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.

PSXIII-12 Exploring nutritional differences of canola seeds and bio-processing co-products (meals, pellets) from different processing plants/companies in Canada and China for dairy cattle

Canola was created as a low erucic acid and low glucosinolate seed, to produce high quality oil for human consumption and meal for use in livestock feed. China is an important user of Canadian canola products (seeds, oil, and meal). The extraction of the oil from the seed produces a co-product called canola meal. This meal is rich in protein and is used as a protein source in animal diets. However, differences in the characteristics of the seeds, or processing methods during oil extraction may affect the quality of this co-product. Plus, the synthesis of tissues and milk is related to the amino acids available to the animal for absorption in the small intestine. This study aimed to determine if there are significant differences in the intestinal digestibility (in vitro) of CP and DM between canola seeds and meals from different companies in Canada and to determine if there are significant differences between them in Canada and China. The three-step procedure was applied on residues from a 12-hour rumen incubation in fistulated dairy cows to estimate the intestinal digestibility of CP and DM. There were significant differences (P < 0.05) for TDDM (Total digestible dry matter) and IDP (intestinal digestibility of protein) of the meals between countries. The samples from China had higher TDDM (83.76% versus 81.53%, P = 0.018), while Canada’s had higher IDP (68.51% versus 65.28%, P = 0.016). No significant differences were observed within countries. Based on the material analyzed during this study, it is safe to affirm that there are no significant differences in the digestibility of DM and CP between Canada and China. It was concluded that the quality of the canola seeds or meals produced in both Canada and China were similar when used in dairy rations. Key words: canola seeds and bio-processing co-products (meals, pellets), nutritional differences, dairy cows

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.

Vigor difference during storage and germination in Indian mustard explained by reactive oxygen species and antioxidant enzymes

Canola-type genotypes in Indian mustard (Brassica juncea) are a new kind of quality resource developed for their low levels of erucic acid (<2%) and glucosinolate (<30 μmole/g defatted meal) contents. Single-zero (low erucic acid) and double-zero (low erucic acid and glucosinolate content) genotypes of Indian mustard have less vigor. Conventional genotypes (high erucic acid and glucosinolate contents) havea significantly higher seedling vigor index-II (SVI-II) and single-zero genotypes have a significantly higher SVI-I, whereas double-zero genotypes have been observed to have a significantly lower SVI-I and SVI-II. To know the possible reasons for the differences in vigor, the seed quality parameters, reactive oxygen species (ROS) contents (superoxide radicals (O2−·) and hydrogen peroxide), lipid peroxidation, and antioxidant enzyme activity were examined. In the dry seeds, the conventional genotypes revealed lower ROS contents and higher catalase and peroxidase enzyme activity. This trend was reversed in the double-zero genotypes, which could be the reason why they were more susceptible to oxidative damage. During seed germination, an increase in the ROS contents, and corresponding increase in antioxidant enzyme activity, was noticed, which was highest in the conventional genotypes, followed by the single-zero genotypes. Double-zero genotypes showed the lowest increase in ROS contents and antioxidant enzyme activity during this period. This meant that the required attributes were met for maintaining oxidative balance within the cells and triggering physiological activities to reach high vigor. This study proposed 2 causes for the poor vigor of the double-zero genotypes; first, in the dry seeds, the ROS remained high due to low antioxidant activity (ROS scavengers) and the second was less generation of O2−· during germination.

Evaluation of the Risks of Contaminating Low Erucic Acid Rapeseed with High Erucic Rapeseed and Identification of Mitigation Strategies

High erucic acid rapeseed (HEAR) oil is under increasing demand for various industrial applications. However, many growers are concerned that if they grow the crop, they will not be able to revert to other rapeseed varieties in the future due to the risk of erucic acid (EA) contamination of the harvested seed and inability to maintain acceptable erucic acid thresholds. This review considered published literature and, using the same criteria as that used to contain transgenic crops, aimed to identify the key risks of erucic acid contamination, broadly prioritise them and identify pragmatic mitigation options. Oilseed rape has a number of traits that increase the risk of low erucic acid rapeseed (LEAR) crops being contaminated with EA from HEAR varieties. The quantity of seed produced and the potential for seed dormancy coupled with partial autogamy (self-fertilisation) facilitate the establishment and persistence of volunteer and feral populations. The large quantities of pollen produced when the crop is in flower mean there is also a high potential for cross-pollination. Self-sown volunteer plants represent the highest potential contamination risk, followed by the presence of arable weeds (e.g., wild mustard) whose seeds are also high in EA. Other risks arise from the cross-pollination of compatible wild relatives and the mixing of seed prior to sowing. It is important that both HEAR and LEAR varieties are appropriately managed since risks and their potential for mitigation arise throughout the entire LEAR crop production process. The length of rotation, type of tillage, cultivar choice, buffer zones, effective weed management and basic machinery hygiene are all factors that can reduce the risk of erucic acid contamination of LEAR crops and maintain the required thresholds.

Effects of bio- and chemical-organic fertilizers on yield, some physiological traits and fatty acids composition of canola

Effects of bio- and chemical-organic fertilizers on some physiological traits and fatty acids composition in canola (Brassica napus L.) was studied employing a factorial RCBD design in 2016. Experimental factors included four bio-fertilizers levels, such as (i) no bio-fertilizer (B1), (ii) seed inoculation with Nitrobacter (B2), (iii) Pseudomonas (B3), (iv) Azosprillum (B4) and five levels of nitrogen and farmyard manure (FYM) viz. (i) 100% nitrogen (F1), (ii) 25% FYM+75% nitrogen (F2), (iii) 50% FYM+50% nitrogen (F3), (iv) 75% FYM +25% nitrogen (F4) and (v) 100% FYM (F5). The unsaturated fatty acids (oleic acids, linoneic, linoleni and palminoleic acid) increased at 50% N+50% FMY along with bio-fertilizer compared to the control, while it was found vice versa in case of saturated fatty acids (palmetic and arashidic acid). Application of 100% FYM decreased oil (low erucic acid) and enhances the seed protein. Application of 50% FYM + 50% nitrogen along with Pseudomonas treated plants caused an increase of about 111% in seed yield of canola in comparison to that found in B1F5.

Coexistence in Oilseed Rape: Effect of Donor Variety Type and Discarding Field Edges

Oilseed rape is one of the most important sources of vegetable oil worldwide. Approximately 24% of the world’s total cultivation area in 2015 was planted with genetically modified (GM) varieties. Until now the cultivation of GM oilseed rape in the EU is not approved since coexistence of GM and non-GM oilseed rape is a matter of significant public concern. One reason is the differing information about pollen-mediated gene flow in this crop species. Therefore, in a 3-year field trial we investigated gene flow using a GM-free marker system consisting of a high erucic acid oilseed rape (HEAR) variety as pollen donor and a low erucic acid oilseed rape (LEAR) variety as pollen recipient. Donor and receptor fields were equally-sized (75 by 100 m or 0.75 ha) and separated by an isolation distance of 20 m clover-grass. Two different HEAR varieties, a hybrid variety and a line variety, were compared as pollen donor. Generally, outcrossing rates at 1 m field depth were significantly highest. A significant decrease in outcrossing was observed in the first donor-facing 20 m of recipient fields, with no further significant decrease in field depths > 20 m. Outcrossing rates of the total field harvest never exceeded 0.25%. Due to irregular patterns of outcrossing, a separated harvest of the receptor field edge facing the donor plot only marginally reduced the GM content in the total harvest.