Search

Spontaneous fertility reversion has been documented in cytoplasmic male sterile (CMS) plants of several species, influenced in frequency by nuclear genetic background. In this study, we found that MutS HOMOLOG1 (MSH1) mediates fertility reversion via substoichiometric shifting (SSS) of the CMS-associated mitochondrial Open Reading Frame 220 (ORF220), a process that may be regulated by pollination signalling in Brassica juncea. We show that plants adjust their growth and development in response to unsuccessful pollination. Measurable decrease in MSH1 transcript levels and evidence of ORF220 SSS under non-pollination conditions suggest that this nuclear-mitochondrial interplay influences fertility reversion in CMS plants in response to physiological signals. Suppression of MSH1 expression induced higher frequency SSS in CMS plants than occurs normally. Transcriptional analysis of floral buds under pollination and non-pollination conditions, and the response of MSH1 expression to different sugars, supports the hypothesis that carbon flux is involved in the pollination signalling of fertility reversion in CMS plants. Our findings suggest that facultative gynodioecy as a reproductive strategy may incorporate environmentally responsive genes like MSH1 as an "on-off" switch for sterility-fertility transition under ecological conditions of reproductive isolation., (© 2020 John Wiley & Sons Ltd.)

Background: Brassica nigra (BB), also called black mustard, is grown as a condiment crop in India. B. nigra represents the B genome of U's triangle and is one of the progenitor species of B. juncea (AABB), an important oilseed crop of the Indian subcontinent. We report the genome assembly of B. nigra variety 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 of B. 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 the B. nigra pseudochromosomes. 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 of B. nigra and B. juncea revealed 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 the B. nigra genome 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 for B. nigra pseudochromosomes, taking the B. rapa pseudochromosome nomenclature as the reference.

Heavy metal pollution seriously impairs crop production and poses serious concerns for human health. Exogenous application of biomolecules has been efficiently tested for enhancing plant resistance to metal toxicity. Current study evaluates the possible effect of 5-aminolevulinic acid (ALA) in Brassica juncea L. seedlings subjected to lead (Pb) stress. Our results showed that shoot length, root length and chlorophyll contents were significantly recovered in Pb stressed seedlings after ALA application, accompanied by reduction in the Pb accumulation. Significant reduction in the contents of reactive oxygen species (ROS) like superoxide anion, hydrogen peroxide and malondialdehyde were also observed in ALA treated seedlings under Pb stress. Furthermore, we also noticed enhancement in the activities of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), glutathione-S-transferase (GST) and dehydroascorbate reductase (DHAR). We further noticed that ALA upregulated the expression of SOD (7.30 folds), POD (6.11 folds), CAT (3.52 folds), DHAR (6.42 folds), GR (6.04 folds), and GST (5.58 folds) under the Pb stress. However, RBOH1 (gene involved in ROS generation) and CHLASE (chlorophyllase) expressions were reduced in ALA treated seedlings grown under Pb stress (RBOH1 expression decreased to 3.44 from 6.50 fold and CHLASE expression decreased to 2.97 from 5.58 fold). Phenolic contents were increased in the presence of ALA and expression of genes like CHS (chalcone synthase; 7.50 fold) and PAL (phenylalanine ammonia lyase; 4.77 fold) was also stimulated by ALA under Pb stress. Furthermore, contents of the Krebs cycle metabolites (fumarate, succinate, malate and citrate) were also enhanced accompanied by upregulated expression of genes like CS (citrate Synthase; 8.13 fold), SUCLG1 (succinyl CoA ligase 1; 7.40 fold), SDH (succinate dehydrogenase; 5.10 fold) and FH (fumarate hydratase; 5.65 fold). In conclusion, current investigation revealed that ALA attenuated Pb toxicity by modulating the transcription patterns of key enzymes involved in plant defense system., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Heat shock transcription factor (HSF) is ubiquitous in the whole biological world and plays an important role in regulating growth and development and responses to environment stress. In this study, a total of 60 HSF transcription factors in Brassica juncea genome were identified and analyzed. Phylogenetic analysis showed that HSF genes were divided into three groups namely: A, B, and C, of which group A was further divided into nine subgroups (A1-A9). The analysis of gene structure and conserved motifs showed that some homologous genes are highly conserved. There was strong conservative microcollinearity among Brassica rapa , B. juncea , and Brassica oleracea , which provides a basis for studying the replication of gene families. Moreover, the results revealed that the promoter regions of BjuHSF genes were rich in cis -elements related to growth and development, hormone signal, and stress response. The prediction of protein interaction results showed that HSFs could interact with multiple transcription factors and proteins in the genome, while functional annotation revealed that BjuHSF genes were involved in many biological processes. The expression patterns of BjuHSF genes were analyzed by qPCR, and the results showed that these genes were closely linked to stress response, hormones, and development process. These results are a foundation for further analysis of the regulation mechanism of HSF gene family.

Purple leaves are rich in health-protecting anthocyanins and food colorants in Brassica juncea. But the causal gene, which is related to leaf color formation, have not been reported in B. juncea. Anthocyanins mainly accumulated throughout the adaxial and abaxial epidermal leaf cells of purple leaves. A genetic analysis indicated that an incompletely dominant gene controls the purple leaf trait in B. juncea. Furthermore, the BjPur gene, which increased anthocyanin accumulation in purple-leaf mustard, was cloned. Blast and phylogenetic analyses revealed that BjPur encodes a new R2R3-MYB transcription factor. Sequence analysis of two alleles revealed a DNA sequence insertion in the first intron of BjPur in green leaves parent line (LY) when compared with the BjPur gene in the purple-leaf parent line (ZY). And this insertion greatly reduced the transcription of BjPur in green leaves. In purple-leaf plants, the transcript level of BjPur was significantly higher in leaves than in roots, stems, siliques, and flower buds. Additionally, molecular markers linked to leaf color were developed to distinguish different genotypes of B. juncea. These results will be helpful for the genetic improvement of the purple leaf color in B. juncea.

Key Message: Two BjuGα proteins exhibit conserved GTP-binding and GTP-hydrolysis activities, and function in maintaining overall plant architecture and controlling multiple yield-related traits in the oilseed Brassica juncea. Heterotrimeric G-protein (Gα, Gβ and Gγ) are key signal transducers, well characterized in model plants Arabidopsis and rice. However, our knowledge about the roles played by G-proteins in regulating various growth and developmental traits in polyploid crops, having a complex G-protein signalling network, is quite sparse. In the present study, two Gα encoding genes (BjuA.Gα1 and BjuB.Gα1) were isolated from the allotetraploid Brassica juncea, a globally cultivated oilseed crop of the Brassicaceae family. BjuGα1 genes share a close evolutionary relationship, and the encoded proteins exhibit highly conserved G-protein activities while showing expression differentiation, wherein BjuA.Gα1 was the highly abundant transcript during plant growth and developmental stages. RNAi based suppression of BjuGα1 displayed compromised effects on most of the tested vegetative and reproductive parameters, particularly plant height (32-58%), flower and siliques dimensions, and seed weight (11-13%). Further, over-expression of a constitutively active Gα, lacking the GTPase activity, produced plants with increased height, organ size and seed weight (7-25%), without altering seed quality traits like fatty acid composition, glucosinolates, oil and protein contents. Our study demonstrates that BjuGα1 proteins control overall plant architecture and multiple yield-related traits in the oilseed B. juncea, suggesting that BjuGα1 could be a promising target for crop improvement.

