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Cryptochrome 2 (CRY2) perceives blue/UV-A light and regulates photomorphogenesis in plants. However, besides Arabidopsis, CRY2 has been functionally characterized only in native species of japonica rice and tomato. In the present study, the BnCRY2a, generating a relatively longer cDNA and harboring an intron in its 5'UTR, has been characterized in detail. Western blot analysis revealed that BnCRY2a is light labile and degraded rapidly by 26S proteasome when seedlings are irradiated with blue light. For functional analysis, BnCRY2a was over-expressed in Brassica juncea, a related species more amenable to transformation. The BnCRY2a over-expression (BnCRY2a

SummaryBrassica carinata(BBCC) commonly referred to as Ethiopian mustard is a natural allotetraploid containing the genomes ofBrassica nigra(BB) andBrassica oleracea(CC). It is an oilseed crop endemic to the Northeastern regions of Africa. Although it is grown in a limited manner,B. carinatais of value as it is resistant/highly tolerant to most of the pathogens affecting cultivated Brassica species of the U’s triangle that are grown worldwide as oilseed and vegetable crops. We report a chromosome-scale genome assembly ofB. carinataaccession HC20 using long-read Oxford Nanopore and Illumina sequencing and BioNano optical maps. The assembly has a scaffold N50 of ~39.8 Mb and covers ~1.11 Gb of the genome. We compared the available long-read genome assemblies of the six species of the U’s triangle and found a highly conserved gene number and collinearity suggesting thatB. juncea(AABB),B. napus(AACC), andB. carinataare strict allopolyploids. We cataloged the nucleotide-binding and leucine-rich repeat immune receptor (NLR) repertoire ofB. carinataresulting in the identification of 465 NLRs. We investigated the extent and nature of early generation genomic interactions between the subgenomes ofB. carinataandB. junceain interspecific crosses between the two species. We found that C chromosome additions are well tolerated, with homoeologous exchanges occurring between the A and C genomes. Based on the genomic interactions, we propose strategies to utilize the interspecific crosses for transferring disease resistance fromB. carinatatoB. junceaand other Brassica species.

Glucosinolate content in the Brassica oilseed crops has been reduced to the globally accepted Canola quality levels, making the seed meal useful as a protein-rich animal feed. However, the overall lower glucosinolate content in such plants renders them vulnerable to pests. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes to develop ideal oilseed mustard lines with the desired low levels of seed glucosinolates while maintaining high glucosinolate levels in leaves and pod walls, without affecting the seed yield and seed quality traits. The low-seed and high-leaf glucosinolate mustard lines with improved nutritional and defense traits will raise its international trade potential both as oilseed and feed crop.

Brassica juncea (AABB, 2n=36), commonly called mustard is an allopolyploid crop of recent origin but has considerable morphological and underlying genetic variation. An F1-derived doubled haploid (F1DH) population developed from a cross between a Indian oleiferous line, Varuna, and a Chinese stem type vegetable mustard, Tumida showed significant variability for some key plant architectural traits, including four stem strength-related traits, stem diameter, plant height, branch initiation height, number of primary branches (Pbr), and days to flowering (Df). Multi-environment QTL analysis identified twenty environmentally stable QTL for the nine plant architectural traits. Both Tumida and Varuna contain some positive QTL that can be used to breed superior ideotypes in mustard. A QTL cluster on LG A10 contained environmentally stable QTL for seven architectural traits. This region also contained overlapping environmentally stable major QTL (phenotypic variance ≥ 10%) for Df and Pbr, with Tumida contributing the trait enhancing alleles for both the traits. Since early flowering is critical for the cultivation of mustard in the Indian subcontinent, this QTL cannot be used for the improvement of Pbr in the Indian gene pool lines. Conditional QTL analysis for Pbr identified the QTL for improvement of Pbr without negative effects on Df. The environmentally stable QTL were projected on the genome assemblies of Tumida and Varuna for the identification of candidate genes. These findings provide insights into the genetics of plant architectural traits in two diverse gene pools of B. juncea and provide opportunities for improvement of the plant architecture through marker-assisted introgressions.Key MessageGenetic mapping of some key plant architectural traits in a vegetable type and an oleiferous B. juncea cross revealed environmentally stable QTL and candidate genes for breeding more productive ideotypes.

