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.

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.

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.

Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle.

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.

Phytoglobin 3 appears to be ubiquitous in plants, yet there has been dearth of evidence for their potent physiological functions. Previous crystallographic studies suggest a potential NO dioxygenase like activity of Arabidopsis phytoglobin 3 (AHb3). The present work examined the in vivo function of AHb3 in plant physiology and its role in biotic stress using Arabidopsis- Sclerotinia sclerotorium pathosystem. The gene was found to be ubiquitously expressed in all plant tissues, with moderately increased expression in roots. Its expression was induced upon NO, H2O2 and biotic stress. A C-terminal tagged GFP version of the wild type protein revealed its enhanced accumulation in the guard cells. AHb3-GFP was found to be partitioned majorly into the nucleus while residual amounts were present in the cytoplasm. The loss of function AHb3 mutant exhibited reduced root length and fresh weight. AHb3 knockout lines also displayed enhanced susceptibility towards the S. sclerotiorum. Interestingly, these lines displayed enhanced ROS accumulation upon pathogen challenge as suggested by DAB staining. Furthermore, enhanced/decreased NO accumulation in AHb3 knockout/overexpression lines upon treatment with multiple NO donors suggests a potent NO dioxygenase like activity for the protein. Taken together, our data indicate that AHb3 play a crucial role in regulating root length as well as in mediating defense response against S. sclerotiorum, possibly by modulating NO and ROS levels.

Heterotrimeric G-proteins are one of the highly conserved signal transducers across phyla. Despite the obvious importance of G-proteins in controlling various plant growth and environmental responses, there is no information describing the regulatory complexity of G-protein networks during pathogen response in a polyploid crop. Here, we investigated the role of extra-large G-proteins (XLGs) in the oilseed crop Brassica juncea, which has inherent susceptibility to the necrotrophic fungal pathogen Sclerotinia sclerotiorum. The allotetraploid B. juncea genome contains multiple homologs of three XLG genes (two BjuXLG1, five BjuXLG2, and three BjuXLG3), sharing a high level of sequence identity, gene structure organization, and phylogenetic relationship with the progenitors' orthologs. Quantitative reverse transcription PCR analysis revealed that BjuXLGs have retained distinct expression patterns across plant developmental stages and on S. sclerotiorum infection. To determine the role of BjuXLG genes in the B. juncea defence response against S. sclerotiorum, RNAi-based suppression was performed. Disease progression analysis showed more rapid lesion expansion and fungal accumulation in BjuXLG-RNAi lines compared to the vector control plants, wherein suppression of BjuXLG3 homologs displayed more compromised defence response at the later time point. Knocking down BjuXLGs caused impairment of the host resistance mechanism to S. sclerotiorum, as indicated by reduced expression of defence marker genes PDF1.2 and WRKY33 on pathogen infection. Furthermore, BjuXLG-RNAi lines showed reduced accumulation of leaf glucosinolates on S. sclerotiorum infection, wherein aliphatic glucosinolates were significantly compromised. Overall, our data suggest that B. juncea XLG genes are important signalling nodes modulating the host defence pathways in response to this necrotrophic pathogen.

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.