Akshay Kumar Pradhan

SUMMARY: Brassica carinata (BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes of Brassica nigra (BB) and Brassica oleracea (CC). It is an oilseed crop endemic to the northeastern regions of Africa. Although it is under limited cultivation, B. carinata is valuable as it is resistant/highly tolerant to most of the pathogens affecting widely cultivated Brassica species of the U's triangle. We report a chromosome‐scale genome assembly of B. carinata accession HC20 using long‐read Oxford Nanopore sequencing and Bionano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the long‐read genome assemblies of the U's triangle species and found extensive gene collinearity between the diploids and allopolyploids with no evidence of major gene losses. Therefore, B. juncea (AABB), B. napus (AACC), and B. carinata can be regarded as strict allopolyploids. We cataloged the nucleotide‐binding and leucine‐rich repeat immune receptor (NLR) repertoire of B. carinata and, identified 465 NLRs, and compared these with the NLRs in the other Brassica species. We investigated the extent and nature of early‐generation genomic interactions between the constituent genomes of B. carinata and B. juncea in interspecific crosses between the two species. Besides the expected recombination between the constituent B genomes, extensive homoeologous exchanges were observed between the A and C genomes. Interspecific crosses, therefore, can be used for transferring disease resistance from B. carinata to B. juncea and broadening the genetic base of the two allotetraploid species.

Summary: Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR‐edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR‐edited lines displayed a defence response at par or better than that of the wild‐type line. The GTR‐edited lines were equivalent to the wild‐type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low‐seed, high‐leaf glucosinolate lines with an uncompromised defence and yield.

Brassica juncea (AABB, 2n=36), commonly called mustard is an allopolyploid crop of recent origin but has considerable morphological and underlying genetic variation. An F ₁ -derived doubled haploid (F ₁ DH) population developed from a cross between a Indian oleiferous line, Varuna, and a Chinese stem type vegetable mustard, Tumida showed significant variability for some key plant architectural traits, including four stem strength-related traits, stem diameter, plant height, branch initiation height, number of primary branches ( Pbr ), and days to flowering ( Df ). Multi-environment QTL analysis identified twenty environmentally stable QTL for the nine plant architectural traits. Both Tumida and Varuna contain some positive QTL that can be used to breed superior ideotypes in mustard. A QTL cluster on LG A10 contained environmentally stable QTL for seven architectural traits. This region also contained overlapping environmentally stable major QTL (phenotypic variance ≥ 10%) for Df and Pbr , with Tumida contributing the trait enhancing alleles for both the traits. Since early flowering is critical for the cultivation of mustard in the Indian subcontinent, this QTL cannot be used for the improvement of Pbr in the Indian gene pool lines. Conditional QTL analysis for Pbr identified the QTL for improvement of Pbr without negative effects on Df . The environmentally stable QTL were projected on the genome assemblies of Tumida and Varuna for the identification of candidate genes. These findings provide insights into the genetics of plant architectural traits in two diverse gene pools of B. juncea and provide opportunities for improvement of the plant architecture through marker-assisted introgressions. Key Message: Genetic mapping of some key plant architectural traits in a vegetable type and an oleiferous B. juncea cross revealed environmentally stable QTL and candidate genes for breeding more productive ideotypes.

Seed size/weight is a multigenic trait that is governed by complex transcriptional regulatory pathways. An understanding of the genetic basis of seed size is of great interest in the improvement of seed yield and quality in oilseed crops. A global transcriptome analysis was performed at the initial stages of seed development in two lines of Brassica juncea, small-seeded EH-2 and large-seeded PJ. The anatomical analyses revealed significant differences in cell number and cell size in the outer layer of the seed coat between EH-2 and PJ. Pairwise comparisons at each developmental stage identified 5,974 differentially expressed genes (DEGs) between the two lines, of which 954 genes belong to different families of transcription factors. Two modules were found to be significantly correlated with an increased seed size using weighted gene coexpression network analysis. The DEG and coexpression datasets were integrated with the thousand seed weight (Tsw) quantitative trait loci (QTL) mapped earlier in the EPJ (EH-2 × PJ) doubled haploid (DH) population, which identified forty potential key components controlling seed size. The candidate genes included genes regulating the cell cycle, cell wall biogenesis/modification, solute/sugar transport, and hormone signaling. The results provide a valuable resource to widen the current understanding of regulatory mechanisms underlying seed size in B. juncea.