Key Message: Rfo is located on a radish chromosome fragment (~ 108 Kb), which is seated in the middle of a pretty large C genome translocation at the distal region of chromosome A09 of B. juncea. Ogura cytoplasmic male sterility (CMS) is used to produce hybrids in Indian mustard (Brassica juncea L.). Fertility restorers for this CMS were developed by cross-hybridizing B. juncea (AABB; 2n = 36) with B. napus (AACC; 2n = 38) carrying radish Rfo gene. This hybrid production system is normally stable, but many commercial mustard hybrids show male sterile contaminants. We aimed to identify linkage drag associated with Rfo by comparing hybridity levels of 295 handmade CMS x Rfo crosses. Although Rfo was stably inherited, hybridity was < 85 percent in several combinations. Genome re-sequencing of five fertility restorers, mapping sequencing reads to B. juncea reference and synteny analysis with Raphanus sativus D81Rfo genomic region (AJ550021.2) helped to detect ~ 108 Kb of radish chromosome (R) fragment substitution in all fertility restorers. This radish segment substitution was itself located amidst a large C genome translocation on the terminal region of chromosome A09 of B. juncea. The size of alien segment substitution varied from 11.3 (NTCN-R9) to 22.0 Mb (NAJR-102B-R). We also developed an in silico SSR map for chromosome A09 and identified many homoeologous A to the C genome exchanges in the introgressed region. A to the R genome exchanges were rare. Annotation of the substituted fragment showed the gain of many novel genes from R and C genomes and the loss of B. juncea genes from the corresponding region. We have developed a KASPar marker for marker-aided transfer of Rfo and testing hybridity levels in seed production lots.

Main Conclusion: Transgenic technology in Indian mustard has expedited crop improvement programs. Further, there is a need to optimize gene editing protocols and find out the suitable target genes to harvest the benefits of gene editing technology in this important edible oilseed crop. Brassica juncea is an economically and industrially important oilseed crop being grown mainly in India and in some parts of Canada, Russia, China and Australia. Besides being consumed as edible oil, it also has numerous applications in food and paint industry. However, its overall production and productivity are being hampered by a number of biotic and abiotic stress factors. Further, its oil and seedmeal quality needs to be improved for increasing food as well as feed value. However, the lack of resistant crossable germplasm or varieties necessitated the use of genetic engineering interventions in Indian mustard crop improvement. A number of genes conferring resistance to biotic stresses including lectins for aphids' control, chitinase, glucanase and osmotin for disease control and for abiotic stresses, CODA, LEA and ion antiporter genes have been transferred to Indian mustard. Both antisense and RNAi technologies have been employed for improving oil and seedmeal quality. Efforts have been made to improve the phytoremediation potential of this crop through genetic engineering approach. The deployment of barnase/barstar gene system for developing male sterile and restorer lines has really expedited hybrid development programs in Indian mustard. Further, there is a need to optimize gene editing protocols and to find out suitable target genes for gene editing in this crop. In this review paper, authors have attempted to review various genetic transformation efforts carried out in Indian mustard for its improvement to combat biotic and abiotic stress challenges, quality improvement and hybrid development.

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

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., (2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Ethiopian mustard (Brassica carinata A. Braun) is an emerging sustainable source of vegetable oil, in particular for the biofuel industry. The present study exploited genome assemblies of the Brassica diploids, Brassica nigra and Brassica oleracea, to discover over 10,000 genome-wide SNPs using genotype by sequencing of 620 B. carinata lines. The analyses revealed a SNP frequency of one every 91.7 kb, a heterozygosity level of 0.30, nucleotide diversity levels of 1.31 × 10 -05 , and the first five principal components captured only 13% molecular variation, indicating low levels of genetic diversity among the B. carinata collection. Genome bias was observed, with greater SNP density found on the B subgenome. The 620 lines clustered into two distinct sub-populations (SP1 and SP2) with the majority of accessions (88%) clustered in SP1 with those from Ethiopia, the presumed centre of origin. SP2 was distinguished by a collection of breeding lines, implicating targeted selection in creating population structure. Two selective sweep regions on B3 and B8 were detected, which harbour genes involved in fatty acid and aliphatic glucosinolate biosynthesis, respectively. The assessment of genetic diversity, population structure, and LD in the global B. carinata collection provides critical information to assist future crop improvement.

Potential gene flow from transgenic Brassica napus to widely-distributed, cross-compatible weedy relatives has received significant attention. All previous, albeit scarce, research has shown little to no success in producing viable F1 hybrids between B. napus (n = 38) and B. nigra (n = 16). The present study tested the working premise that the propensity for interspecific hybridization is significantly higher between B. napus and wild-growing, B. nigra displaying mixoploidy (n = 32). Controlled hybridization was performed using local, wild-growing B. nigra (♀) x transgenic (Bt Cry1Ac) B. napus (♂). Spontaneous hybridization was performed using the same B. nigra (♀) population x non-transgenic B. napus (♂) under sympatric open-field and greenhouse conditions. The total hybridization frequency, determined by the functional expression of the Bt Cry1Ac endotoxin, was 1.8 % of the F1 hybrids (n = 35). Gene flow from non-transgenic B. napus to B. nigra ranged from 4 to 29 % in F1 hybrids, with combined wind- and wild-insect-mediated pollen dispersal being the most effective. Successful interspecific hybridization is significantly enhanced using mixoploid B. nigra progenitor material. Gene flow rates in F1 hybrids were equivalent to those previously reported between B. napus with B. rapa and B. juncea, respectively, which are at the forefront of risk assessment concerns., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Productivity of Indian mustard (B. juncea), a major oil yielding crop in rapeseed-mustard group is heavily inflicted by mustard aphid, L. erysimi. Mustard aphid, a specialist aphid species on rapeseed-mustard crops, rapidly multiplies and colonizes the plants leading to successful infestation. In contrary, legume specific cowpea aphid, A. craccivora when released on B. juncea plants fails to build up population and thus remains unsuccessful in infestation. In the present study, differential host response of B. juncea to the two aphid species, one being successful insect-pest and the other being unsuccessful on it has been studied based on transcriptome analysis. Differential feeding efficiency of the two aphid species on mustard plants was evident from the amount of secreted honeydews. Leaf-transcriptomes of healthy and infested plants, treated with the two aphid species, were generated by RNA sequencing on Illumina platform and de novo assembly of the quality reads. A comparative assessment of the differentially expressed genes due to treatments revealed a large extent of overlaps as well as distinctness with respect to the set of genes and their direction of regulation. With respect to host-genes related to transcription factors, oxidative homeostasis, defense hormones and secondary metabolites, L. erysimi led to either suppression or limited activation of the transcript levels compared to A. craccivora. Further, a comprehensive view of the DEGs suggested more potential of successful insect-pests towards transcriptional reprogramming of the host. qRT-PCR based validation of randomly selected up- and down-regulated transcripts authenticated the transcriptome data.