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

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

Seed size/weight is a multigenic trait that is governed by complex transcriptional regulatory pathways. An understanding of the genetic basis of seed size is of great interest in the improvement of seed yield and quality in oilseed crops. A global transcriptome analysis was performed at the initial stages of seed development in two lines of

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

The globally cultivated Brassica crops contain high deliverable concentrations of health-promoting glucosinolates. Development of a Visible-Near InfraRed Spectroscopy (Vis-NIRS) calibration to profile different glucosinolate components from 641 diverse Brassica juncea chemotypes was attempted in this study. Principal component analysis of HPLC-determined glucosinolates established the distinctiveness of four B. juncea populations used. Subsequently, modified partial least square regression based population-specific and combined Vis-NIRS models were developed, wherein the combined model exhibited higher coefficient of determination (R2; 0.81–0.97) for eight glucosinolates and higher ratio of prediction determination (RPD; 2.42–5.35) for seven glucosinolates in B. juncea populations. Furthermore, range error ratio (RER > 4) for twelve and RER > 10 for eight glucosinolates make the combined model acceptable for screening and quality control. The model also provided excellent prediction for aliphatic glucosinolates in four oilseed Brassica species. Overall, our work highlights the potential of Vis-NIR spectroscopy in estimating glucosinolate content in the economically important Brassica oilseeds.

Quantitative disease resistance (QDR) is the predominant form of resistance against necrotrophic pathogens. The genes and mechanisms underlying QDR are not well known. In the current study, the Arabidopsis-Alternaria brassicae pathosystem was used to uncover the genetic architecture underlying resistance to A. brassicae in a set of geographically diverse Arabidopsis accessions. Arabidopsis accessions revealed a rich variation in the host responses to the pathogen, varying from complete resistance to high susceptibility. Genome-wide association (GWA) mapping revealed multiple regions to be associated with disease resistance. A subset of genes prioritized on the basis of gene annotations and evidence of transcriptional regulation in other biotic stresses was analysed using a reverse genetics approach employing T-DNA insertion mutants. The mutants of three genes, namely At1g06990 (GDSL-motif lipase), At3g25180 (CYP82G1) and At5g37500 (GORK), displayed an enhanced susceptibility relative to the wild-type. These genes are involved in the development of morphological phenotypes (stomatal aperture) and secondary metabolite synthesis, thus defining some of the diverse facets of quantitative resistance against A. brassicae.

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

Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight. Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0 g to more than 7.0 g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6 g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six ‘consensus’ QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

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.

White rust caused by 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.HighlightBjuWRR1, 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.

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.

We have earlier reported the deployment of an effective pollination control system for hybrid seed production in Brassica juncea based on use of the barnase-barstar system. The current study analyses the stability and performance of four male-sterile (barnase) and 34 fertility restorer (barstar) lines under containment field conditions. Line (barnase) x tester (barstar) crosses were studied for effective fertility restoration over three successive growing seasons by screening the F1 and representative F2 progeny for three major parameters, namely segregation of Basta resistance and sensitive plants, distribution of male-sterile and fertile plants (vis-a-vis segregation of the barnase and the barstar genes) and pollen viability of the restored events. A total of 29 male sterile/restorer combinations out of 53 obtained in the first growing season were stable for their restoration ability when tested for two subsequent growing seasons. Stable restorers with high pollen viability were obtained at a significantly higher frequency for one of the barnase lines, bn 3.6, out of the four lines tested. Among the barstar lines, constructs carrying two transcription units of the barstar gene provided more effective fertility restoration. Quantitative real-time PCR analysis of restored progeny obtained for all the four barnase lines showed that three barnase lines (bn 3.23, bn 3.4 and bn 3.48) had an early and much higher level of barnase expression than the line bn 3.6 indicating that the level of barnase expression in the line bn 3.6 is optimal for better and stable restoration. It was observed that a high barstar : barnase transcript ratio and an extended window of barstar gene expression are critical parameters for the development of stable male-sterile (barnase) and fertility-restorer (barstar) combinations.

Genetic locus for tetralocular ovary ( tet - o ) in Brassica rapa was identified and it was shown that the number of locules and width of silique are associated. Brassica rapa is a highly polymorphic species containing many vegetables and oleiferous types. An interesting group of oleiferous types is the yellow sarson group (subspecies trilocularis) grown mostly in eastern India. This group contains lines that have bilocular ovaries, a defining trait of Brassicaceae, but also lines that have tetralocular ovaries. Yellow sarson lines commonly have high silique width which is further enhanced in the tetralocular types. We mapped the locus influencing tetralocular ovary in B. rapa using three mapping populations (F2, F6 and F7) derived from a cross between Chiifu (subspecies pekinensis, having bilocular ovary) and Tetralocular (having tetralocular ovary). QTL mapping of silique width was undertaken using the three mapping populations and a F2 population derived from a cross between Chiifu and YSPB-24 (a bilocular line belonging to yellow sarson group). Qualitative mapping of the trait governing locule number (tet-o) in B. rapa mapped the locus to linkage group A4. QTL mapping for silique width detected a major QTL on LG A4, co-mapping with the tet-o locus in bilocular/tetralocular cross. This QTL was not detected in the bilocular/bilocular cross. Saturation mapping of the tet-o region with SNP markers identified Bra034340, a homologue of CLAVATA3 of Arabidopsis thaliana, as the candidate gene for locule number. A C → T transition at position 176 of the coding sequence of Bra034340 revealed co-segregation with the tetralocular phenotype. The study of silique related traits is of interest both for understanding evolution under artificial selection and for breeding of cultivated Brassica species.

