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Heavy doses of gamma irradiation can reduce linkage drag by disrupting large sized alien translocations and promoting exchanges between crop and wild genomes. Resistance to mustard aphid (Lipaphis erysimi) infestation was significantly improved in Brassica juncea through B. juncea-B. fruticulosa introgression. However, linkage drag caused by introgressed chromatin fragments has so far prevented the deployment of this resistance source in commercial cultivars. We investigated the patterns of donor chromatin segment substitutions in the introgression lines (ILs) through genomic in situ hybridization (GISH) coupled with B. juncea chromosome-specific oligonucleotide probes. These allowed identification of large chromosome translocations from B. fruticulosa in the terminal regions of chromosomes A05, B02, B03 and B04 in three founder ILs (AD-64, 101 and 104). Only AD-101 carried an additional translocation at the sub-terminal to intercalary position in both homologues of chromosome A01. We validated these translocations with a reciprocal blast hit analysis using shotgun sequencing of three ILs and species-specific contigs/scaffolds (kb sized) from a de novo assembly of B. fruticulosa. Alien segment substitution on chromosome A05 could not be validated. Current studies also endeavoured to break linkage drag by exposing seeds to a heavy dose (200kR) of gamma radiation. Reduction in the size of introgressed chromatin fragments was observed in many M3 plants. There was a complete loss of the alien chromosome fragment in one instance. A few M3 plants with novel patterns of chromosome segment substitutions displayed improved agronomic performance coupled with resistance to mustard aphid. SNPs in such genomic spaces should aid the development of markers to track introgressed DNA and allow application in plant breeding.

Genome wide association studies enabled prediction of many candidate genes for flowering, maturity and plant height under differing day-length conditions. Some genes were envisaged only from derived B. rapa. Flowering and plant height are the key life history traits. These are crucial for adaptation and productivity. Current investigations aimed to examine genotypic differences governing days to flowering, maturity and plant height under contrasting day-length conditions; and identify genomic regions governing the observed phenotypic variations. An association panel comprising 195 inbred lines, representing natural (NR) and derived (DR) forms of Brassica rapa (AA; 2n = 20), was evaluated at two sowing dates and two locations, representing different day-length regimes. Derived B. rapa is a unique pre-breeding material extracted from B. juncea (AABB; 2n = 36). Population structure analysis, using DArT genotypes established derived B. rapa as a genetic resource distinct from natural B. rapa. Genome wide association studies facilitated detection of many trait associated SNPs. Chromosomes A03, A05 and A09 harboured majority of these. Functional annotation of the associated SNPs and surrounding genome space(s) helped to predict 43 candidate genes. Many of these were predicted under specific day-length conditions. Important among these were the genes encoding floral meristem identity (SPL3, SPL15, AP3, BAM2), photoperiodic responses (COL2, AGL18, SPT, NF-YC4), gibberellic acid biosynthesis (GA1) and regulation of flowering (EBS). Some of the predicted genes were detected for DR subpanel alone. Genes controlling hormones, auxins and gibberellins appeared important for the regulation of plant height. Many of the significant SNPs were located on chromosomes harbouring previously reported QTLs and candidate genes. The identified loci may be used for marker-assisted selection after due validation.

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard. Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (No without added N and N100 added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

Hyaloperonospora brassicae causes downy mildew, a major disease of Brassicaceae species. We sequenced the genomes of two H. brassicae isolates of high (Sample B) and low (Sample C) virulence. Sequencing reads were first assembled de novo with software's SOAPdenovo2, ABySS V2.1 and Velvet V1.1 and later combined to create meta-assemblies with genome sizes of 72.762 and 76.950Mb and predicted gene densities of 1628 and 1644 /Mb, respectively. We could annotate 12.255 and 13,030 genes with high proportions (91-92%) of complete BUSCOs for Sample B and C, respectively. Comparative analysis revealed conserved and varied molecular machinery underlying the physiological specialisation and infection capabilities. BLAST analysis against PHI gene database suggested a relatively higher loss of genes for virulence and pathogenicity in Sample C compared to Sample B, reflecting pathogen evolution through differential rates of mutation and selection. These studies will enable identification and monitoring of H. brassicae virulence factors prevailing in-field.