HDA9, a member of the deacetylase family, plays a vital role in regulating plant flowering time through flowering integrator SOC1 and AGL24. However, it remains elusive how HDA9 interacts with SOC1 and AGL24 in flowering time control. Here, HDA9 was cloned in Brassica juncea and then its three active sites were separately replaced with Ala via overlap extension PCR. Thus, mutants of HDA9(D172A), HDA9(H174A) and HDA9(D261A) were constructed and fused into the pGADT7 vector. The yeast one-hybrid assays indicated that HDA9 mutants remained the interactions with the promoters of SOC1 and AGL24. Furthermore, the aforementioned results were confirmed in the dual luciferase assays. Interestingly, the DNA-protein interactions were weakened significantly due to the mutation in the three active sites of HDA9. It suggested that flowering signal integrator SOC1 and AGL24 were regulated by the key amino acid residues of 172th, 174th and 261th in HDA9. Our results provide valuable information for the in-depth study of the biological function and molecular regulation of HDA9 in Brassica juncea flowering time control.

Tuber mustard, which is the raw material of Fuling pickle, is a crop with great economic value. However, during growth and development, tuber mustard is frequently attacked by the pathogen Plasmodiophora brassicae and frequently experiences salinity stress. Jasmonic acid (JA) is a hormone related to plant resistance to biotic and abiotic stress. Jasmonate ZIM domain proteins (JAZs) are crucial components of the JA signaling pathway and play important roles in plant responses to biotic and abiotic stress. To date, no information is available about the characteristics of the JAZ family genes in tuber mustard. Here, 38 BjJAZ genes were identified in the whole genome of tuber mustard. The BjJAZ genes are located on 17 of 18 chromosomes in the tuber mustard genome. The gene structures and protein motifs of the BjJAZ genes are conserved between tuber mustard and Arabidopsis. The results of qRT-PCR analysis showed that BjuA030800 was specifically expressed in root, and BjuA007483 was specifically expressed in leaf. In addition, 13 BjJAZ genes were transiently induced by P. brassicae at 12 h, and 7 BjJAZ genes were induced by salt stress from 12 to 24 h. These results provide valuable information for further studies on the role of BjJAZ genes in the regulation of plant growth and development and in the response to biotic and abiotic stress., Competing Interests: The authors have declared that no competing interests exist.

Auxin signalling is vital for plant growth and development, from embryogenesis to senescence. Recent studies have shown that auxin regulates biological processes by mediating gene expression through a family of functionally original DNA-binding auxin response factors, which exist in a large multi-gene family in plants. However, to date, no information has been available about characteristics of the ARF gene family in Brassica juncea var. tumida. In this study, 65 B. juncea genes that encode ARF proteins were identified in the B. juncea whole-genome, classified into three phylogenetical groups and found to be widely and randomly distributed in the A-and B-genome. Highly conserved proteins were also found within each ortholog based on gene structure and conserved motifs, as well as clustering level. Furthermore, promoter cis-element analysis of BjARFs demonstrated that these genes affect the levels of plant hormones, such as auxin, salicylic, gibberellin acid, MeJA, abscisic acid, and ethylene. Expression analysis showed that differentially expressed BjARF genes were detected during the seedling stage, tumor stem development and the flowering period of B. juncea. Interestingly, we found that BjARF2b&#95;A, BjARF3b&#95;A, BjARF6b&#95;A, and BjARF17a&#95;B were significantly expressed in tumor stem, and an exogenous auxin assay indicated that these genes were sensitive to auxin and IAA signaling. Moreover, eight of the nine BjARF10/16/17 genes and all of the BjARF6/8 genes were involved in post-transcriptional regulation, targeted by Bj-miR160 and Bj-miR167c, respectively. This analysis provides deeper insight of diversification for ARFs and will facilitate further dissection of ARF gene function in B. juncea., Competing Interests: The authors have declared that no competing interests exist.

The involvement of two extremely important signalling molecules, nitric oxide (NO) and abscisic acid (ABA) has been employed by plants to facilitate the adaptive/tolerate response during stressful conditions. However, the interactive role of exogenously applied NO and ABA is very less studied at physiological, biochemical and molecular levels. The present study therefore, evaluated the effects of individual and simultaneous addition of exogenous NO donor SNP (100μM) and ABA (10μM) on photosynthesis, Calvin-Benson cycle enzymes, S-assimilation enzymes, oxidative stress components, and genotoxicity in Brassica juncea cv. Varuna, exposed to polyethylene glycol (PEG)-induced drought stress. Results showed that a loss induced by PEG was significantly surpassed by the application of NO or/and ABA with PEG for chlorophyll content, net photosynthestic rate (Pn), internal CO 2 concentration (Ci), stomatal conductance (gs), transpiration rate (Tr), maximum photosystem II (PSII) efficiency (Fv/Fm), actual PSII efficiency (ΦPSII), intrinsic PSII efficiency (Fv´/ Fm´), photochemical quenching (qP), non-photochemical quenching (NPQ), electron transport chain (ETC), ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo), glyceraldehyde-3-phosphate dehydrogenase (GapDH), phosphoribulokinase (PRK), ATP-sulfurylase (ATP-S), and serine acetyltransferase (SAT) activities. The genomic template stability (GTS) (measured as changes in RAPD profiles) was significantly affected and showed varying degrees of DNA polymorphism, highest in PEG and lowest in PEG + NO and PEG + NO + ABA. Furthermore, the changes in RAPD profiles showed consistent results when compared with various photosynthetic and oxidative parameters. Altogether, this study concluded that supplementation of individual NO and together with ABA was more effective than individual ABA in alleviating PEG-induced drought stress in B. juncea L. seedlings., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We investigated phenotypic variations for pod shattering, pod length and number of seeds per pod in large germplasm collections of Brassica juncea (2n = 36; AABB) and its progenitor species, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Pod shatter resistance was measured as energy required for rupturing a mature dry pod, with a specially fabricated pendulum machine. Rupture energy (RE) ranged from 3.3 to 11.0 mJ in B. juncea. MCP 633, NR 3350 and Albeli required maximum energy to shatter a pod. It ranged from 2.5 to 7.8 mJ for B. rapa with an average of 5.5 mJ. B. nigra possessed easy to rupture pods. Correlation analysis showed strong associations among these traits in B. juncea and B. rapa. Genome wide association studies were conducted with select sets of B. juncea and B. rapa germplasm lines. Significant and annotated associations predict the role of FRUITFULL, MANNASE7, and NAC secondary wall thickening promoting factor (NST2) in the genetic regulation of shatter resistance in B. juncea. NST2 and SHP1 appeared important for pod length and seeds per pod in B. rapa. Candidate gene based association mapping also confirmed the role of SHP1 and NST2 in regulating pod shattering and related pod traits in B. rapa and B. juncea. Footprints of selection were detected in SHP1, SHP2 (B. rapa, B. nigra and B. juncea), RPL (B. rapa) and NAC (B. juncea). Our results provide insights into the genetic architecture of three pod traits. The identified genes are relevant to improving and securing crop productivity of mustard crop.