Identification of the candidate gene responsible for the seed coat colour variation in Brassica juncea was undertaken following an earlier study where two independent loci (BjSc1 and BjSc2) were mapped to two linkage groups, LG A9 and B3 (Padmaja et al. in Theor Appl Genet 111:8-14, 2005). The genome search from BRAD data for the presence of flavonoid genes in B. rapa identified three candidate genes namely, DFR, TT1 and TT8 in the LG A9. Quantitative real-time PCR revealed absence of transcript for the late biosynthetic genes (LBGs) and showed significant reduction of transcript in the TT8 from the developing seeds of yellow-seeded line. While mapping of two DFR genes, the BjuA.DFR and BjuB.DFR did not show perfect co-segregation with the seed coat colour loci, that of the two TT8 genes, BjuA.TT8 and BjuB.TT8 showed perfect co-segregation with the seed coat colour phenotype. The BjuA.TT8 allele from the yellow-seeded line revealed the presence of an insertion of 1,279 bp in the exon 7 and did not produce any transcript as revealed by reverse transcriptase PCR. The BjuB.TT8 allele from the yellow-seeded line revealed the presence of an SNP (C→T) in the exon 7 resulting in a stop codon predicting a truncated protein lacking the C-terminal 8 amino acid residues and produced significantly low level of transcript than its wild-type counterpart. Hence, it is hypothesized that the mutations in both the TT8 genes are required for inhibiting the transcription of LBGs in the yellow-seeded mutant of 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.

Search for genic SSRs from transcriptome sequence data of Brassica juncea and B. nigra and from unigene databank of B. rapa resulted in identification of 20,529 SSRs from 330,827 gene sequences. Identified SSRs (named as unigene microsatellite; UGM) were characterized based on their number of repeats, type of motif and base composition. Primers were designed for 2118 UGMs after in silico polymorphism survey and were tested across 11 different genotypes of B. juncea and B. rapa for PCR amplification and polymorphism. It resulted in clear-cut amplification from 1993 UGMs of which 1319 UGMs showed polymorphism. It was observed that a priori in silico polymorphism survey helped in increasing the polymorphism percentage (6.1–13.2) by PCR amplification. We also report here the development of first SSR-based linkage map of Brassica juncea using a DH mapping population derived from F1 of the cross between the lines EH-2 and Pusajaikisan. The map consisted of 860 markers comprising 462 UGMs, 157 BAC-derived SSRs and 241 intron polymorphic markers and covered a total genetic length of 2073.6 cM. The study also reports an interesting observation of appearance of novel non-parental bands in the mapping population from 18 SSRs and 13 IP markers. Search of Arabidopsis genes orthologous to their primer sequences showed that 11 of such Arabidopsis genes code for proteins involved in various abiotic stresses. The highly transferable genic SSRs developed in the study would serve as valuable resources for comparative genome mapping, mapping of qualitative and quantitative traits, gene cloning and marker-assisted breeding in Brassica species.

Summary Polyploidy, the possession of multiple sets of chromosomes, has been a predominant factor in the evolution and success of the angiosperms. Although artificially formed allopolyploids show a high rate of genome rearrangement, the genomes of cultivars and germplasm used for crop breeding were assumed stable and genome structural variation under the artificial selection process of commercial breeding has remained little studied. Here, we show, using a repurposed visualization method based on transcriptome sequence data, that genome structural rearrangement occurs frequently in varieties of three polyploid crops (oilseed rape, mustard rape and bread wheat), meaning that the extent of genome structural variation present in commercial crops is much higher than expected. Exchanges were found to occur most frequently where homoeologous chromosome segments are collinear to telomeres and in material produced as doubled haploids. The new insights into genome structural evolution enable us to reinterpret the results of recent studies and implicate homoeologous exchanges, not deletions, as being responsible for variation controlling important seed quality traits in rapeseed. Having begun to identify the extent of genome structural variation in polyploid crops, we can envisage new strategies for the global challenge of broadening crop genetic diversity and accelerating adaptation, such as the molecular identification and selection of genome deletions or duplications encompassing genes with trait‐controlling dosage effects.

White rust caused by Albugo candida (Pers.) Kuntze is a major disease of the oilseed mustard Brassica juncea. Almost all the released varieties of B. juncea in India are highly susceptible to the disease. This causes major yield losses. Hence, there is an urgent need to identify genes for resistance to white rust and transfer these to the existing commercial varieties through marker-assisted breeding. While the germplasm belonging to the Indian gene pool is highly susceptible to the disease, the east European germplasm of B. juncea is highly resistant. In the present study, we have tagged two independent loci governing resistance to A. candida race 2V in two east European lines, Heera and Donskaja-IV. Two doubled haploid populations were used; the first population was derived from a cross between Varuna (susceptible Indian type) and Heera (partially resistant east European line) and the second from a cross between TM-4 (susceptible Indian type) and Donskaja-IV (fully resistant east European line). In both the resistant lines, a single major locus was identified to confer resistance to white rust. In Heera, the resistance locus AcB1-A4.1 was mapped to linkage group A4, while in Donskaja-IV, the resistant locus AcB1-A5.1 was mapped to linkage group A5. In both the cases, closely linked flanking markers were developed based on synteny between Arabidopsis and B. juncea. These flanking markers will assist introgression of resistance-conferring loci in the susceptible varieties.