Timely transition to flowering, maturity and plant height are important for agronomic adaptation and productivity of Indian mustard (B. juncea), which is a major edible oilseed crop of low input ecologies in Indian subcontinent. Breeding manipulation for these traits is difficult because of the involvement of multiple interacting genetic and environmental factors. Here, we report a genetic analysis of these traits using a population comprising 92 diverse genotypes of mustard. These genotypes were evaluated under deficient (N75), normal (N100) or excess (N125) conditions of nitrogen (N) application. Lower N availability induced early flowering and maturity in most genotypes, while high N conditions delayed both. A genotyping-by-sequencing approach helped to identify 406,888 SNP markers and undertake genome wide association studies (GWAS). 282 significant marker-trait associations (MTA's) were identified. We detected strong interactions between GWAS loci and nitrogen levels. Though some trait associated SNPs were detected repeatedly across fertility gradients, majority were identified under deficient or normal levels of N applications. Annotation of the genomic region (s) within ± 50 kb of the peak SNPs facilitated prediction of 30 candidate genes belonging to light perception, circadian, floral meristem identity, flowering regulation, gibberellic acid pathways and plant development. These included over one copy each of AGL24, AP1, FVE, FRI, GID1A and GNC. FLC and CO were predicted on chromosomes A02 and B08 respectively. CDF1, CO, FLC, AGL24, GNC and FAF2 appeared to influence the variation for plant height. Our findings may help in improving phenotypic plasticity of mustard across fertility gradients through marker-assisted breeding strategies.

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

Brassicas are widely used for different purposes as vegetable oil, condiments, vegetables, biofuels, and animal feed. The cultivated rapeseed-mustard species are adapted in diverse agro-climatic situation ranged from warmer semitropical to cooler temperate regions of the world represented in China, Canada, India, Germany, the UK, France, Australia, Poland, and the USA. Cytoplasmic male sterility (CMS) is one of the extensively studied and useful examples of nucleo-cytoplasmic interactions. CMS is attributed by failure of plant to produce functional/viable pollen grain. CMS is a consequence of conflict/miscoordination between the organelles (mitochondria) and nuclear genes that arises due to instinctively mutation mt DNA and production of alloplasmic substitutions with widely differentiated wild spp. In this chapter, we will discuss the various cytoplasmic male sterility systems with fertility restorer (Rf) genes and advancement in the understanding of fertility restoration mechanism of fertility restorer genes.

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

Brassica rapa L. (AA; 2n = 20) store considerable amounts of oil and protein reserves in the seeds. Genetic enhancement of these seed storage products is sought to meet specific requirements of the producers and consumers of edible oils, biofuels, animal feed and other industrial products. This necessitates the introduction of new genetic variation and a greater understanding of the trait genetics. We tested 195 germplasm lines of B. rapa for oil, protein and glucosinolates under the environment conditions differing for day length and ambient temperatures during development and maturation of the seeds. This helped to document phenotypic variations for these traits and also identify accessions with extreme phenotypes. DArT genotyping and genome wide association studies enabled detection of SNP markers within or near the genes that are associated directly or indirectly with the biosynthesis of oil (LTPG5, FRK7, SSE1, LACS8, LTPG33), protein (VTI1B, TSB2, SESA4, bZIP1, PPI1, K18J17.13) and glucosinolates (IMD1, MYB119, ILR3, IQD1, ADS2, SOT18). We also envisaged a gene, PLAT2. It is a member of PLAT-plant-stress subgroup of proteins. PLAT2 can be a target for breeding efforts aimed at improving abiotic stress tolerance. Introduction of novel allelic diversity may be more desirable for the development of designer genotypes rather than the simple repackaging of the genetic variation extant in the crop gene pools. It is for the first time that an attempt has been made to characterize allelic diversity extant in the ‘A’ sub genome of B. juncea for its deployment in B. rapa genepool.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N0) and agrnomically recommened dose (100 kg/ha) of N application (N100). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