The exploitation of heterosis through hybrid breeding is one of the major breeding objectives for productivity increase in crop plants. This research analyzes the genetic basis of heterosis in Brassica juncea by using a doubled haploid (DH) mapping population derived from F₁ between two heterotic inbred parents, one belonging to the Indian and the other belonging to the east European gene pool, and their two corresponding sets of backcross hybrids. An Illumina Infinium Brassica 90K SNP array-based genetic map was used to identify yield influencing quantitative trait loci (QTL) related to plant architecture, flowering, and silique- and seed-related traits using five different data sets from multiple trials, allowing the estimation of additive and dominance effects, as well as digenic epistatic interactions. In total, 695 additive QTL were detected for the 14 traits in the three trials using five data sets, with overdominance observed to be the predominant type of effect in determining the expression of heterotic QTL. The results indicated that the design in the present study was efficient for identifying common QTL across multiple trials and populations, which constitute a valuable resource for marker-assisted selection and further research. In addition, a total of 637 epistatic loci were identified, and it was concluded that epistasis among loci without detectable main effects plays an important role in controlling heterosis in yield of B. juncea.

Background: Brassica nigra(BB), also called black mustard, is grown as a condiment crop in India.B. nigrarepresents the B genome of U’s triangle and is one of the progenitor species ofB. juncea(AABB), an important oilseed crop of the Indian subcontinent. We report the genome assembly ofB. nigravariety Sangam.Results: The genome assembly was carried out using Oxford Nanopore long-read sequencing and optical mapping. A total of 1549 contigs were assembled, which covered ~ 515.4 Mb of the estimated ~ 522 Mb of the genome. The final assembly consisted of 15 scaffolds that were assigned to eight pseudochromosomes using a high-density genetic map ofB. nigra. Around 246 Mb of the genome consisted of the repeat elements; LTR/Gypsy types of retrotransposons being the most predominant. The B genome-specific repeats were identified in the centromeric regions of theB. nigrapseudochromosomes. A total of 57,249 protein-coding genes were identified of which 42,444 genes were found to be expressed in the transcriptome analysis. A comparison of the B genomes ofB. nigraandB. juncearevealed high gene colinearity and similar gene block arrangements. A comparison of the structure of the A, B, and C genomes of U’s triangle showed the B genome to be divergent from the A and C genomes for gene block arrangements and centromeric regions.Conclusions: A highly contiguous genome assembly of theB. nigragenome reported here is an improvement over the previous short-read assemblies and has allowed a comparative structural analysis of the A, B, and C genomes of the species belonging to the U’s triangle. Based on the comparison, we propose a new nomenclature forB. nigrapseudochromosomes, taking theB. rapapseudochromosome nomenclature as the reference.

Summary: Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species—B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single‐molecule real‐time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short‐read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping‐by‐sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome‐scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.

The development of more productive crops will be key to addressing the challenges that climate change, population growth and diminishing resources pose to global food security. Advanced ‘omics techniques can help to accelerate breeding by facilitating the identification of genetic markers for use in marker‐assisted selection. Here, we present the validation of a new Associative Transcriptomics platform in the important oilseed crop Brassica juncea. To develop this platform, we established a pan‐transcriptome reference for B. juncea, to which we mapped transcriptome data from a diverse panel of B. juncea accessions. From this panel, we identified 355 050 single nucleotide polymorphism variants and quantified the abundance of 93 963 transcripts. Subsequent association analysis of functional genotypes against a number of important agronomic and quality traits revealed a promising candidate gene for seed weight, BjA.TTL, as well as additional markers linked to seed colour and vitamin E content. The establishment of the first full‐scale Associative Transcriptomics platform for B. juncea 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.

Brassica juncea (AABB; genome size ∼920 Mb), commonly referred to as mustard, is a natural allopolyploid of two diploid species – B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ∼100x PacBio single-molecule real-time (SMRT) reads providing contigs with an N50 value of >5Mb. Assembled contigs were corrected and scaffolded with BioNano optical mapping. Three different linkage maps containing a large number of GBS markers were developed and used to anchor scaffolds/contigs to the 18 linkage groups of B. juncea . The resulting chromosome-scale assembly is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content, and gene block associations. Both A and B genomes contain highly fragmented gene block arrangements. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The genome assembly reported here will provide a fillip to the breeding work on oleiferous types of mustard that are grown extensively in the dry land areas of South Asia and elsewhere.