Fine mapping of six seed glucosinolate QTL (J2Gsl1, J3Gsl2, J9Gsl3, J16Gsl4, J17Gsl5 and J3Gsl6) (Ramchiary et al. in Theor Appl Genet 116:77-85, 2007a) was undertaken by the candidate gene approach. Based on the DNA sequences from Arabidopsis and Brassica oleracea for the different genes involved in the aliphatic glucosinolate biosynthesis, candidate genes were amplified and sequenced from high to low glucosinolate Brassica juncea lines Varuna and Heera, respectively. Of the 20 paralogues identified, 17 paralogues belonging to six gene families were mapped to 12 of the 18 linkage groups of B. juncea genome. Co-mapping of candidate genes with glucosinolate QTL revealed that the candidate gene BjuA.GSL-ELONG.a mapped to the QTL interval of J2Gsl1, BjuA.GSL-ELONG.c, BjuA.GSL-ELONG.d and BjuA.Myb28.a mapped to the QTL interval of J3Gsl2, BjuA.GSL-ALK.a mapped to the QTL interval of J3Gsl6 and BjuB.Myb28.a mapped to the QTL interval of J17Gsl5. The QTL J9Gsl3 and J16Gsl4 did not correspond to any of the mapped candidate genes. The functionality and contribution of different candidate genes/QTL was assessed by allelic variation study using phenotypic data of 785 BC(4)DH lines. It was observed that BjuA.Myb28.a and J9Gsl3 contributed significantly to the base level glucosinolate production while J16Gsl4, probably GSL-PRO, BjuA.GSL-ELONG.a and BjuA.GSL-ELONG.c contributed to the C3, C4 and C5 elongation pathways, respectively. Three A genome QTL: J2Gsl1harbouring BjuA.GSL-ELONG.a, J3Gsl2 harbouring both BjuA.GSL-ELONG.c and BjuA.Myb28.a and J9Gsl3, possibly the 'Bronowski genes', were identified as most important loci for breeding low glucosinolate B. juncea. We observed two-step genetic control of seed glucosinolate in B. juncea mainly effected by these three A genome QTL. This study, therefore, provides clues to the genetic mechanism of 'Bronowski genes' controlling the glucosinolate trait and also provides efficient markers for marker-assisted introgression of low glucosinolate trait in B. juncea.

Seed glucosinolate content in Brassica juncea is a complex quantitative trait. A recurrent selection backcross (RSB) method with a doubled haploid (DH) generation interspersing backcross generations was used for the introgression of low glucosinolate alleles from an east European gene pool B. juncea line, Heera into an Indian gene pool variety, Varuna. Phenotypic comparisons among the DH populations derived from early to advanced backcrosses revealed a shift in the mean values for various glucosinolates with the advancement of backcrossing, indicating a change in the selective values of the alleles with change in the genetic background due to the existence of epistasis and context dependencies. QTL mapping for various seed glucosinolates from early (F(1)DH) and advanced generation (BC(4)DH) populations confirmed the presence of epistasis and context dependency. The common QTL detected in both F(1)DH and BC(4)DH changed their R (2) values from the former to the later generation. Some of the QTL detected in the F(1)DH became irrelevant in the BC(4)DH population. Further, new QTL were detected in the BC(4)DH population for various glucosinolates. A validation study on a population of low glucosinolate DH lines derived from all the backcross generations of the RSB breeding programme revealed that the QTL detected in BC(4)DH were the 'true' QTL. Using glucosinolate as an example, the study provides strong evidence for the importance of the RSB method for the identification of the 'true' QTL which would be significant for marker assisted introgression of a complex quantitative trait whose expression is influenced by epistatic interactions.

Tea is an important health drink which contributes significantly to the economy of Asian and African countries. India contributes about 29 % of the world’s total tea production where majority of the tea cultivated is Assam type (native to Assam, India). Apart from India, Assam type contributes to the majority of tea grown in tropical regions of Sri Lanka and Kenya as well. We have developed the first genetic linkage map in Indian beveragial tea using 234 DNA markers (AFLP and RAPD). The plausibility of the linkage group formation was compared using two-way pseudo-testcross approach (using maximum likelihood algorithm) and integrated approaches (using regression algorithm). Both the approaches generated linkage maps containing 18 linkage groups. The composition and order of markers in the corresponding linkage groups in both the maps showed significant congruence. Maximum likelihood algorithm could map 78.6 % of the markers with total map length of 8527.5 and 45.3 cM average distance between each marker. Regression algorithm could map 73.5 % of the total markers with total map length of 2051.7 and 14.96 cM average distance between each marker. Drought is the major abiotic stress responsible for heavy yield losses in cultivated tea all over the world. The present map was used to map the locus, AFLP_CS_87 that segregates with drought tolerance phenotype in both the maps.

