Genomic Regions Associated With Seed Meal Quality Traits in Brassica napus Germplasm.

The defatted Brassica napus (rapeseed) meal can be high-protein feed for livestock as the protein value of rapeseed meal is higher than that of the majority of other vegetable proteins. Extensive work has already been carried out on developing canola rapeseed where the focus was on reducing erucic acid and glucosinolate content, with less consideration to other antinutritional factors such as tannin, phytate, sinapine, crude fiber, etc. The presence of these antinutrients limits the use and marketing of rapeseed meals and a significant amount of it goes unused and ends up as waste. We investigated the genetic architecture of crude protein, methionine, tryptophan, total phenols, ß-carotene, glucosinolates (GLSs), phytate, tannins, sinapine, and crude fiber content of defatted seed meal samples by conducting a genome-wide association study (GWAS), using a diversity panel comprising 96 B. napus genotypes. Genotyping by sequencing was used to identify 77,889 SNPs, spread over 19 chromosomes. Genetic diversity and phenotypic variations were generally high for the studied traits. A total of eleven genotypes were identified which showed high-quality protein, high antioxidants, and lower amount of antinutrients. A significant negative correlation between protein and limiting amino acids and a significant positive correlation between GLS and phytic acid were observed. General and mixed linear models were used to estimate the association between the SNP markers and the seed quality traits and quantile-quantile (QQ) plots were generated to allow the best-fit algorithm. Annotation of genomic regions around associated SNPs helped to predict various trait-related candidates such as ASP2 and EMB1027 (amino acid biosynthesis); HEMA2, GLU1, and PGM (tryptophan biosynthesis); MS3, CYSD1, and MTO1 (methionine biosynthesis); LYC (ß-carotene biosynthesis); HDR and ISPF (MEP pathway); COS1 (riboflavin synthesis); UGT (phenolics biosynthesis); NAC073 (cellulose and hemicellulose biosynthesis); CYT1 (cellulose biosynthesis); BGLU45 and BGLU46 (lignin biosynthesis); SOT12 and UGT88A1 (flavonoid pathway); and CYP79A2, DIN2, and GSTT2 (GLS metabolism), etc. The functional validation of these candidate genes could confirm key seed meal quality genes for germplasm enhancement programs directed at improving protein quality and reducing the antinutritional components in B. napus.

Association Mapping of Seed Quality Traits Under Varying Conditions of Nitrogen Application in Brassica juncea L. Czern & Coss.

Indian mustard (Brassica juncea) is a major source of vegetable oil in the Indian subcontinent. The seed cake left after the oil extraction is used as livestock feed. We examined the genetic architecture of oil, protein, and glucosinolates by conducting a genome-wide association study (GWAS), using an association panel comprising 92 diverse genotypes. We conducted trait phenotyping over 2 years at two levels of nitrogen (N) application. Genotyping by sequencing was used to identify 66,835 loci, covering 18 chromosomes. Genetic diversity and phenotypic variations were high for the studied traits. Trait performances were stable when averaged over years and N levels. However, individual performances differed. General and mixed linear models were used to estimate the association between the SNP markers and the seed quality traits. Population structure, principal components (PCs) analysis, and discriminant analysis of principal components (DAPCs) were included as covariates to overcome the bias due to the population stratification. We identified 16, 23, and 27 loci associated with oil, protein, and glucosinolates, respectively. We also established LD patterns and haplotype structures for the candidate genes. The average block sizes were larger on A-genome chromosomes as compared to the B- genome chromosomes. Genetic associations differed over N levels. However, meta-analysis of GWAS datasets not only improved the power to recognize associations but also helped to identify common SNPs for oil and protein contents. Annotation of the genomic region around the identified SNPs led to the prediction of 21 orthologs of the functional candidate genes related to the biosynthesis of oil, protein, and glucosinolates. Notable among these are: LACS5 (A09), FAD6 (B05), ASN1 (A06), GTR2 (A06), CYP81G1 (B06), and MYB44 (B06). The identified loci will be very useful for marker-aided breeding for seed quality modifications in B. juncea.

The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil.

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very-long-chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn-2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.

Validation of an updated Associative Transcriptomics platform for the polyploid crop species Brassica napus by dissection of the genetic architecture of erucic acid and tocopherol isoform variation in seeds.

An updated platform was developed to underpin association genetics studies in the polyploid crop species Brassica napus (oilseed rape). Based on 1.92 × 1012 bases of leaf mRNAseq data, functional genotypes, comprising 355 536 single-nucleotide polymorphism markers and transcript abundance were scored across a genetic diversity panel of 383 accessions using a transcriptome reference comprising 116 098 ordered coding DNA sequence (CDS) gene models. The use of the platform for Associative Transcriptomics was first tested by analysing the genetic architecture of variation in seed erucic acid content, as high-erucic rapeseed oil is highly valued for a variety of applications in industry. Known loci were identified, along with a previously undetected minor-effect locus. The platform was then used to analyse variation for the relative proportions of tocopherol (vitamin E) forms in seeds, and the validity of the most significant markers was assessed using a take-one-out approach. Furthermore, the analysis implicated expression variation of the gene Bo2g050970.1, an orthologue of VTE4 (which encodes a γ-tocopherol methyl transferase converting γ-tocopherol into α-tocopherol) associated with the observed trait variation. The establishment of the first full-scale Associative Transcriptomics platform for B. napus enables rapid progress to be made towards an understanding of the genetic architecture of trait variation in this important species, and provides an exemplar for other crops.

Discovery and mapping of Brassica juncea Sdt 1 gene associated with determinate plant growth habit.

KEY MESSAGE: Newly discovered determinate plant growth habit in Brassica juncea is simply inherited and can help in architectural restructuring of Brassica oilseeds. Brassica juncea is naturally indeterminate. This growth habit tends to accentuate intra-plant competition for resources within the plant canopy, leading to unfilled seeds, immature pods and tip sterility. Recent identification of plants with determinate growth habit is expected to open up new avenues for plant architectural modifications in crop Brassicas. Plants with determinate plant growth habit were identified in progenies of resynthesized B. juncea as a de novo variation. F1 plants, developed from crosses of determinate mustard with natural indeterminate genotypes were indeterminate, indicating the dominance of indeterminacy. F2 and F3 segregation revealed monogenic recessive inheritance in the progenies studied. Gene for determinacy (Sdt 1 ) was mapped to the linkage group 15 of B. juncea. Sdt 1 was flanked by SSR markers SJ6842 and Ni4-A10 at distances of 15.9 cM and 14.0 cM, respectively. Determinate progenies showed significant variation for plant height, flowering time and productivity. There appeared to be no adverse association in terms of lower pod density, productivity or oil content. Determinacy was under control of single recessive gene, mapped to the linkage group 15 of B. juncea. Determinate progenies with high agronomic performance were identified.