As a member of the large Brassicaceae family, yellow mustard (Sinapis alba L.) has been used as an important gene pool for the genetic improvement of cash crops in Brassicaceae. Understanding the phylogenetic relationship between Sinapis alba (S. alba) and other Brassicaceae crops can provide guidance on the introgression of its favorable alleles into related species. The chloroplast (cp) genome is an ideal model for assessing genome evolution and the phylogenetic relationships of complex angiosperm families. Herein, we de novo assembled the complete cp genome of S. alba by integrating the PacBio and Illumina sequencing platforms. A 153,760 bp quadripartite cycle without any gap was obtained, including a pair of inverted repeats (IRa and IRb) of 26,221 bp, separated by a large single copy (LSC) region of 83,506 bp and a small single copy (SSC) region of 17,821 bp. A total of 78 protein-coding genes, 30 tRNA genes, and four rRNA genes were identified in this cp genome, as were 89 simple sequence repeat (SSR) loci of 18 types. The codon usage analysis revealed a preferential use of the Leu codon with the A/U ending. The phylogenetic analysis using 82 Brassicaceae species demonstrated that S. alba had a close relationship with important Brassica and Raphanus species; moreover, it likely originated from a separate evolutionary pathway compared with the congeneric Sinapis arvensis. The synonymous (Ks) and non-synonymous (Ks) substitution rate analysis showed that genes encoding "Subunits of cytochrome b/f complex" were under the lowest purifying selection pressure, whereas those associated with "Maturase", "Subunit of acetyl-CoA", and "Subunits of NADH-dehydrogenase" underwent relatively higher purifying selection pressures. Our results provide valuable information for fully utilizing the S. alba cp genome as a potential genetic resource for the genetic improvement of Brassica and Raphanus species., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Microbes isolated from hyperaccumulating plants have been reported to be effective in achieving higher phytoextraction efficiency. The plant growth-promoting bacteria (PGPB) SaMR12 from the cadmium (Cd)/zinc hyperaccumulator Sedum alfredii Hance could promote the growth of a non-host plant, oilseed rape, under Cd stress. However, the effect of SaMR12 on Brasscia juncea antioxidative response under Cd exposure was still unclear., Results: A hydroponic experiment was conducted to study the effects of Sphingomonas SaMR12 on its non-host plant Brassica juncea (L.) Czern. under four different Cd treatments. The results showed that SaMR12 could colonize and aggregate in the roots and then move to the shoots. SaMR12 inoculation promoted plant growth by up to 71% in aboveground biomass and 81% in root biomass over that of the non-inoculated plants. SaMR12-inoculated plants significantly enhanced root Cd accumulation in the 10 and 20 μM Cd treatments, with 1.72- and 0.86-fold increases, respectively, over that of the non-inoculated plants. SaMR12 inoculation not only decreased shoot hydrogen peroxide (H 2 O 2 ) content by up to 38% and malondialdehyde (MDA) content by up to 60% but also reduced proline content by 7-30% in shoots and 17-32% in roots compared to the levels in non-inoculated plants. Additionally, SaMR12 inoculation promoted the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and facilitated the relative gene expression levels of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in the glutathione (GSH)-ascorbic acid (AsA) cycle., Conclusions: The results demonstrated that, under Cd stress, SaMR12 inoculation could activate the antioxidative response of B. juncea by decreasing the concentrations of H 2 O 2 , MDA and proline, increasing the activities of antioxidative enzymes, and regulating the GSH-AsA cycle. These results provide a theoretical foundation for the potential application of hyperaccumulator endophytic bacteria as remediating agents to improve heavy metal tolerance within non-host plant species, which could further improve phytoextraction efficiency.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes ( ACTIN , UBQ , EF-1α , UBC , IF-4α , TUB , PP2A, and HIS ), which were screened from the genome and transcriptome data in Brassica juncea . Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes ( BjuB045330 and BjuA003219 ) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

The genome in situ hybridization (GISH) technique has become important for deciphering the organization of the constituent genomes in the allopolyploid plants that comprise many of the crop species. This technique comprises using the nuclear DNA from the constituent genomes as probes that have been labeled separately with different nucleotides that can be identified by using secondary antibodies. The Brassica family includes a range of mustard species with diverse phytochemical and morphological profile, hence making it an important plant family in agriculture. Meiosis is a specialized cellular division which brings the homologous chromosomes together and creates recombinants via pairing and synapsis during its early phase. Transfer of the genetic material within homoelog pairs creates novelty in subsequent generations which hold promise for improving the agriculture sector. This chapter is concerned with developing a GISH technique that discriminates between the constituent genomes in the allopolyploid B. juncea, in order to study meiosis.

Chitinases belong to the group of Pathogenesis-related (PR) proteins that provides protection against fungal pathogens. This study presents the, genome-wide identification and characterization of chitinase gene family in two important oilseed crops B. juncea and C. sativa belonging to family Brassicaceae. We have identified 47 and 79 chitinase genes in the genomes of B. juncea and C. sativa, respectively. Phylogenetic analysis of chitinases in both the species revealed four distinct sub-groups, representing different classes of chitinases (I-V). Microscopic and biochemical study reveals the role of reactive oxygen species (ROS) scavenging enzymes in disease resistance of B. juncea and C. sativa. Furthermore, qRT-PCR analysis showed that expression of chitinases in both B. juncea and C. sativa was significantly induced after Alternaria brassicae infection. However, the fold change in chitinase gene expression was considerably higher in C. sativa compared to B. juncea, which further proves their role in C. sativa disease resistance to A. brassicae. This study provides comprehensive analysis on chitinase gene family in B. juncea and C. sativa and in future may serve as a potential candidate for improving disease resistance in B. juncea through transgenic approach., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Alternaria blight is one of the most devastating diseases of rapeseed-mustard caused by a necrotrophic fungus Alternaria brassicae. Lack of satisfactory resistance resource in Brassica is still a main obstruction for developing resistance against Alternaria. In this study, we have selected Brassica juncea, Sinapis alba and Camelina sativa to understand and unravel the mechanism of disease resistance against Alternaria. Histopathological studies showed early onset of necrosis in B. juncea (1 dpi) and delayed in S. alba (2 dpi) and C. sativa (3 dpi) respectively. Early and enhanced production of hydrogen peroxide (H 2 O 2 ) was observed in C. sativa and S. alba (6 hpi) when compared to B. juncea (12 hpi). An increase in catalase activity was observed in both C. sativa (36 % at 6 hpi) and S. alba (15 % at 12 hpi), whereas it significantly decreased in B. juncea at 6 hpi (23 %), 12 hpi (30 %) and 24 hpi (8 %). Gene expression analysis showed induction of PR-3 and PR-12 genes only in C. sativa and S. alba when compared to B. juncea suggesting their vital role for Alternaria resistance. In contrast, SA marker genes were significantly expressed in B. juncea only which provides evidence of hormonal cross talk in B. juncea during Alternaria infection thereby increasing its susceptibility., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Background: Narrow genetic base, complex allo-tetraploid genome and presence of repetitive elements have led the discovery of single nucleotide polymorphisms (SNPs) in Brassica juncea (AABB; 2n = 4x = 36) at a slower pace. Double digest RAD (ddRAD) - a genome complexity reduction technique followed by NGS was used to generate a total of 23 million paired-end reads from three genotypes each of Indian (Pusa Tarak, RSPR-01 and Urvashi) and Exotic (Donskaja IV, Zem 1 and EC287711) genepools., Results: Sequence data analysis led to the identification of 10,399 SNPs in six genotypes at a read depth of 10x coverage among the genotypes of two genepools. A total of 44 hyper-variable regions (nucleotide variation hotspots) were also found in the genome, of which 93% were found to be a part of coding genes/regions. The functionality of the identified SNPs was estimated by genotyping a subset of SNPs on MassARRAY® platform among a diverse set of B. juncea genotypes. SNP genotyping-based genetic diversity and population studies placed the genotypes into two distinct clusters based mostly on the place of origin. The genotypes were also characterized for six morphological traits, analysis of which revealed a significant difference in the mean values between Indian and Exotic genepools for six traits. The association analysis for six traits identified a total of 45 significant marker-trait associations on 11 chromosomes of A- and B- group of progenitor genomes., Conclusions: Despite narrow diversity, the ddRAD sequencing was able to identify large number of nucleotide polymorphisms between the two genepools. Association analysis led to the identification of common SNPs/genomic regions associated between flowering and maturity traits, thereby underscoring the possible role of common chromosomal regions-harboring genes controlling flowering and maturity in Brassica juncea.