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607-614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5’ and 3’ RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2 20%, C18:2 between 12% and 20%) and C18:2

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high x low erucic acid F(1) hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 ( FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes ( FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild ( E1E2) and mutant ( e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (2%) and E1E1E2E2 identified individuals with highest erucic acid content (40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica species.

A high-density genetic linkage map of Brassica juncea (2n = 36) was constructed with 996 AFLP (amplified fragment length polymorphism) and 33 RFLP (restriction fragment length polymorphism) markers using a F1-derived doubled-haploid (DH) population of 123 individuals. This mapping population was developed by crossing a well-adapted, extensively grown Indian variety Varuna and a canola quality line Heera. The two lines are highly divergent and contain a number of contrasting qualitative and quantitative traits of high agronomic value. AFLPs were generated by the use of restriction enzymes EcoRI or PstI in combination with either MseI or TaqI. Using 91 primer pairs, a total of 1,576 parental polymorphic bands were detected of which 996 were used for mapping. In addition, 33 RFLP markers, developed from genomic clones of B. napus, were added to the map. The segregation of each marker and linkage analysis was performed using the program JoinMap version 2.0. The 1,029 mapped-markers were aligned in 18 linkage groups, which is the haploid chromosome number of the species, at LOD values ranging from 5 to 8. The total map length was 1,629 cM with an average marker interval of 3.5 cM. AFLP markers generated by EcoRI were more clustered, whereas PstI markers showed more extensive distribution. A set of 26 primer pairs (9 EcoRI/ MseI, 6 EcoRI/ TaqI, 6 PstI/ MseI and 5 PstI/ TaqI) generating 385 markers were identified for AFLP-based whole-genome selection as these markers covered 96% of the genome mapped with the 91 primer pairs. The map developed in the present study could be used for dissection and the transfer of agronomically important traits and favourable QTLs from ill-adapted exotic germplasm to cultivated Indian varieties.

Analysis of the glucosinolate content and composition by high-pressure liquid chromatography indicated that varieties of Brassica juncea bred and grown in India have a high glucosinolate content characterized by the presence of 2-propenyl (allyl) and 3-butenyl as the major and 4-pentenyl as the minor fractions. In contrast, the B. juncea germplasm from other countries is characterized by the presence of 2-propenyl as the major glucosinolate fraction, trace amounts of 3-butenyl and a total lack of the 4-pentenyl types. In order to transfer the low glucosinolate trait to Indian B. juncea, the inheritance of total glucosinolates was investigated using doubled haploid (DH) populations derived from F1 (DH1) and BC1 (BC1DH) of a cross between ‘Varuna’ (the most widely cultivated high glucosinolate variety of India) and ‘Heera’ (a non-allyl type low glucosinolate line). A total of 752 DH1 and 1263 BC1DH gave rise to seven and 11 low glucosinolate (containing less than 18 μmol/g seed) individuals, respectively. On the basis of the frequency of the low glucosinolate individuals, the total glucosinolate was found to be under the control of seven genes. There was presence of both allyl and non-allyl types in DH1 and BC1DH low-glucosinolate individuals and absence of 3-butenyl glucosinolate in some of the BC1DH low glucosinolate individuals, indicating segregation for these fractions in the population. The size of the segregating DH population proved to be crucial for precise determination of the number of genes controlling the trait. Because of the large number of genes involved, incorporation of low glucosinolate trait in Indian B. juncea should be approached through doubled haploid (DH) breeding.

QTL mapping by two DH mapping populations deciphered allelic variations for five different seed glucosinolate traits in B. juncea. Allelic variations for five different seed glucosinolate (GS) traits, namely % propyl, % butyl, % pentyl, aliphatics and total GS content were studied through QTL analysis using two doubled haploid (DH) mapping populations. While the high GS parents in two populations differed in their profiles of seed aliphatic GS, the low GS parents were similar. Phenotypic data of seed GS traits from three environments of the two populations were subjected to QTL analysis. The first population (referred to as DE population) detected a total of 60 QTL from three environments which upon intra-population meta-QTL analysis were merged to 17 S-QTL (Stable QTL) and 15 E-QTL (Environment QTL). The second population (referred to as VH population) detected 58 QTL from the three environments that were merged to 15S-QTL and 16E-QTL. In both the populations, majority of S-QTL were detected as major QTL. Inter-population meta-analysis identified three C-QTL (consensus QTL) formed by merging major QTL from the two populations. Candidate genes of GS pathway were co-localized to the QTL regions either through genetic mapping or through in silico comparative analysis. Parental allelic variants of QTL or of the co-mapped candidate gene(s) were determined on the basis of the significantly different R 2 values of the component QTL from the two populations which were merged to form C-QTL. The results of the study are significant for marker-assisted transfer of the low GS trait and also for developing lines with lower GS than are present in Brassica juncea.

