Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes-SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.

Brassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra.

Increasing oil content in oilseed mustard (Brassica juncea) is a major breeding objective-more so, in the lines that have "0" erucic acid content (< 2% of the seed oil) as earlier studies have shown negative pleiotropic effect of erucic acid loci on the oil content, both in oilseed mustard and rapeseed. We report here QTL analysis of oil content in eight different mapping populations involving seven different parents-including a high oil content line J8 (~49%). The parental lines of the mapping populations contained wide variation in oil content and erucic acid content. The eight mapping populations were categorized into two sets-five populations with individuals segregating for erucic acid (SE populations) and the remaining three with zero erucic acid segregants (ZE populations). Meta-analysis of QTL mapped in individual SE populations identified nine significant C-QTL, with two of these merging most of the major oil QTL that colocalized with the erucic acid loci on the linkage groups A08 and B07. QTL analysis of oil content in ZE populations revealed a change in the landscape of the oil QTL compared to the SE populations, in terms of altered allelic effects and phenotypic variance explained by ZE QTL at the "common" QTL and observation of "novel" QTL in the ZE background. The important loci contributing to oil content variation, identified in the present study could be used in the breeding programmes for increasing the oil content in high erucic and "0" erucic backgrounds.

Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea.

Alternaria brassicae, a necrotrophic fungal pathogen, causes Alternaria blight, one of the most important diseases of oleiferous Brassica crops. The current study utilized Arabidopsis as a model to decipher the genetic architecture of defense against A. brassicae. Significant phenotypic variation that was largely genetically determined was observed among Arabidopsis accessions in response to pathogen challenge. Three biparental mapping populations were developed from three resistant accessions viz. CIBC-5, Ei-2, and Cvi-0 and two susceptible accessions - Gre-0 and Zdr-1 (commonly crossed to CIBC-5 and Ei-2). A total of six quantitative trait locus (QTLs) governing resistance to A. brassicae were identified, five of which were population-specific while one QTL was common between all the three mapping populations. Interestingly, the common QTL had varying phenotypic contributions in different populations, which can be attributed to the genetic background of the parental accessions. The presence of both common and population-specific QTLs indicate that resistance to A. brassicae is quantitative, and that different genes may mediate resistance to the pathogen in different accessions. Two of the QTLs had moderate-to-large effects, one of which explained nearly 50% of the variation. The large effect QTLs may therefore contain genes that could play a significant role in conferring resistance even in heterologous hosts.

Bhut jolokia, commonly known as Ghost chili, a native Capsicum species found in North East India was recorded as the naturally occurring hottest chili in the world by the Guinness Book of World Records in 2006. Although few studies have reported variation in pungency content of this particular species, no study till date has reported detailed expression analysis of candidate genes involved in capsaicinoids (pungency) biosynthesis pathway and other fruit metabolites. Therefore, the present study was designed to evaluate the diversity of fruit morphology, fruiting habit, capsaicinoids and other metabolite contents in 136 different genotypes mainly collected from North East India. Significant intra and inter-specific variations for fruit morphological traits, fruiting habits and 65 fruit metabolites were observed in the collected Capsicum germplasm belonging to three Capsicum species i.e., Capsicum chinense (Bhut jolokia, 63 accessions), C. frutescens (17 accessions) and C. annuum (56 accessions). The pungency level, measured in Scoville Heat Unit (SHU) and antioxidant activity measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay showed maximum levels in C. chinense accessions followed by C. frutescens accessions, while C. annuum accessions showed the lowest value for both the traits. The number of different fruit metabolites detected did not vary significantly among the different species but the metabolite such as benzoic acid hydroxyl esters identified in large percentage in majority of C. annuum genotypes was totally absent in the C. chinense genotypes and sparingly present in few genotypes of C. frutescens. Significant correlations were observed between fruit metabolites capsaicin, dihydrocapsaicin, hexadecanoic acid, cyclopentane, α-tocopherol and antioxidant activity. Furthermore, comparative expression analysis (through qRT-PCR) of candidate genes involved in capsaicinoid biosynthesis pathway revealed many fold higher expression of majority of the genes in C. chinense compared to C. frutescens and C. annuum suggesting that the possible reason for extremely high pungency might be due to the higher level of candidate gene(s) expression although nucleotide variation in pungency related genes may also be involved in imparting variations in level of pungency.