Alternaria blight, caused by Alternaria brassicae , causes considerable yield loss in Brassica crops. While several blight-resistant varieties have been developed using resistance sources from host germplasm, none of them are entirely successful in imparting durable resistance. This has prompted the exploration of novel gene pools of nonhost plant species. Nonhost resistance (NHR) is a durable form of resistance, comprising pre- and postinvasion layers of defense. We aimed to identify the molecular basis of NHR to A. brassicae and identify the layers of NHR operating in a nonhost, chickpea ( Cicer arietinum ). To elucidate the layers of NHR operating against A. brassicae , we compared the histopathology and infection patterns of A. brassicae in C. arietinum and Brassica juncea . Delayed conidial germination, impeded hyphal growth, suppressed appressorium formation, and limited hyphal penetration occurred in the nonhost plant compared with the host plant, implying the involvement of the preinvasion layer of NHR in C. arietinum. Next, we investigated the molecular basis of robust NHR, in C. arietinum challenged with A. brassicae , by microarray-based global transcriptome profiling. Genes involved in stomatal closure, cuticular wax biosynthesis, cell-wall modification, and secondary metabolite production (contributing to preinvasion NHR) as well as reactive oxygen species (ROS) and cell death (contributing to postinvasion NHR) were found to be upregulated. Consistent with transcriptomic analysis, the morpho-pathological analysis revealed stomatal closure, ROS accumulation, and localized cell death in C. arietinum as the defense strategies against A. brassicae. Thus, we identified NHR-contributing genes with potential applications in blight resistance gene transfer to B. juncea .

Arsenic is a widespread toxic metalloid that is classified as a class I carcinogen known to cause adverse health effects in humans. In the present study, we investigated arsenic accumulation potential and comparative gene expression in Indian mustard. The amount of arsenic accumulated in shoots varied in the range of 15.99-1138.70 mg/kg on a dry weight basis among five cultivars. Comparative expression analysis revealed 10 870 significantly differentially expressed genes mostly belonging to response to stress, metabolic processes, signal transduction, transporter activity, and transcription regulator activity to be up-regulated, while most of the genes involved in photosynthesis, developmental processes, and cell growth were found to be down-regulated in arsenic-treated tissues. Further, pathway analysis using the KEGG Automated Annotation server (KAAS) revealed a large-scale reprogramming of genes involved in genetic and environmental information processing pathways. Top pathways with maximum KEGG orthology hits included carbon metabolism (2.5%), biosynthesis of amino acids (2.1%), plant hormone signal transduction (1.4%), and glutathione metabolism (0.6%). A transcriptomic investigation to understand the arsenic accumulation and detoxification in Indian mustard will not only help to improve its phytoremediation efficiency but also add to the control measures required to check bioaccumulation of arsenic in the food chain.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a major disease of crop brassicas, with inadequate variation for resistance in primary gene pools. We utilized a wild Brassicaceae species with excellent resistance against stem rot to develop a set of B. juncea - B. fruticulosa introgression lines (ILs). These were assessed for resistance using a highly reproducible stem inoculation technique against a virulent pathogen isolate. Over 40% of ILs showed higher levels of resistance. IL-43, IL-175, IL-215, IL-223 and IL-277 were most resistant ILs over three crop seasons. Sequence reads (21x) from the three most diverse ILs were then used to create B. juncea pseudomolecules, by replacing SNPs of reference B. juncea with those of re-sequenced ILs. Genotyping by sequencing (GBS) was also carried out for 88 ILs. Resultant sequence tags were then mapped on to the B. juncea pseudomolecules, and SNP genotypes prepared for each IL. Genome wide association studies helped to map resistance responses to stem rot. A total of 13 significant loci were identified on seven B. juncea chromosomes (A01, A03, A04, A05, A08, A09 and B05). Annotation of the genomic region around identified SNPs allowed identification of 20 candidate genes belonging to major disease resistance protein families, including TIR-NBS-LRR class, Chitinase, Malectin/receptor-like protein kinase, defensin-like (DEFL), desulfoglucosinolate sulfotransferase protein and lipoxygenase. A majority of the significant SNPs could be validated using whole genome sequences (21x) from five advanced generation lines being bred for Sclerotinia resistance as compared to three susceptible B. juncea germplasm lines. Our findings not only provide critical new understanding of the defensive pathway of B. fruticulosa resistance, but will also enable development of marker candidates for assisted transfer of introgressed resistant loci in to agronomically superior cultivars of crop Brassica.

Organic selenium (Se), specifically Se-methylselenocysteine (MeSeCys), has demonstrated potential effects in human disease prevention including cancer and the emerging ameliorating effect on Alzheimer's disease. In plants, selenocysteine methyltransferase (SMT) is the key enzyme responsible for MeSeCys formation. In this study, we first isolated a novel SMT gene, designated as BjSMT, from the genome of a known Se accumulator, Brassica juncea L. BjSMT shows high sequence (amino acid) similarity with its orthologues from Brassica napus and Brassica oleracea var. oleracea, which can use homocysteine (HoCys) and selenocysteine (SeCys) as substrates. Similar to its closest homologues, BjSMT also possesses a conserved Thr187 which is involved in transferring a methyl group to HoCys by donating a hydrogen bond, suggesting that BjSMT can methylate both HoCys and SeCys substrates. Using quantitative real-time PCR (qRT-PCR) technology and BjSMT-transformed tobacco (Nicotiana tabacum) plants, we observed how BjSMT responds to selenite [Se(IV)] and selenate [Se(VI)] stress in B. juncea, and how the phenotypes of BjSMT-overexpressing tobacco cultured under selenite stress are affected. BjSMT expression was nearly undetectable in the B. juncea plant without Se exposure, but in the plant leaves it can be rapidly and significantly up-regulated upon a low level of selenite stress, and enormously up-regulated upon selenate treatment. Overexpression of BjSMT in tobacco substantially enhanced tolerance to selenite stress manifested as significantly higher fresh weight, plant height, and chlorophyll content than control plants. In addition, transgenic plants exhibited low glutathione peroxidase activity in response to a lower dose of selenite stress (with a higher dose of selenite stress resulting in a high activity response) compared with the controls. Importantly, the BjSMT-transformed tobacco plants accumulated a high level of Se upon selenite stress, and the plants also had significantly increased MeSeCys production potential in their leaves. This first study of B. juncea SMT demonstrates its potential applications in crop MeSeCys biofortification and phytoremediation of Se pollution., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The Salt Overly Sensitive (SOS) pathway in Arabidopsis thaliana plays important roles in maintaining appropriate ion homeostasis in the cytoplasm and regulating plant tolerance to salinity. However, little is known about the details regarding SOS family genes in the tuber mustard crop (Brassica juncea var. tumida). Here, 12 BjSOS family genes were identified in the B. juncea var. tumida genome including two homologous genes of SOS1, one and three homologs of SOS2 and SOS3, two homologs of SOS4, two homologs of SOS5 and two homologs of SOS6, respectively. The results of conserved motif analysis showed that these SOS homologs contained similar protein structures. By analyzing the cis-elements in the promoters of those BjSOS genes, several hormone- and stress-related cis-elements were found. The results of gene expression analysis showed that the homologous genes were induced by abiotic stress and pathogen. These findings indicate that BjSOS genes play crucial roles in the plant response to biotic and abiotic stresses. This study provides valuable information for further investigations of BjSOS genes in tuber mustard., Competing Interests: The authors have declared that no competing interests exist.