The multiple BjuCYP83A1 genes formed as a result of polyploidy have retained cell-, tissue-, and condition-specific transcriptional sub-functionalization to control the complex aliphatic glucosinolates biosynthesis in the allotetraploid Brassica juncea. Glucosinolates along with their breakdown products are associated with diverse roles in plant metabolism, plant defense and animal nutrition. CYP83A1 is a key enzyme that oxidizes aliphatic aldoximes to aci-nitro compounds in the complex aliphatic glucosinolate biosynthetic pathway. In this study, we reported the isolation of four CYP83A1 genes named BjuCYP83A1-1, -2, -3, and -4 from allotetraploid Brassica juncea (AABB genome), an economically important oilseed crop of Brassica genus. The deduced BjuCYP83A1 proteins shared 85.7-88.4 % of sequence identity with A. thaliana AtCYP83A1 and 84.2-95.8 % among themselves. Phylogenetic and divergence analysis revealed that the four BjuCYP83A1 proteins are evolutionary conserved and have evolved via duplication and hybridization of two relatively simpler diploid Brassica genomes namely B. rapa (AA genome) and B. nigra (BB genome), and have retained high level of sequence conservation following allopolyploidization. Ectopic over-expression of BjuCYP83A1-1 in A. thaliana showed that it is involved mainly in the synthesis of C4 aliphatic glucosinolates. Detailed expression analysis using real-time qRT-PCR in B. juncea and PromoterBjuCYP83A1-GUS lines in A. thaliana confirmed that the four BjuCYP83A1 genes have retained ubiquitous, overlapping but distinct expression profiles in different tissue and cell types of B. juncea, and in response to various elicitor treatments and environmental conditions. Taken together, this study demonstrated that transcriptional sub-functionalization and coordinated roles of multiple BjuCYP83A1 genes control the biosynthesis of aliphatic glucosinolates in the allotetraploid B. juncea, and provide a framework for metabolic engineering of aliphatic glucosinolates in economically important Brassica species.

Background 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. Results RNA isolated from two lines of B. juncea – Varuna (Indian type) and Heera (east European type) – was sequenced using Illumina paired end sequencing technology, and assembled using the Velvet de novo programme. A and B genome specific contigs were identified in two steps. First, by aligning contigs against the B. rapa protein database (available at BRAD), and second by comparing percentage identity at the nucleotide level with B. rapa CDS and B. nigra transcriptome. 135,693 SNPs were recorded in the assembled partial gene models of Varuna and Heera, 85,473 in the A genome and 50,236 in the B. Using KASpar technology, 999 markers were added to an earlier intron polymorphism marker based map of a B. juncea Varuna x Heera DH population. Many new gene blocks were identified in the B genome. A number of SNP markers covered single copy homoeologues of the A and B genomes, and these were used to identify homoeologous blocks between the two genomes. Comparison of the block architecture of A and B genomes revealed extensive differences in gene block associations and block fragmentation patterns. Conclusions Sufficient SNP markers are available for general and specific -region fine mapping of crosses between lines of two diverse B. juncea gene pools. Comparative gene block arrangement and block fragmentation patterns between A and B genomes support the hypothesis that the two genomes evolved from independent hexaploidy events. Electronic supplementary material The online version of this article (doi: 10.1186/1471-2164-15-396) contains supplementary material, which is available to authorized users.

AFLP markers were employed to assess the genetic diversity amongst 21 established natural and nine synthetic varietes and lines of Brassica juncea originating from Asia, Australia, Canada, Eastern Europe and Russia. Six of the synthetics used for diversity studies have been developed recently. Twenty one EcoRI/MseI-based AFLP primer pairs generated a total of 1251 scorable fragments among the 30 genotypes studied, of which 778 bands were polymorphic with an average of 37 polymorphic bands per primer pair. On the basis of the similarity coefficients (F value), cluster analysis was performed using the UPGMA method. The 30 B. juncea lines could be grouped into three distinct clusters. All the Indian, Chinese and previously developed synthetics formed one cluster (cluster A), the recently developed synthetics formed a separate cluster (cluster B) and the lines from Australia, Canada, Eastern Europe and Russia formed the third cluster (cluster C). A majority of the lines were uniquely identified by one or more primer pairs due to the presence or absence of variety specific band(s). Four primer pairs were found to be most informative, since these uniquely identified all the genotypes assayed. These four primer pairs, could therefore be used as fingerprinting primers for varietal identification.

