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.

Morphological Formation, Fatty Acid Profile, and Molecular Identification of Some Landraces of Ethiopian Brassica as a Promising Crop to Support Breeding Programs

There has been an increased interest in oilseed crops for agro-industry research and development breeding programs to secure sustainable food and agriculture. The introgression of exotic genotypes of oilseed Brassica into cultivated relatives is inevitable in the genetic improvement of oilseed crops. This experimental attempt aimed to characterize the morphological and molecular basis for the identification and characterization of some Brassica genotypes. Fatty acid profile, yield, and morphology are under genetic control and can be used to identify genotypes. Characterization and identification were fulfilled for five accessions from Brassica spp. Plant height, height of first branch, number of branches and pods per plant, seed yield per plant, average pod length, number of seeds per pod, protein and oil contents (%), and fatty acid profile were examined. Besides, the relationship between seed yield and seed yield-contributing characteristics was estimated, as well as the phylogenetic relationship of the internal transcribed spacer (ITS). The genotypes varied significantly for all examined traits, taking into account the most important traits: seed yield per plant and oil content. For example, oil content in the samples ranged between 41.1 and 49.3%. Path analysis results showed a high and positive direct effect between each number of primary branches and the number of pods per plant with seed yield per plant (0.48). The morphological and molecular observations suggest that the Fay1, Fay3, Fay4, and Fay6 accessions belong to Brassica rapa, while Fay2 belongs to Brassica carinata. It can be concluded based on the present findings that the Fay3 genotype with the highest oil content and the lowest erucic acid content compared to the other genotypes can be proposed as a potential donor for future breeding programs for oil production and quality, while Fay1 can be utilized as donor to increase the seed yield per plant.

Interactive Effects of Nitrogen and Sulfur Nutrition on Growth, Development, and Physiology of Brassica carinata A. Braun and Brassica napus L.

Brassica carinata (carinata) has emerged as a potential biofuel source due to its high erucic acid content, making it desirable for various industrial applications. Nitrogen (N) and sulfur (S) are required as primary sources of nutrition for growth and development in different oilseed crops and their utilization is interdependent. The purpose of the study was to analyze the interactive effect of N and S nutrition on the growth and other physiological activities of carinata and B. napus (napus). Four treatments, i.e., optimum NS (+N+S, 100% N and 100% S); N limited (−N+S, 0% N, 100% S); S limited (+N−S, 100% N, 0% S), and NS limited (−N−S, 0% N and 0% S) of N and S in full-strength Hoagland solution were imposed in the current study. Effect of different NS treatments was observed on vegetative traits such as number of primary and secondary branches, total leaf area, total biomass production and allocation, and physiological traits such as production of photosynthetic pigments, net photosynthesis, electron transport, and other aspects for both carinata and napus. The traits of stem elongation, number of nodes, node addition rate, internode length, number of primary and secondary branches were 60%, 36%, 50%, 35%, 56%, and 83% lower, respectively, in napus in comparison to carinata. Different NS treatments also positively influenced the production of photosynthetic pigments such as chlorophyll (Chl) a and b and carotenoids in carinata and napus. The concentration of Chla was 11% higher in napus in comparison to carinata. The rate of net photosynthesis, electron transport, and fluorescence was 12%, 8%, and 5% higher based on overall value, respectively, in napus compared to carinata. On the other hand, the overall value for stomatal conductance decreased by 5% in napus when compared to carinata. Different growth-related traits such as vegetative (plant height, node number, internode length, leaf area, number of primary and secondary branches), reproductive (pod number, pod length, seeds per pod), and photosynthetic capacity in oilseed brassicas are correlated with the final seed and oil yield and chemical composition which are of economic importance for the adoption of the crop. Thus, the analysis of these traits will help to determine the effect of NS interaction on crop productivity of carinata and napus.

Determination of oil content, linolenic and erucic acids contents in false flax seeds using IRspectrometry

Spectroscopy of near infrared reflection (NIRS) was used for estimation of biochemical indicators in seeds of false flax. The purpose of our work was to develop calibrating models for IR-analyzer MATRIXI for determination of weight percentage of oil, linolenic and erucic acids contents in oil of seeds in unbroken seeds of false flax (winter and spring forms). The researches were conducted in the biochemistry laboratory on false flax samples cultivated in 2016–2020 in the different environments of the Russian Federation. Oil content was determined with NMR-analyzer АМV 1006М according to the technique described in the State Standard 8.597-2010, percentage contents of linolenic and erucic acids in oil was estimated on the gas chromatograph “Chromatech – Kristal 5000” with an automatic dipper on a capillary column SolGelWax 30 m × 0.25 mm × 0.5 µcm. The best indicators of quality of the calibrating models (root mean square error of prediction, coefficient of determination, and meaning of a residual deflection of prediction for a rank reflected on a figure) were obtained by oil content (RMSEP = 0.20%, R 2 = 99.3, and RPD = 12.3), linolenic acid content (RMSEP = 0.35%, R 2 = 98.8, and RPD = 9.2) and erucic acid content (RMSEP = 0.14%, R 2 = 85.7, and RPD = 2.6). In a program OPUS LAB, we received a method “False flax 51” based on the developed calibrating models for a routine analysis for determination of oil content, linolenic and erucic acids contents in oil in the unbroken seeds of false flax in an average (9–20 g) in a cuvette with diameter of 51 mm. this method allows conducting express-estimation of false flax seeds for breeding traits with performance of more than 100 sample per seven hours.

