Development and Validation of Kompetitive Allele-Specific PCR Assays for Erucic Acid Content in Indian Mustard [Brassica juncea (L.) Czern and Coss.]

Brassica juncea L. is the most widely cultivated oilseed crop in Indian subcontinent. Its seeds contain oil with very high concentration of erucic acid (≈50%). Of late, there is increasing emphasis on the development of low erucic acid varieties because of reported association of the consumption of high erucic acid oil with cardiac lipidosis. Erucic acid is synthesized from oleic acid by an elongation process involving two cycles of four sequential steps. Of which, the first step is catalyzed by β-ketoacyl-CoA synthase (KCS) encoded by the fatty acid elongase 1 (FAE1) gene in Brassica. Mutations in the coding region of the FAE1 lead to the loss of KCS activity and consequently a drastic reduction of erucic acid in the seeds. Molecular markers have been developed on the basis of variation available in the coding or promoter region(s) of the FAE1. However, majority of these markers are not breeder friendly and are rarely used in the breeding programs. Present studies were planned to develop robust kompetitive allele-specific PCR (KASPar) assays with high throughput and economics of scale. We first cloned and sequenced FAE1.1 and FAE1.2 from high and low erucic acid (<2%) genotypes of B. juncea (AABB) and its progenitor species, B. rapa (AA) and B. nigra (BB). Sequence comparisons of FAE1.1 and FAE1.2 genes for low and high erucic acid genotypes revealed single nucleotide polymorphisms (SNPs) at 8 and 3 positions. Of these, three SNPs for FAE1.1 and one SNPs for FAE1.2 produced missense mutations, leading to amino acid modifications and inactivation of KCS enzyme. We used SNPs at positions 735 and 1,476 for genes FAE1.1 and FAE1.2, respectively, to develop KASPar assays. These markers were validated on a collection of diverse genotypes and a segregating backcross progeny. KASPar assays developed in this study will be useful for marker-assisted breeding, as these can track recessive alleles in their heterozygous state with high reproducibility.

Development of B. carinata with super-high erucic acid content through interspecific hybridization

Key message Disomic alien chromosome addition Brassica carinata lines with super-high erucic acid content were developed through interspecific hybridization with B. juncea and characterized using molecular, cytological and biochemical techniques. Abstract Brassica carinata [A.] Braun (BBCC, 2n = 34) is a climate-resilient oilseed. Its seed oil is high in erucic acid (> 40%), rendering it well suited for the production of biofuel and other bio-based applications. To enhance the competitiveness of B. carinata with high erucic B. napus (HEAR), lines with super-high erucic acid content were developed through interspecific hybridization. To this end, a fad2B null allele from Brassica juncea (AABB, 2n = 36) was introgressed into B. carinata, resulting in a B. carinata fad2B mutant with erucic acid levels of over 50%. Subsequently, the FAE allele from B. rapa spp. yellow sarson (AA, 2n = 20) was transferred to the fad2B B. carinata line, yielding lines with erucic acid contents of up to 57.9%. Molecular analysis using the Brassica 90 K Illumina Infinium™ SNP genotyping array identified these lines as disomic alien chromosome addition lines, with two extra A08 chromosomes containing the BrFAE gene. The alien chromosomes from B. rapa were clearly distinguished by molecular cytogenetics in one of the addition lines. Analysis of microspore-derived offspring and hybrids from crosses with a CMS B. carinata line showed that the transfer rate of the A08 chromosome into male gametes was over 98%, resulting in almost completely stable transmission of an A08 chromosome copy into the progeny. The increase in erucic acid levels was accompanied by changes in the proportions of other fatty acids depending on the genetic changes that were introduced in the interspecific hybrids, providing valuable insights into erucic acid metabolism in Brassica.

Evolve Roundup Ready® high erucic acid, low glucosinolate hybrid summer rape

Evolve is the world’s first ogu INRA (Institut national de la recherche agronomique) CMS (cytoplasmic male sterility) hybrid summer rape (Brassica napus L.) Roundup Ready® high erucic acid, low glucosinolate cultivar. On average, Evolve yielded 12.4% more seed, 6 g kg−1 more seed oil, and 5 g kg−1 less meal protein than Red River 1861, an open-pollinated Roundup Ready® high erucic acid, low glucosinolate summer rape. Evolve has an erucic acid content of 52.3% in isolated field trials of high erucic acid rapeseed genotypes and is adapted to the southern B. napus growing regions of western Canada.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver

Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague–Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal β-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal β-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal β-oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid–induced hepatic steatosis and related metabolic disorders.

EFFECT OF TEMPERATURE AND EXCESS AIR RATIO ON COMBUSTION PROCESS OF MIXTURES OF ERUCIC RAPESEED

The paper presents results of investigations into diesel engine fuelled with high erucic acid rapeseed oil and its mixture with diesel, petrol and ethanol at different proportions. The study was performed in a chamber with constant volume depending on temperature and air pressure and the coefficient of excess air. The main purpose of performed tests was to determine the effect of various parameters on processes of spontaneous combustion and combustion of fuels. During the study basic parameters of combustion, e.g. auto-ignition delay, greatest pressure and contractual time were compared. Studies show that increased pressure injection improves combustion process for all test fuels, and injection pressure most strongly affect the combustion process of rapeseed oil and its mixtures with ethanol.

HYHEAR 1 hybrid Roundup Ready™ high erucic acid, low glucosinolate summer rape

McVetty, P. B. E., Cuthbert, J. L., Marwede, V., Paulmann, W., Sass, O., Duncan, R. W., Fernando, W. G. D., Li, G. and Zelmer, C. D. 2014. HYHEAR 1 hybrid Roundup Ready™ high erucic acid, low glucosinolate summer rape. Can. J. Plant Sci. 94: 453–455. HYHEAR 1 summer rape (Brassica napus L.) is the world's first hybrid Roundup Ready™ high erucic acid, low glucosinolate cultivar. On average, HYHEAR 1 yielded 33% more seed, 23 g kg−1 more seed oil but 17 g kg −1 less meal protein than MillenniUM 03 high erucic acid, low glucosinolate, open pollinated population summer rape. HYHEAR 1 has an erucic acid content of 52.2% in isolated field trials of HEAR lines and is adapted to the southern B. napus growing regions of western Canada.