Alloplasmic lines of cultivated Brassica species with B. oxyrrhina cytoplasm are male-sterile and suffer from severe chlorosis. We developed male-sterile lines corrected for chlorosis by fusing protoplasts of CMS B. juncea (AABB) with ’oxy’ cytoplasm and normal B. oleracea (CC). A large number of male-sterile AABBCC somatic hybrids with desirable organelle combinations, i.e. chloroplasts of B. oleracea and mitochondria with recombinant genomes, were recovered. While no recombination was observed in the chloroplast genome, the mitochondrial genome showed extensive recombination that resulted in the appearance of totally novel banding patterns in some of the hybrids. Hybrids with a parental-type mitochondrial genome as well as recombinant patterns close to either of the parental types were also obtained. Using AABBCC somatic hybrids as bridging material, we transferred the desirable organelle combinations to B. juncea (AABB), B. napus (AACC), and B. carinata (BBCC). Many of these lines are now at advanced stages of backcrossing and show stable inheritance of the CMS character and do not suffer from chlorosis.

Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum × B. napus, B. maurorum × B. carinata, B. maurorum × B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques.

Alloplasmic Brassica juncea was synthesized by repeatedly backcrossing amphidiploid of Erucastrum gallicum × B. juncea with pollen of B. juncea. The E. gallicum cytoplasm was also transferred to B. napus and B. carinata through alloplasmic (E. gallicum) B. juncea. RFLP analyses of organelle DNA confirmed the presence of E. gallicum cytoplasm in all the alloplasmics. The cytoplasm of E. gallicum, though alien, did not induce stable cytoplasmic male sterility in any of the oilseed brassicas. Many useful variants such as early flowering and dwarf phenotypes were isolated in the alloplasmics of B. napus and B. carinata. These morphotypes provide useful genetic variability for the improvement of crop brassicas.

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.

The objective of this study was to broaden the genetic base in oleiferous Brassica juncea by resynthesis, using 10 diverse parental lines of oleiferous B. rapa and two lines of B. nigra of both Indian and exotic origin. Out of 14 crosses attempted using B. rapa as the female parent, eight were successful. Embryo rescue was necessary to obtain interspecific plants. A total of 29 fertile interspecific plants were obtained after colchicine treatment. In the S 2 generation, the expression of component characters in the majority of the resynthesized plants showed a negative trend. The resynthesized B. juncea lines are being maintained through repeated selfing and selection at each generation for desirable plant types. This process will continue till the progeny lines of the desirable plants achieve uniformity.

Eighteen genotypes of Brassica napus were crossed to a cytoplasmic male sterile (CMS) line of B. napus BO 15 carrying B. tournefortii cytoplasm ('tour' cytoplasm). Fourteen genotypes were found to be stable maintainers of the 'tour' CMS. Of the remaining four genotypes, GSL-1 and 'Asahi-natane' were found to be heterozygous and 'Mangun' and 'Yudal' were homozygous for the restorer gene. Analysis of the F 1 and F 2 progenies of (CMS) BO 15 ×'Mangun' and (CMS) BO 15 ×'Yudal' showed that fertility restoration is controlled by a single dominant gene. The availability of a number of stable maintainer lines and the simple inheritance pattern of fertility restorer gene makes 'tour' CMS a useful system for hybrid seed production in rapeseed.

Brassica carinata (BBCC), a potential oilseed crop for dry land agriculture, is sensitive to high temperatures during germination and early stages of growth, which thereby restricts the possibility of using the residual soil moisture available after the rainy season for its cultivation. To overcome this problem, a three-genome hybrid, RCBB, was synthesized using Raphanus sativus (RR) and Brassica oleracea (CC) as donor sources for the desired heat tolerance. Protoplasts of RC hybrids obtained through sexual crosses between R. sativus (female) and B. oleracea (male) were fused with protoplasts of Brassica nigra (BB) to produce RCBB somatic hybrids. The hybrid colonies regenerated with an average frequency of 7.6%. Twelve out of 36 hybrids grown to maturity were characterized for their nuclear and organelle genomes. While all the hybrids showed the presence of B. nigra chloroplasts, 10 of the hybrids showed B. nigra-specific mitochondria and two had Raphanus-specific mitochondria. The somatic hybrids could be backcrossed to B. carinata.

