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.

Genetic mapping of some key plant architecture traits in Brassica juncea using a doubled haploid population derived from a cross between two distinct lines: vegetable type Tumida and oleiferous Varuna.

KEY MESSAGE: Genetic mapping of some key plant architectural traits in a vegetable type and an oleiferous B. juncea cross revealed QTL and candidate genes for breeding more productive ideotypes. Brassica juncea (AABB, 2n = 36), commonly called mustard, is an allopolyploid crop of recent origin but contains considerable morphological and genetic variation. An F1-derived doubled haploid population developed from a cross between an Indian oleiferous line, Varuna, and a Chinese stem type vegetable mustard, Tumida showed significant variability for some key plant architectural traits-four stem strength-related traits, stem diameter (Dia), plant height (Plht), branch initiation height (Bih), number of primary branches (Pbr), and days to flowering (Df). Multi-environment QTL analysis identified twenty Stable QTL for the above-mentioned nine plant architectural traits. Though Tumida is ill-adapted to the Indian growing conditions, it was found to contribute favorable alleles in Stable QTL for five architectural traits-press force, Dia, Plht, Bih, and Pbr; these QTL could be used to breed superior ideotypes in the oleiferous mustard lines. A QTL cluster on LG A10 contained Stable QTL for seven architectural traits that included major QTL (phenotypic variance ≥ 10%) for Df and Pbr, with Tumida contributing the trait-enhancing alleles for both. 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, however, identified other QTL which could be used for the improvement of Pbr without affecting Df. The Stable QTL intervals were mapped on the genome assemblies of Tumida and Varuna for the identification of candidate genes.

A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes.

Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species-B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single-molecule real-time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short-read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping-by-sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome-scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.

A highly contiguous genome assembly of Brassica nigra (BB) and revised nomenclature for the pseudochromosomes.

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

Correction to: Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae - a pathogen of the Brassicaceae family.

Following the publication of this article [1], the authors reported that the captions of Figs. 2 and 3 were published in the incorrect order, whereby they mismatch with their corresponding images. The figures are reproduced in the correct sequence with the correct captions in this Correction article. The original article has been corrected.

Near-Complete Genome Assembly of Alternaria brassicae-A Necrotrophic Pathogen of Brassica Crops.

Alternaria brassicae, a necrotrophic fungal pathogen, causes Alternaria blight, an important disease of brassica crops. Although many Alternaria spp. have been sequenced, no genome information is available for A. brassicae, a monotypic lineage within the Alternaria genus. A highly contiguous genome assembly of A. brassicae has been generated using Nanopore MinION sequencing with an N50 of 2.98 Mb, yielding nine full chromosome-level sequences. This study adds to the current genomic resources available for the genus Alternaria and will provide opportunities for further analyses to unravel the mechanisms underlying pathogenicity of this important necrotrophic pathogen.

Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae - a pathogen of the Brassicaceae family.

BACKGROUND: Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. RESULTS: We also sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea using Nanopore MinION sequencing for additional comparative analyses within the Alternaria genus. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae, a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae-specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae. CONCLUSION: The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae.

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.

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.

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.

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.

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.

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 in some agronomically important oleiferous lines of Brassica rapa and their use for genome-wide linkage mapping and specific-region fine mapping.

BACKGROUND: Brassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra. RESULTS: RNA-seq was carried out on the RNA isolated from young inflorescences containing unopened floral buds, floral axis and small leaves, using Illumina paired-end sequencing technology. Sequence assembly was carried out using the Velvet de-novo programme and the assembled contigs were organised against Chiifu gene models, available in the BRAD-CDS database. RNA-seq confirmed the presence of more than 17,000 single-copy gene models described in the BRAD database. The assembled contigs and the BRAD gene models were analyzed for the presence of SSRs and SNPs. While the number of SSRs was limited, more than 0.2 million SNPs were observed between Chiifu and the three oleiferous lines. Assays for SNPs were designed using KASPar technology and tested on a F7-RIL population derived from a Chiifu x Tetra cross. The design of the SNP assays were based on three considerations - the 50 bp flanking region of the SNPs should be strictly similar, the SNP should have a read-depth of ≥7 and no exon/intron junction should be present within the 101 bp target region. Using these criteria, a total of 640 markers (580 for genome-wide mapping and 60 for specific-region mapping) marking as many genes were tested for mapping. Out of 640 markers that were tested, 594 markers could be mapped unambiguously which included 542 markers for genome-wide mapping and 42 markers for fine mapping of the tet-o locus that is involved with the trait tetralocular ovary in the line Tetra. CONCLUSION: A large number of SNPs and PSVs are present in the transcriptome of B. rapa lines for genome-wide linkage mapping and specific-region fine mapping. Criteria used for SNP identification delivered markers, more than 93% of which could be successfully mapped to the F7-RIL population of Chiifu x Tetra cross.

QTL mapping of yield-associated traits in Brassica juncea: meta-analysis and epistatic interactions using two different crosses between east European and Indian gene pool lines.

Genetic analysis of 12 yield-associated traits was undertaken by dissection of quantitative trait loci (QTL) through meta-analysis and epistatic interaction studies in Brassica juncea. A consensus (integrated) map in B. juncea was constructed using two maps. These were VH map, developed earlier in the laboratory by using a DH population from the cross between Varuna and Heera (Pradhan et al. in Theor Appl Genet 106:607-614, 2003; Ramchiary et al. in Theor Appl Genet. 115:807-817, 2007; Panjabi et al. in BMC Genomics 9:113, 2008), and the TD map, developed in the present study using a DH population of 100 lines from the cross between TM-4 and Donskaja-IV. The TD map was constructed with 911 markers consisting of 585 AFLP, 8 SSR and 318 IP markers covering a total genome length of 1,629.9 cM. The consensus map constructed by using the common markers between the two maps contained a total of 2,662 markers and covered a total genome length of 1,927.1 cM. Firstly, QTL analysis of 12 yield-associated traits was undertaken for the TD population based on three-environment phenotypic data. Secondly, the three-environment phenotypic data for the same 12 quantitative traits generated by Ramchiary et al. (2007) were re-analyzed for the QTL detection in the VH map. Comparative analysis identified both common and population-specific QTL. The study revealed the presence of QTL clusters on LG A7, A8 and A10 in both TD and VH maps. Meta-analyses resolved 187 QTL distributed over nine linkage groups of TD and VH maps into 20 meta-QTL. Maximum resolution was recorded for the LG A10 wherein all the 54 QTL were mapped to a single meta-QTL within a confidence interval of 3.0 cM. Digenic epistatic interactions of QTL in both TD and VH maps revealed substantial additive × additive interactions showing a higher frequency of Type 1 and Type 2 interactions than Type 3 interactions. Some of the loci interacted with more than one locus indicating the presence of higher order epistatic interactions. These findings provided some detailed insight into the genetic architecture of the yield-associated traits in B. juncea.

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.

Eliminating expression of erucic acid-encoding loci allows the identification of "hidden" QTL contributing to oil quality fractions and oil content in Brassica juncea (Indian mustard).

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.

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.

Molecular mapping reveals two independent loci conferring resistance to Albugo candida in the east European germplasm of oilseed mustard Brassica juncea.

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.