A chromosome-scale assembly of Brassica carinata (BBCC) accession HC20 containing resistance to multiple pathogens and an early generation assessment of introgressions into B. juncea (AABB).

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

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed, high-leaf glucosinolate oilseed mustard with uncompromised defence and yield.

Glucosinolate content in the two major oilseed Brassica crops-rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 µmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 µmoles/g DW to as low as 6.21 µmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.

Characterization of Cry2 genes (CRY2a and CRY2b) of B. napus and comparative analysis of BnCRY1 and BnCRY2a in regulating seedling photomorphogenesis.

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 (BnCRY2aOE) transgenics developed short hypocotyl and expanded cotyledons, accumulated more anthocyanin in light-grown seedlings, and displayed early flowering on maturity. Early flowering in BnCRY2aOE transgenics was coupled with the up-regulation of many flowering-related genes such as FT. The present study also highlights the differential light sensitivity of cry1 and cry2 in controlling hypocotyl elongation growth in Brassica. BnCRY2aOE seedlings developed much shorter hypocotyl under the low-intensity of blue light, while BnCRY1OE seedling hypocotyls were shorter under the high-intensity blue light, compared to untransformed seedlings.

Comparative Analysis of Seed Transcriptome and Coexpression Analysis Reveal Candidate Genes for Enhancing Seed Size/Weight in Brassica 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 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.

Genetic Analysis of Heterosis for Yield Influencing Traits in Brassica juncea Using a Doubled Haploid Population and Its Backcross Progenies.

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.

A comprehensive Vis-NIRS equation for rapid quantification of seed glucosinolate content and composition across diverse Brassica oilseed chemotypes.

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.

BjuWRR1, a CC-NB-LRR gene identified in Brassica juncea, confers resistance to white rust caused by Albugo candida.

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.

QTL Landscape for Oil Content in Brassica juncea: Analysis in Multiple Bi-Parental Populations in High and "0" Erucic Background.

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.

Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes.

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

Genetic Architecture of Resistance to Alternaria brassicae in Arabidopsis thaliana: QTL Mapping Reveals Two Major Resistance-Conferring Loci.

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.

Genetic dissection of seed weight by QTL analysis and detection of allelic variation in Indian and east European gene pool lines of Brassica juncea.

KEY MESSAGE: 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.

Extensive homoeologous genome exchanges in allopolyploid crops revealed by mRNAseq-based visualization.

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.

BjuB.CYP79F1 Regulates Synthesis of Propyl Fraction of Aliphatic Glucosinolates in Oilseed Mustard Brassica juncea: Functional Validation through Genetic and Transgenic Approaches.

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

Deciphering allelic variations for seed glucosinolate traits in oilseed mustard (Brassica juncea) using two bi-parental mapping populations.

KEY MESSAGE: 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.

Genomic origin, expression differentiation and regulation of multiple genes encoding CYP83A1, a key enzyme for core glucosinolate biosynthesis, from the allotetraploid Brassica juncea.

MAIN CONCLUSION: 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.

Tetralocular ovary and high silique width in yellow sarson lines of Brassica rapa (subspecies trilocularis) are due to a mutation in Bra034340 gene, a homologue of CLAVATA3 in Arabidopsis.

KEY MESSAGE: 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.

RNA-seq based SNPs for mapping in Brassica juncea (AABB): synteny analysis between the two constituent genomes A (from B. rapa) and B (from B. nigra) shows highly divergent gene block arrangement and unique block fragmentation patterns.

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.

Two plastid DNA lineages--Rapa/Oleracea and Nigra--within the tribe Brassiceae can be best explained by reciprocal crosses at hexaploidy: evidence from divergence times of the plastid genomes and R-block genes of the A and B genomes of Brassica juncea.

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.

Natural mutations in two homoeologous TT8 genes control yellow seed coat trait in allotetraploid Brassica juncea (AABB).

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.