Brassica juncea (AABB) is an allotetraploid species containing genomes of B. rapa (AA) and B. nigra (BB). It is a major oilseed crop in South Asia, and grown on approximately 6-7 million hectares of land in India during the winter season under dryland conditions. B. juncea has two well defined gene pools--Indian and east European. Hybrids between the two gene pools are heterotic for yield. A large number of qualitative and quantitative traits need to be introgressed from one gene pool into the other. This study explores the availability of SNPs in RNA-seq generated contigs, and their use for general mapping, fine mapping of selected regions, and comparative arrangement of gene blocks on B. juncea A and B genomes.

White rust, causal agent oomycete Albugo candida, is a significant disease of the cultivated Brassica species. The Indian gene pool lines of oilseed mustard, Brassica juncea, are highly susceptible to the pathogen. Resistance to A. candida has been reported in the east European gene pool lines of mustard and mapped to LG A4 in line Heera and LG A5 in line Donskaja-IV. A new resistance-conferring locus to A. candida isolate AcB1 has been mapped to LG A6 of B. juncea line Tumida-a Chinese vegetable type mustard using an F1DH mapping population that has been developed from a Tumida × Varuna (susceptible Indian gene pool line) cross. A molecular map containing 8,303 genic and GBS markers was used to map the resistance trait to an interval of 63.0 cM-70.8 cM on LG A6. Genome assemblies of Tumida and Varuna were used to find the genes present within the flanking markers discerned by genetic mapping. The most likely candidate gene in the mapped interval is BjuA046215, a CC-NBS-LRR (CNL) type R gene that encodes a protein with all the specific subdomains of the proteins encoded by such genes. Alleles of BjuA046215 in Varuna and other lines of the Indian and the east European gene pools encode proteins that have truncated LRR domains. Analysis of the syntenic regions in some of the Brassicaceae genomes and phylogenetic analysis of CNL type R genes showed BjuA046215 to be closely related to a recently described white rust resistance-conferring R gene BjuWRR1 in B. juncea Donskaja-IV, both belonging to the CNL-D group of R genes. Related R genes in Arabidopsis thaliana confer resistance to another oomycete, Peronospora parasitica.

Brassica species (tribe Brassiceae) belonging to U's triangle--B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC)--originated via two polyploidization rounds: a U event producing the three allopolyploids, and a more ancient b genome-triplication event giving rise to the A-, B-, and C-genome diploid species. Molecular mapping studies, in situ hybridization, and genome sequencing of B. rapa support the genome triplication origin of tribe Brassiceae, and suggest that these three diploid species diversified from a common hexaploid ancestor. Analysis of plastid DNA has revealed two distinct lineages--Rapa/Oleracea and Nigra--that conflict with hexaploidization as a single event defining the tribe Brassiceae. We analysed an R-block region of A. thaliana present in six copies in B. juncea (AABB), three copies each on A- and B-genomes to study gene fractionation pattern and synonymous base substitution rates (Ks values). Divergence time of paralogues within the A and B genomes and homoeologues between the A and B genomes was estimated. Homoeologous R blocks of the A and B genomes exhibited high gene collinearity and a conserved gene fractionation pattern. The three progenitors of diploid Brassicas were estimated to have diverged approximately 12 mya. Divergence of B. rapa and B. nigra, calculated from plastid gene sequences, was estimated to have occurred approximately 12 mya, coinciding with the divergence of the three genomes participating in the b event. Divergence of B. juncea A and B genome homoeologues was estimated to have taken place around 7 mya. Based on divergence time estimates and the presence of distinct plastid lineages in tribe Brassiceae, it is concluded that at least two independent triplication events involving reciprocal crosses at the time of the b event have given rise to Rapa/Oleracea and Nigra lineages.

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.