SQUAMOSA promoter-binding protein-like (SPL), as plant specific transcription factors, is involved in many plant growth and development processes. However, there is less systematical study for SPL transcription factor in B. juncea (Cruciferae). Here, a total of 59 SPL genes classified into eight phylogenetic groups were identified in B. juncea, highly conserved within each ortholog were also found based on gene structure, conserved motif, as well as clustering level. In addition, clustering of SPL domain showed that two zinc finger-like structures and NLS segments were identified in almost of BjuSPLs. Analyzed of putative cis-elements for BjuSPLs demonstrated that SPL transcription factors were involved in adverse environmental changes, such as light, plant stresses and phytohormones response. Expression analysis showed that differentially expressed SPL genes were identified in flower and stem development of Cruciferae; such as BjuSPL3a-B, BjuSPL2b&#95;B and BjuSPL2c&#95;A were significantly expressed in flower; BjuSPL 3b&#95;B and BjuSPL10a&#95;A were significantly expressed in stem node (VP: vegetative period). Moreover, 28 of the 59 BjuSPLs were found involved in their posttranscriptional regulation targeted by miR156. We demonstrated that miR156 negatively regulated BjuSPL10a&#95;A and BjuSPL3b&#95;B to act for stem development in B. juncea., Competing Interests: The authors have declared that no competing interests exist.

Heavy metal removal by phytoremediation bears a great potential to decontaminate soils and Brassica juncea L. (Indian mustard) seems to be a possible candidate species for this purpose. A field experiment was conducted to compare the efficiency of eighty Indian mustard cultivars for phytoextraction of cadmium (Cd) and lead (Pb) from bimetal contaminated soil. Our results indicated that total Cd and Pb concentrations in the shoots and roots were in the range of 2.43 ± 0.00 to 0.31 ± 0.02 mg/kg and 2.94 ± 0.05 to 0.44 ± 0.03 mg/kg and 5.33 ± 0.76 to 0.47 ± 0.20 mg/kg and 3.78 ± 0.06 to 0.16 ± 0.08 mg/kg. Significant differences based on the translocation factors indicated that root-to-shoot transfer is higher for Pb (3.87 ± 0.12 to 0.48 ± 0.03) than Cd (3.38 ± 0.05 to 0.22 ± 0.01). Furthermore, significant correlations between dry weights, Cd and Pb concentrations and uptake in both shoots and roots were observed, but translocation factor showed a negative correlation with roots, but not in shoots. Among 80 genotypes of Indian mustard IM-25, IM-13 and IM-65 for Cd and IM-79, IM-24 and IM-32 for Pb seems to perform well for phytoextraction. The results of the field experiment suggest that certain Brassica juncea L. cultivars are suitable for removal of Cd and Pb in low to moderately contaminated soils., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. Joint QTL mapping and RNA-sequencing analyses reveal one candidate gene of IIL1 (LOC106416451) for GSL metabolism in B. juncea. These results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea., Competing Interests: The authors have declared that no competing interests exist.

Indian mustard (Brassica juncea L.) was more tolerance to Cs than some sensitive plants, such as Arabidopsis thaliana and Vicia faba, and may have a special detoxification mechanism. In this study, the effects on reactive oxygen species (ROS) content, the antioxidant enzyme system and chelation system in Indian mustard were studied by observing different plant physiological responses. In addition, we focused on the analysis of gene regulatory networks related to ROS formation, ROS scavenging system, and other stress-response genes to Cs exposure using a transcriptome-sequencing database. The results showed that ROS and malonaldehyde content in seedlings increased significantly in Cs-treatment groups. The enzyme activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were increased, and the synthesis of antioxidants glutathione, phytochelatin and metallothionein also increased under Cs treatment. Further analysis showed that ROS formation pathways were primarily the photosynthetic electron transport chain process and photorespiration process in the peroxisome. Antioxidant enzyme systems and the respiratory burst oxidase homolog protein-mediated signal transduction pathway played a key role in ROS scavenging. In summary, one of the mechanisms of tolerance and detoxification of Indian mustard to Cs was that it enhanced the scavenging ability of antioxidant enzymes to ROS, chelated free Cs ions in cells and regulated the expression of related disease-resistant genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Response of drought susceptible (DS) genotype Pusa Karishma LES-39 and drought tolerant (DT) mustard genotype NPJ-124, to rhizobacterial inoculation under water deficit stress, was compared in the present study to determine the influence of inoculants on biochemical and physiological attributes of these two different genotypes. Inoculation was observed to improve root and shoot dry weight in both the genotypes, although better results were observed in the DS genotype. There was variation in the response of the two genotypes to rhizobacterial inoculation, under water deficit stress. Significant improvement in most of the physiological and biochemical parameters including antioxidative enzyme activities of the DS genotype; with concomitant decrease in starch content, accumulation of H 2 O 2 and lipid peroxidation upon inoculation of rhizobacteria was observed. In contrast, there was improvement in only few physiological and biochemical parameters in the DT genotype in response to inoculation with rhizobacteria. There was significant increase in catalase enzyme activity along with concomitant decrease in lipid peroxidation. Thus, drought susceptibility of the mustard genotypes, NPJ-124 and Pusa Karishma LES-39, determined their physiological, biochemical and antioxidative responses to rhizobacterial inoculation under water deficit stress. Expression of drought stress responsive genes belonging to ABA-dependent (RD20 and RD26) and ABA-independent (DREB2 and DREB1-2) pathways was studied in the DS genotype. Expression of DREB2 and DREB1-2 genes was considerably enhanced due to inoculation under water deficit stress; indicating that in Bacillus-mediated priming for drought stress tolerance, in this genotype, ABA-independent pathway probably played key role in enhancing tolerance to drought stress., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Concomitant increase of auxin-responsive factors ARF16 and ARF17 , along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola , revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola -inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4 , ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea . Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola .

