rapeseed meal finds its application in animal feed and various industrial purposes, but its improvement is of great interest, especially for non-ruminants and poultry feed. To be able to manipulate the quality and quantity of seed protein in oilseed rape, understanding genetic architecture of seed storage protein (SSPs) synthesis and accumulation in this crop species is of great interest. For this, application of modern molecular breeding tools such as whole genome sequencing, genotyping, association mapping, and genome editing methods implemented in oilseed rape seed protein improvement would be of great interest. This review examines current knowledge and opportunities to manipulate of SSPs in oilseed rape to improve its quality, quantity and digestibility.

Antioxidant (Tocopherol and Canolol) Content in Rapeseed Oil Obtained from Roasted Yellow-Seeded Brassica napus.

In this study, the effect of temperature (140, 160, 180 °C) and roasting time (5, 10, 15 min) on the bioactive compound content (canolol, tocopherol and plastochromanol-8) of cold-pressed oil from yellow-seeded rapeseed lines of different colors was investigated. Roasting increased the peroxide value in the seed oils compared to the oils from the control samples. However, roasting did not affect the acid values of the oils, which were 1.15-1.47 and 1.30-1.40 mg KOH/g, for line PN1 03/1i/14 (yellow seeds) and line PN1 563/1i/14 (brown seeds), respectively. In this study, the seeds of line PN1 03/1i/14 were characterized by different changes in canolol content during roasting than the seeds of PN1 563/1i/14. There was a 90-fold increase in canolol for the line PN1 03/1i/14 (768.26 µg/g) and a 46-fold increase for the line PN1 563/1i/14 (576.43 µg/g). Changes in tocopherol and PC-8 contents were also observed. There was an increase in the contents of γ-T and PC-8 in the oils obtained from the seeds roasted at 180 °C for 10 and 15 min. γ-T content increased by 17-18% after 15 min of roasting, whereas the PC-8 content increased twofold.

Genome-Wide Association Study of Genetic Control of Seed Fatty Acid Biosynthesis in Brassica napus.

Fatty acids and their composition in seeds determine oil value for nutritional or industrial purposes and also affect seed germination as well as seedling establishment. To better understand the genetic basis of seed fatty acid biosynthesis in oilseed rape (Brassica napus L.) we applied a genome-wide association study, using 91,205 single nucleotide polymorphisms (SNPs) characterized across a mapping population with high-resolution skim genotyping by sequencing (SkimGBS). We identified a cluster of loci on chromosome A05 associated with oleic and linoleic seed fatty acids. The delineated genomic region contained orthologs of the Arabidopsis thaliana genes known to play a role in regulation of seed fatty acid biosynthesis such as Fatty acyl-ACP thioesterase B (FATB) and Fatty Acid Desaturase (FAD5). This approach allowed us to identify potential functional genes regulating fatty acid composition in this important oil producing crop and demonstrates that this approach can be used as a powerful tool for dissecting complex traits for B. napus improvement programs.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress.

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Determination of fatty acid composition in seed oil of rapeseed (Brassica napus L.) by mutated alleles of the FAD3 desaturase genes.

One of the goals in oilseed rape programs is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute - NRI, in Poznan, Poland, and allele-specific SNP markers were designed for monitoring of two statistically important single nucleotide polymorphisms detected by SNaPshot analysis in two FAD3 desaturase genes, BnaA.FAD3 and BnaC.FAD3, respectively. Strong negative correlation between the presence of mutant alleles of the genes and linolenic acid content was revealed by analysis of variance. In this paper we present detailed characteristics of the markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil, as well as by calculation of the phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker. The obtained percentage of variation in fatty acid composition was considerable only for linolenic acid content and equaled 35.6% for BnaA.FAD3 and 39.3% for BnaC.FAD3, whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously. Our results revealed high specificity of the markers for effective monitoring of the wild-type and mutated alleles of the Brassica napus FAD3 desaturase genes in the low linolenic mutant recombinants in breeding programs.

Genetic divergence is not the same as phenotypic divergence.

Far too often, phenotypic divergence has been misinterpreted as genetic divergence, and based on phenotypic divergence, genetic divergence has been indicated. We have attempted to disprove this statement and call for the differentiation of phenotypic and genotypic variation.

Antioxidant capacity, total phenolics, glucosinolates and colour parameters of rapeseed cultivars.

The antioxidant capacity of twenty nine rapeseed varieties was determined by using ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods. Mean FRAP (3190-6326µmol Trolox/100g) and DPPH (3194-6346µmol Trolox/100g) values for methanolic extracts of rapeseed cultivars did not differ significantly. Moreover, the total content of phenolics (756-1324mg sinapic acid/100g), glucosinolates (4.2-87.5µmol/g, respectively), erucic acid (0.0-56.1%) and colour parameters of the studied rapeseed cultivars were analysed. Antioxidant capacity determined by FRAP and DPPH methods correlated significantly with total phenolic content (TPC) in rapeseed cultivars (r=0.9332, 0.9339, p<0.001). Also, significant, inverse correlations were found between antioxidant capacity, total phenolics and luminosity (L(∗)) or red colour intensity (a(∗)) of rapeseed cultivars. Principal component analysis (PCA) allowed the rapeseed varieties to be differentiated based on their antioxidant capacities, total amounts of phenolics, glucosinolates, erucic acid and colour parameters.