A set of 96 Brassica juncea–Erucastrum cardaminoides introgression lines (ILs) were developed with genomic regions associated with Sclerotinia stem rot (Sclerotinia sclerotiorum) resistance from a wild Brassicaceous species E. cardaminoides. ILs were assessed for their resistance responses to stem inoculation with S. sclerotiorum, over three crop seasons (season I, 2011/2012; II, 2014/2015; III, 2016–2017). Initially, ILs were genotyped with transferable SSR markers and subsequently through genotyping by sequencing. SSR based association mapping identified six marker loci associated to resistance in both A and B genomes. Subsequent genome-wide association analysis (GWAS) of 84 ILs recognized a large number of SNPs associated to resistance, in chromosomes A03, A06, and B03. Chromosomes A03 and A06 harbored the maximum number of resistance related SNPs. Annotation of linked genomic regions highlighted an array of resistance mechanisms in terms of signal transduction pathways, hypersensitive responses and production of anti-fungal proteins and metabolites. Of major importance was the clustering of SNPs, encoding multiple resistance genes on small regions spanning approximately 885 kb region on chromosome A03 and 74 kb on B03. Five SNPs on chromosome A03 (6,390,210-381) were associated with LRR-RLK (receptor like kinases) genes that encode LRR-protein kinase family proteins. Genetic factors associated with pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) were predicted on chromosome A03, exhibiting 11 SNPs (6,274,763-994). These belonged to three R-Genes encoding TIR-NBS-LRR proteins. Marker trait associations (MTAs) identified will facilitate marker assisted introgression of these critical resistances, into new cultivars of B. juncea initially and, subsequently, into other crop Brassica species.

Seed size and seed metabolites have been the targets of direct or indirect selection during domestication and subsequent crop breeding. Understanding these traits and associated genetics can prove very useful for plant translational research. Large germplasm assemblage (235) of Brassica juncea and its progenitor species (B. rapa and B. nigra) was evaluated to establish seed trait variations for seed size and seed metabolites. Seeds were smallest in B. nigra and largest in B. juncea. Australian B. juncea and Indian B. rapa var brown sarson types averaged more seed oil content. Seed size and oil content were generally higher in modern cultivars in comparison to the land races. Allelic diversity for known associated genes for seed-size and oil-content (AP2, ARF2, TTG2, GRF2, GL2, CYP78A5, CYP78A6, MINI3, IKU2, IKU1, BRI1, DGAT, GPDH, LPAAT, GPAT and DA1) was studied so as to infer the effect of domestication on seed traits. Three genes (IKU1, IKU2, AP2) in B. rapa, two (TTG2 and GL2) in B. nigra and two (IKU1 and GRF2) in natural B. juncea were identified as targets of selection on the basis of Fst outlier and/or sequence diversity tests. We report parallel divergence for seed traits between B. juncea and B. rapa. Directional selection appeared stronger for seed-size as compared to correlated seed metabolites. Positive selection on seed-size is likely to have played a significant role in structuring regional variation in the germplasm.

A core subset of genetically fixed lines was selected through stratified sampling to represent the allelic variations within Brassica juncea gene pools. It included germplasm accessions from India, China, and Australia, as well as B. Juncea introgression lines carrying alien genetic variation and/or cytoplasm from wild crucifers, namely Diplotaxis cardaminoides, Diplotaxis tenuisiliqua, and Erucastrum abyssinicum. The aim of the study was to infer the influence of underlying cytoplasm on population structure and trait variation. Molecular characterization using nuclear SSR markers (158) and chloroplast SSR markers (9) revealed interesting germplasm patterns and trait variations. Diversity groups obtained using distance-based clustering were not fully compliant with structure analysis, especially for the nuclear genetic variation. Association between population structure and phenotypic variation was indicated by significant interactions between chlorotype and seed/biological yields. Thus, there may be a need to factor in the impact of background cytoplasm on population structure in association studies of crops where different cytoplasmic lineages are known.