Quantitative real-time PCR (qRT-PCR) is an efficient method to estimate the gene expression levels but the accuracy of its result largely depends on the stability of the reference gene. Many studies have reported considerable variation in the expression of reference genes (RGs) in different tissue and conditions. Therefore, screening for appropriate RGs with stable expression is crucial for functional analysis of the target gene. Two closely related crucifers Brassica juncea (cultivated) and Camelina sativa (wild) respond differently towards various abiotic and biotic stress where C. sativa exhibits higher tolerance to various stress. Comparative gene expression analysis of the target genes between two such species is the key approach to understand the mechanism of a plant's response to stress. However, using an unsuitable RG can lead to misinterpretation of expression levels of the target gene in such studies. In this investigation, the stability of seven candidate RGs including traditional housekeeping genes (HKGs) and novel candidate RGs were identified across diverse sample sets of B. juncea and C. sativa representing- hormone treated, wounded, Alternaria brassicae inoculated and combination treated samples (exogenous hormone treatment followed by A. brassicae inoculation). In this investigation, we identified stable RGs in both the species and the most suitable RGs to perform an unbiased comparative gene expression analysis between B. juncea and C. sativa. Results revealed that TIPS41 and PP2A were identified as the overall best performing RGs in both the species. However, the most suitable RG for each sample subset representing different condition must be individually selected. In Hormone treated and wounded samples TIPS41 expressed stably in both the species and in A. brassicae inoculated and combination treatment performance of PP2A was the best. In this study, for the first time, we have identified and validated stable reference gene in C. sativa for accurate normalization of gene expression data., Competing Interests: The authors have declared that no competing interests exist.

To analyze the differences between high- and low-accumulation plants in cesium (Cs) uptake and its related mechanism, Brassica juncea (a hyperaccumulation plant for Cs) and Vicia faba (a low-accumulation plant for Cs) were selected as comparative experimental materials. The contributions to Cs uptake of a K-transporter-mediated high-affinity transport system and a K-channel-mediated low-affinity transport system in the two plants were compared and analyzed. The difference between the two plants in the mechanism of Cs uptake was further analyzed using transcription sequence technology. The results show that the transfer characteristics of Cs in the two plants had a similar distribution relationship with K. The contribution rate of the K-channel pathway to Cs uptake was 32.00% in the V. faba seedling roots, which was significantly higher than for B. juncea (9.81%) (P < 0.01); the contribution rate of the K-transporter pathway to Cs uptake of the B. juncea seedlings was 32.08%, which was significantly higher than that of the V. faba seedlings (17.13%)(P < 0.05). Other uptake pathways also mediated the uptake of Cs by roots in B. juncea and V. faba (contribution rate: 54.92-60.09% and 42.18-59.73%, respectively). The transcriptome sequencing results confirmed that Cs-induced treatment significantly inhibited the expression of the K-transporter protein and K-channel protein-related genes in the V. faba roots, but it had no significant effect on the expression of related genes in the B. juncea roots. Thus, one reason for the significant difference between the two plant in the accumulation of Cs is that Cs inhibited the expression of related transporter protein genes in the V. faba roots., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Heterosis refers to the superior performance of F1 hybrids over their respective parental inbred lines. Although the genetic and expression basis of heterosis have been previously investigated, the metabolic basis for this phenomenon is poorly understood. In a preliminary morphological study in Brassica juncea, we observed significant heterosis at the 50% flowering stage, wherein both the growth and reproduction of F1 reciprocal hybrids were greater than that of their parents. To identify the possible metabolic causes or consequences of this heterosis, we carried out targeted LC-MS analysis of 48 primary (amino acids and sugars) and secondary metabolites (phytohormones, glucosinolates, flavonoids, and phenolic esters) in five developmental tissues at 50% flowering in hybrids and inbred parents. Principal component analysis (PCA) of metabolites clearly separated inbred lines from their hybrids, particularly in the bud tissues. In general, secondary metabolites displayed more negative heterosis values in comparison to primary metabolites. The tested primary and secondary metabolites displayed both additive and non-additive modes of inheritance in F1 hybrids, wherein the number of metabolites showing an additive mode of inheritance were higher in buds and siliques (52.77-97.14%) compared to leaf tissues (47.37-80%). Partial least regression (PLS) analysis further showed that primary metabolites, in general, displayed higher association with morphological parameters in F1 hybrids. Overall, our results are consistent with a resource-cost model for heterosis in B. juncea, where metabolite allocation in hybrids appears to favor growth, at the expense of secondary metabolism.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T 1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al. 2014, Funct Integr Genomics 14:307-317).

Key Message: BjuWRR1, a CNL-type R gene, was identified from an east European gene pool line of Brassica juncea and validated for conferring resistance to white rust by genetic transformation. White rust caused by the oomycete pathogen Albugo candida is a significant disease of crucifer crops including Brassica juncea (mustard), a major oilseed crop of the Indian subcontinent. Earlier, a resistance-conferring locus named AcB1-A5.1 was mapped in an east European gene pool line of B. juncea-Donskaja-IV. This line was tested along with some other lines of B. juncea (AABB), B. rapa (AA) and B. nigra (BB) for resistance to six isolates of A. candida collected from different mustard growing regions of India. Donskaja-IV was found to be completely resistant to all the tested isolates. Sequencing of a BAC spanning the locus AcB1-A5.1 showed the presence of a single CC-NB-LRR protein encoding R gene. The genomic sequence of the putative R gene with its native promoter and terminator was used for the genetic transformation of a susceptible Indian gene pool line Varuna and was found to confer complete resistance to all the isolates. This is the first white rust resistance-conferring gene described from Brassica species and has been named BjuWRR1. Allelic variants of the gene in B. juncea germplasm and orthologues in the Brassicaceae genomes were studied to understand the evolutionary dynamics of the BjuWRR1 gene.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N 0 ) and agrnomically recommened dose (100 kg/ha) of N application (N 100 ). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