In Vitro Production of Somaclones with Decreased Erucic Acid Content in Indian Mustard [Brassica juncea (Linn.) Czern&Coss]

Brassica junceais a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

Applicability of the German Food Based Dietary Guidelines for Infancy to Estimate Exposure to Substances in Food – The Example of Erucic Acid

Objectives The objective of this study was to use the recommended total daily food amounts of the German ‘Dietary Scheme for the first year of life’ (for the first time) to calculate the potential exposure of substances in the infant diet from a public health perspective. Erucic acid was taken as a concrete example, because the European Food Safety Agency (EFSA) recently issued a Tolerable Daily Intake (TDI) for erucic acid with a special note that vulnerable groups, especially young children, may be particularly at risk of exceeding the TDI. Methods The age of about 1 month with the highest consumption per kg body weight was chosen for scenarios for the phase of exclusive milk feeding, the age of about 8 months for the CF phase. Principle of calculation was: erucic acid exposure = food consumption volume per kg bodyweight per day x erucic acid concentration in food (from European studies for breastmilk, EFSA food samples for formula, current German market analysis for rapeseed oil, EU food law). We calculated 6 scenarios for the milk phase (4 formula-feeding, 2 breastfeeding) and 8 scenarios for the CF phase (5 CF + formula-feeding, 3 CF + breastfeeding). Results Out of the 14 scenarios, only 3 resulted in exposures in the safe range below the TDI (range 4.4.– 6.0 mg/kg BW). All scenarios assuming either high consumption (p95) or high concentration led to high exceedances of the TDI (range 7.5–26.2 mg/kg BW), especially when using maximum values of EU law for formula or vegetable oils (33.6 and 43.2 mg/kg BW respectively). Conclusions The Dietary Scheme proved to be a suitable frame to calculate the potential exposure of substances in foods forming the daily infant diet. The results give cause for concern, as the calculated high exposure to erucic acid in the scenarios occurs during infancy as a particularly sensitive developmental period and results from the food-based recommendations as public health guidelines. Considering the scarcity of data, in addition to the proven low erucic acid concentrations in rapeseed oil in Germany, it is imperative to analyze the erucic acid content of mature breast milk and formula as well. Funding Sources Union for the Promotion of Oil and Protein Plants (UFOP, Berlin, Germany).

Genetic variability and regression analysis for yield and quality traits in Brassica napus

An experiment was conducted to estimate the genetic variability and regression analysis for yield and quality traits in a set of eight brassica genotypes in a randomized complete block design with three replications at The University of Agriculture Peshawar, during 2011-12 cropping season. Significant genetic variability was recorded among genotypes for plant height, pods main receme-1, pod length, seed yield plant-1, 1000-seed weight, seeds pod-1, oil content, protein content, glucosinolate content, erucic acid content, oleic acid content and linolenic acid content except for primary branch plant-1 and pods plant-1 which showed non-significant differences. Genotypes, AUP-401, AUP-402, AUP-404 and AUP-407 showed potential performance for glucosinolate content (55.1 µmol g-1), seed yield plant-1 (23.93 g), pods plant-1 (268.83), protein contents (21.1 %), erucic acid contents (42.7 %) and oil contents (53.3 %). Seed yield plant-1 was taken as a dependent variable to study the effects of many morphological yield traits on it. Branches plant-1, pods main raceme-1, pods plant-1, and seed pod-1 showed a positive effect on seed yield plant-1, while 1000-seed weight, pod length, and plant height showed a negative effect on seed yield plant-1. Seed yield plant-1 as an independent variable showed a negative effect on oil content. Protein content as an independent variable showed a positive effect on erucic acid content, while oil contents as independent variables show a negative effect on protein contents, respectively. The developed germplasm could be used in the upcoming brassica breeding programs to release high yielding, disease resistance, stress tolerance, and insect resistance varieties, which will eventually upsurge the productivity and decrease the gap between demand and domestic production of the brassica crop.