Indian mustard (Brassica juncea (L) Czern & Coss) is naturally indeterminate. Plants with determinate inflorescence were first discovered in the self progenies of B. juncea (AABB; 2n = 36), resynthesized by combining A-genome from B. napus (AACC; 2n = 38) and B-genome from B. carinata (BBCC; 2n = 34). In the determinate plants, apical meristems were transformed into pods. In contrast, the inflorescence in the indeterminate plants continues to grow indefinitely while producing peripheral flowers. Genotyping and morphological characterization of newly developed determinate gene pool (A8) showed a rapid emergence and progression of genetic and phenotypic alterations which continued even after seven generations of selfing. A large number of determinate genotypes (125) were evaluated to establish agronomic potential of determinate B. juncea. A high proportion of determinate genotypes outperformed the best indeterminate checks, suggesting that critical productivity related traits like pod number, seed size and oil content were not a function of indeterminacy. As the gene for determinacy has also been introgressed in B. napus and B. carinata, the stage is now set for future breeding to aim at crop architectural modifications through determinacy in all three types of oilseed Brassicas grown in the world. Our studies emphasized the role of polyploidy as a major force of differentiation at both genotypic and phenotypic level. Although, there was no direct correlation between evolution of genetic and phenotypic diversities following polyploidization. We suggest the use of polyploidy as a plant breeding tool to benefit from de novo variation rather than restricting its use as a method to overcome sterility following wide hybridization alone.

We used a Diversity Fixed Foundation Set comprising 48 inbred lines of Brassica juncea and representing all the adaption zones of the crop for association mapping. Extensive phenotypic variations were observed for all the grain yield components and root traits under both irrigated and restricted moisture conditions. The genotypes differed in their responses to moisture stress. Trait averages declined numerically under restricted moisture conditions when compared to a standard irrigation schedule. Canonical analysis demonstrated the importance of primary branches and seed size as the traits of significance for drought susceptibility index. Microsatellite markers (158), representing all the 18 chromosomes, were used to assess the population structure, linkage disequilibrium (LD) in the association panel and marker-trait associ- ations (MTA's). A comparison of four association models (general linear model/GLM(Q-matrix/Q), mixed linear model: MLM(Q?kinship matrix/K), GLM (principal components/PC) and MLM(PC ? K)) showed that GLM(PC) and MLM(PC ? K), incorpo- rating principal components and kinship matrix, were the best models. Maximum proportions of significant results were observed in the models GLM(PC) and MLM(PC ? K). MLM was preferred as there were fewer false positives than GLM. Thirteen significant associations were detected between the molecular markers and agronomic traits. Of these, seven were identified under normal moisture conditions, and six under restricted moisture conditions. Marker-trait associations included four markers associated with grain yield, three with seed size, two with secondary branches and one marker each with plant height, root diameter and root length. A single marker SB1822-1, was repeatedly detected for seed size and grain yield, and was localized at 17.5 cM (centiMorgans) on chromosome B3. Marker SB3872-3 revealed a signif- icant effect under normal moisture conditions on seed size (R 2 % = 15.16) at 60.9 cM on chromosome B5 during the first year. Among the favorable alleles, SB1822-1 had the average positive phenotypic effect for seed size and grain yield. Marker cnu316-3 had maximum positive phenotypic effects on grain yield.

• Premise of the study: Brassica juncea is a major source of edible oil in the Indian subcontinent and northern China. It is also used as a root and leaf vegetable in China and as a condiment in Europe and America. There is a long-standing view that B. juncea originated from multiple hybridization events between B. rapa and B. nigra and that hybridizations were always unidirectional with B. rapa as the cytoplasmic donor. These conclusions were, however, centered primarily on nuclear markers.• Methods: Two hundred forty-six accessions of B. juncea, B. rapa, and B. nigra were genotyped using chloroplast and nuclear simple sequence repeat (SSR) markers.• Key results: A structure analysis assigned B. juncea germplasm (122) into three major groups based on plasmotype variation. The bulk of Indian B. juncea genotypes were grouped along with Chinese and Australian accessions. This plasmotype was absent in sampled accessions of B. rapa (97), B. nigra (27), and other wild crucifers (10). The second group of B. juncea included East European genotypes and four accessions from India. It showed unambiguous homology with the predominant B. nigra plasmotype. The neighbor joining tree produced seven subgroups, arranged into two broad lineages. The first lineage included Indian, Australian, and Chinese B. juncea genotypes; it was associated with wild species belonging to the "rapa" lineage. Nuclear SSR marker-based analyses were largely supportive of results from chloroplast SSR analyses.• Conclusions: Based on these results, we provide the first report that B. juncea originated several times with both B. rapa and B. nigra as cytoplasmic donors in separate hybridization events.