Plants have ability to adapt themselves through altering their growth process. In the present study, we examined exogenous application of nitric oxide (NO) on nitrogen metabolism and auxin (PIN) gene expression, and its possible role in alleviation of arsenic (As) toxicity in Brassica juncea seedlings. Seven days old hydroponically grown B. juncea seedlings were exposed to As III (150 μM), Sodium nitroprusside (NO donor, 100 μM), As III  + SNP and control (without metal)for 48 h. Experimental results revealed that As III stress: enhanced the level of nitrite, NiR activity, NO 3 - and NH 4 + content as well as NADH-GOGAT activity; but GDH level decreased; enhanced content of amino acids; upregulated gene expression level of N metabolism and downregulated polar auxin transporter genes (PIN); inhibited plant growth and morphological parameters; increased MDA, H 2 O 2, cysteine, proline content, enzymatic antioxidants (SOD, CAT, APX; GSH, TT, NPT); and decreased nutrient content. As III  + SNP combination reduced the accumulation of As; improved growth; chlorophyll, protein and mineral nutrient content by scavenging ROS generation; maintained amino acids content; downregulated expression of N metabolism genes and upregulated expression of auxin transporter (PIN) genes . Additional biochemical data depicts reduction in the level of nitrogen related enzymatic activities, and other stress related parameters. Overall, this study provides an integrated view that exogenous SNP (NO donor) supplementation alleviated the inhibitory role of As III in B. juncea seedlings by altering nutrients, amino acids and auxin redistribution via expression of nitrogen and PIN gene profiling., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sucrose plays pivotal role in energy metabolism and regulating gene expression of several physiological processes in higher plants. Here, fourteen sucrose synthase (SUS) genes have been identified in the allotetraploid genome of Indian mustard, Brassica juncea. The identified SUS genes in B. juncea (BjSUS) were derived from the two-progenitor species, B. rapa and B. nigra. Intron-exon analysis indicated loss or gain of 1-3 introns in diversification of SUS gene family. Phylogenetic analysis revealed discrete evolutionary paths for the BjSUS genes, originating from three ancestor groups, SUS I, SUS II and SUS III. Gene expression study revealed significant variability in expression of the BjSUS paralogs across the different tissues. BjSUS genes showed transcriptional activation in response to defense hormones and a late response to wounding. Tissue and temporal specificity of expression revealed importance of specific SUS paralogs at different developmental stages and under different stress conditions. The study highlighted differential involvement of SUS paralogs in sucrose metabolism across the tissues and stress-responses, in a major oilseed crop B. juncea., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Branching is a major determinant of crop yield, and enables vigorous shoot growth and the production of a dense canopy. Phytochrome A signal transduction 1 (PAT1) positively regulates phytochrome A signal transduction in response to light, but its effects on branching remain unknown. In this study, we mapped PAT1, and revealed a previously unknown role related to branching and flowering in leafy Brassica juncea. Earlier and increased branching was observed when PAT1 expression was down-regulated, implying that PAT1 negatively regulates shoot branching. Additionally, down-regulated PAT1 expression reversed the inhibited branching induced by far-red light, suggesting PAT1 is involved in the shade avoidance response. PAT1 negatively regulated branching only after bud initiation. The observed interaction between PAT1 and BRC1 implied that PAT1 influences bud outgrowth in a BRC1-dependent manner. Biochemical and genetic evidence indicate that PAT1 directly interacts with CONSTANS-LIKE 13 (COL13), which negatively regulates flowering, with the resulting PAT1-COL13 complex mediating shoot branching and flowering. Our findings reveal a new crosstalk modality between phytochrome signalling and flowering pathways during the regulation of shoot branching and flowering. The data presented herein may be useful for future studies involving the editing of the GRAS family transcription factor PAT1 gene to enhance crop productivity and enable earlier harvesting., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Abscisic acid (ABA) plays important roles in multiple physiological processes, such as plant response to stresses and plant development. The ABA receptors pyrabactin resistance (PYR)/ PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) play a crucial role in ABA perception and signaling. However, little is known about the details regarding PYL family genes in Brassica juncea var. tumida. Here, 25 PYL family genes were identified in B. juncea var. tumida genome, including BjuPYL3, BjuPYL4s, BjuPYL5s, BjuPYL6s, BjuPYL7s, BjuPYL8s, BjuPYL10s, BjuPYL11s, and BjuPYL13. The results of phylogenic analysis and gene structure showed that the PYL family genes performed similar gene characteristics. By analyzing cis -elements in the promoters of those BjuPYLs , several hormone and stress related cis -elements were found. The results of gene expression analysis showed that the ABA receptor homologous genes were induced by abiotic and biotic stress. The tissue-specific gene expression patterns of BjuPYLs also suggested those genes might regulate the stem swelling during plant growth. These findings indicate that Bj uPYLs are involved in plant response to stresses and organ development. This study provides valuable information for further functional investigations of PYL family genes in B. juncea var. tumida., Competing Interests: The authors declare that they have no competing interests for this research.

Centromeres are indispensable functional units of chromosomes. The evolutionary mechanisms underlying the rapid evolution of centromeric repeats, especially those following polyploidy, remain unknown. In this study, we isolated centromeric sequences of Brassica nigra, a model diploid progenitor (B genome) of the allopolyploid species B. juncea (AB genome) and B. carinata (BC genome) by chromatin immunoprecipitation of nucleosomes containing the centromere-specific histone CENH3. Sequence analysis detected no centromeric satellite DNAs, and most B. nigra centromeric repeats were found to originate from Tyl/copia-class retrotransposons. In cytological analyses, six of the seven analyzed repeat clusters had no FISH signals in A or C genomes of the related diploid species B. rapa and B. oleracea. Notably, five repeat clusters had FISH signals in both A and B subgenomes in the tetraploid B. juncea. In the tetraploid B. carinata, only CL23 displayed three pairs of signals in terminal or interstitial regions of the C-derived chromosome, and no evidence of colonization of CLs onto C-subgenome centromeres was found in B. carinata. This observation suggests that centromeric repeats spread and proliferated between genomes after polyploidization. CL3 and CRB are likely ancient centromeric sequences arising prior to the divergence of diploid Brassica which have detected signals across the genus. And in allotetraploids B. juncea and B. carinata, the FISH signal intensity of CL3 and CRB differed among subgenomes. We discussed possible mechanisms for centromeric repeat divergence during Brassica speciation and polyploid evolution, thus providing insights into centromeric repeat establishment and targeting.

Indian hedge mustard (Sisymbrium orientale) (IHM) is an important broadleaf weed across southern Australia. Resistance to sulfonylurea (SU) herbicides that inhibit acetohydroxyacid synthase (AHAS) is extensive in Australia, but resistance to imidazolinone (IMI) herbicides has only been reported recently. The AHAS-mutation profile of 65 IHM populations collected randomly from cropped fields was investigated to better understand the extent and types of resistance present. Resistance to SU herbicides was present in 40% of the populations and resistance to IMI herbicides in 11%. Mutations were identified in SoAHAS by sequence analysis, and included previously reported amino-acid substitutions at Pro197 and Trp574, but also new substitutions at Pro197 and Asp376 for this species. One population with possible non-target-site resistance was identified. Germination studies with fresh seed found no significant effect by mutations in SoAHAS on germination; however, population factors had a large effect on germination in S. orientale. Resistance to AHAS-inhibiting herbicides in populations of S. orientale is endowed by mutations in SoAHAS in all but one population examined. Mutations at Pro197 conferring resistance to SU herbicides were most common, while mutations at Trp574 that provide resistance to IMI herbicides are also present., (Copyright © 2019. Published by Elsevier Inc.)

Background: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F 1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC 1 and 3R:1S in the F 2 , which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis., Results: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819., Conclusion: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study.

Background: Alloplasmic lines, in which the nuclear genome is combined with wild cytoplasm, are often characterized by cytoplasmic male sterility (CMS), regardless of whether it was derived from sexual or somatic hybridization with wild relatives. In this study, we sequenced and analyzed the mitochondrial genomes of five such alloplasmic lines in Brassica juncea., Results: The assembled and annotated mitochondrial genomes of the five alloplasmic lines were found to have virtually identical gene contents. They preserved most of the ancestral mitochondrial segments, and the same candidate male sterility gene (orf108) was found harbored in mitotype-specific sequences. We also detected promiscuous sequences of chloroplast origin that were conserved among plants of the Brassicaceae, and found the RNA editing profiles to vary across the five mitochondrial genomes., Conclusions: On the basis of our characterization of the genetic nature of five alloplasmic mitochondrial genomes, we speculated that the putative candidate male sterility gene orf108 may not be responsible for the CMS observed in Brassica oxyrrhina and Diplotaxis catholica. Furthermore, we propose the potential coincidence of CMS in alloplasmic lines. Our findings lay the foundation for further elucidation of male sterility gene.