Divergence of 25 accessions of Brassica juncea of Indian, CIS (Commonwealth of Independent States, former USSR) and synthetic origin was studied by D2 analysis. On the basis of divergence, ten accessions were selected and crossed in a diallel fashion without reciprocals to study the combining ability and heterosis. None of the accessions was found to be a good general combiner for all the nine quantitative characters that were studied. Significant heterosis over better parent for single plant yield was recorded in CIS x Indian and synthetic x CIS crosses (5 each) followed by Indian x synthetic types (3). The analysis of component characters showed that the mean performance of the majority of hybrids was intermediate for five out of six yield attributing traits, thus exhibiting dominance or partial dominance effect. To estimate the contribution of such yield attributing traits towards heterosis for yield, a comparison was made among three parameters viz. heterosis over mid parent (MP), better parent (BP) and better yielding parent (BYP) of the concerned hybrid. It was observed that estimation of heterosis from BYP was a more accurate method to determine the contribution of component characters towards yield heterosis than the analysis based on MP and BP. From the component character analysis, it was concluded that characters like number of primary and secondary branches, number of siliqua per plant and siliqua density contributed significantly towards heterosis in yield. Plot level yield trials of two selected hybrids (Skorospieka II x RH30 and Donskaja IV x Varuna) over two growing seasons revealed 29.4 to 91.8% heterosis over BYP.

Brassica juncea (AABB) is a natural allopolyploid of Brassica rapa (AA) and Brassica nigra (BB). B. juncea group contains both oilseed types and vegetable types. Extensive phenotypic and molecular marker-based studies on oilseed types of B. juncea have identified two major divergent gene pools, the Indian gene pool and the east European gene pool. Hybrids between the lines of these two gene pools are heterotic for yield, and such hybrids have now been commercialized in India. The two gene pools are significantly contrasting in major agronomic traits like seed size, pod size, pod number, height and oil content. A major future challenge in heterosis breeding is to transfer superior traits from one gene pool to the other while retaining the overall genetic divergence. This would allow further increases in yield. Recent work has provided reasonable number of markers for molecular tagging of both qualitative and quantitative traits and comparative mapping with model crucifer Arabidopsis and other Brassica species. Current work involves development of “00” canola-quality lines and their agronomic trials, disease resistance and QTL introgression. As the crop holds great promise for dryland agriculture in India and many other such areas around the globe, a concerted international effort is required on germplasm exchange, genetics, genomics and breeding of B. juncea.

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2),fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.

'Polima' cytoplasmic male sterility (CMS) was transferred from 'Polima' Brassica napus ISN 706' to five different cultivars of Brassica campestris ('Pusa kalyani', Pant toria', 'Candle', Tobin' and 'ATC 94211') by repeated backcrossing. It was observed that, while 'Polima' CMS manifested complete and stable male sterility in the nuclear backgrounds of Pusa kalyani', Pant toria', and Tobin', the cultivars Candle' and 'ATC 94211' possessed the restorer gene for this CMS in the heterozygous condition. An analysis of F 1 and F 2 generations of Polima' Pusa kalyani' × Candle' and 'Polima' 'Pusa kalyani' x 'ATC 94211' revealed that restoration is controlled by a single dominant gene. Identification of stable maintainers and restorers of Polima' CMS could facilitate the development of hybrid varieties in B. campestris.

Chloroplast (cp) and mitochondrial (mt) DNA restriction profiles of a cytoplasmic male sterile (CMS) line of Brassica juncea and its maintainer line were compared and found to be markedly different. Comparison of cpDNA restriction profiles of fifty different species of genus Brassica and some allied genera showed that the cpDNA profiles of CMS lines were similar to that of B. tournefortii for twenty different restriction endonucleases. This CMS system is thus not of spontaneous origin as reported earlier, but is alloplasmic in nature. Comparison of restriction profiles of mtDNA of B. tournefortii and CMS lines revealed some differences which might either be due to changes in DNA pattern during the transfer, or, due to the cytoplasm coming from a B. tournefortii line different from the one used in this study.

Background Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP) markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome) and A. thaliana and analyzed the arrangement of 24 (previously described) genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes. Results IP markers were highly efficient and generated easily discernable polymorphisms on agarose gels. Comparative analysis of the segmental organization of the A and B genomes of B. juncea (present study) with the A and B genomes of B. napus and B. nigra respectively (described earlier), revealed a high degree of colinearity suggesting minimal macro-level changes after polyploidization. The ancestral block arrangements that remained unaltered during evolution and the karyotype rearrangements that originated in the Oleracea lineage after its divergence from Rapa lineage were identified. Genomic rearrangements leading to the gain or loss of one chromosome each between the A-B and A-C lineages were deciphered. Complete homoeology in terms of block organization was found between three linkage groups (LG) each for the A-B and A-C genomes. Based on the homoeology shared between the A, B and C genomes, a new nomenclature for the B genome LGs was assigned to establish uniformity in the international Brassica LG nomenclature code. Conclusion IP markers were highly effective in generating comparative relationships between Arabidopsis and various Brassica species. Comparative genomics between the three Brassica lineages established the major rearrangements, translocations and fusions pivotal to karyotype diversification between the A, B and C genomes of Brassica species. The inter-relationships established between the Brassica lineages vis-à-vis Arabidopsis would facilitate the identification and isolation of candidate genes contributing to traits of agronomic value in crop Brassicas and the development of unified tools for Brassica genomics.