Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition.

Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re-sequenced panel of 1133 M2 generation rapeseed plants and analysed large-scale deletions, single nucleotide variants and small insertion-deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large-scale impacts can also be detected using transcriptome re-sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21-base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics-led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops.

Genetic analysis of the variation for mineral accumulation in the leaves and seeds of natural germplasm of Brassica rapa L. (AA) and the its derived forms extracted from an allotetraploid B.juncea L.(AABB).

Brassica rapa L. (2n = 20; AA) is a vegetable and oilseed crop that is grown all over the world. Its leaves, shoots, and seeds store significant amounts of minerals. We used inductively coupled plasma-optical emission spectroscopy (ICP-OES) to determine the concentrations of eleven minerals in the leaves and seeds of 195 advanced generation inbred lines, of which 92 represented natural (NR) B. rapa and the remaining 103 were derived (DR) from a set of mother genotypes originally extracted from an allotetraploid B. juncea (2n = 36; AABB). The inbred lines differed for the composition of leaf and seed minerals. Leaf concentrations of N, K, Zn, and Se were higher in the DR subpanel as compared to NR subpanel, along with high seed accumulations of K and Se. DArT genotyping and genome wide association mapping led to the identification of SNPs associated with leaf and seed mineral compositions. Chromosomes A03, A05, and A10 harboured the most associated loci. Annotations of the regions adjacent to respective GWAS peaks allowed prediction of genes known for acquisition, transport, and accumulation of minerals and heavy metal detoxification. Transcriptome analysis revealed differential expression patterns of the predicted candidates, with most genes either down-regulated in derived genotypes relative to natural forms or their expression being comparable between the two. General downregulation may be a consequence of extracting B. rapa from allotetraploid B. juncea through genome resection. Some of the identified SNPs may be used as DNA markers for breeding programmes designed to modify the leaf and seed mineral compositions.

Novel Resistances Provide New Avenues to Manage Alternaria Leaf Spot (Alternaria brassicae) in Canola (Brassica napus), Mustard (B. juncea), and Other Brassicaceae Crops.

Alternaria leaf spot (Alternaria brassicae) can be a devastating disease in canola (Brassica napus) and mustard (B. juncea), but there are no highly effective host resistances available. Screening of 150 diverse Brassicaceae varieties under glasshouse conditions highlighted important novel resistances. In particular, Camelina sativa '4076' and Diplotaxis erucoides 'Wasabi Rocket' had complete resistance across disease assessment parameters (leaf incidence [%LDI]; severity [%LAD]; consequent defoliation [%LCI]). The next most resistant varieties were C. sativa 'CSA' (%LDI 0.6; %LAD 0.4), '4144' (%LDI 1.2; %LAD 0.5), '405' (%LDI 1.7; %LAD 0.7), C. sativa '3274' (%LDI 2.5; %LAD 0.8), Carrichtera annua 'CAN3' (%LDI 7.7; %LAD 4.0), and Sisymbrium irio 'London Rocket' (%LDI 2.1; %LAD 0.8), all with %LCI values of 0. Other genotypes showing high-level resistance included S. erysimoides 'SER 4' (%LDI 11.8; %LAD 5.6; %LCI 0) and D. cardaminoides 'Wild Rocket' (%LDI 15.5; %LAD 7.2; %LCI 0), and those showing moderate resistance were Brassica carinata 'ML-EM-1' (Rungwe), B. insularis 'Moris', B. napus 'ZY006', B. oxyrrhina 'BOX1', B. oleracea var. capitata 'Sugarloaf', B. tournefortii 'CN01-104-2', and Sinapis alba 'Concerta' with %LDI 21.6 to 29.8, %LAD 12.8 to 21.0, and %LCI 0 to 5.7. In particular, B. napus 'ZY006' for canola and B. oleracea var. capitata 'Sugarloaf' can now be directly utilized (i.e., without crossing impairment) for Brassica species and vegetable breeding programs, respectively. While all B. juncea genotypes were susceptible, there were some less susceptible varieties from India in comparison with genotypes from Australia or China. The most susceptible test genotype was Rapistrum sativus (%LDI 89.4; %LAD 83.9; %LCI 71.0), highlighting the value of the resistances identified. These findings not only highlight a range of novel resistances against A. brassicae for canola, mustard, and other diverse Brassicaceae breeding programs to develop resistant commercial varieties, but also emphasize highly susceptible varieties to avoid in both breeding programs and commercial situations conducive to Alternaria leaf spot.

Host-induced gene silencing reveals the role of Sclerotinia sclerotiorum oxaloacetate acetylhydrolase gene in fungal oxalic acid accumulation and virulence.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T3 transformants with significantly higher resistance to S. sclerotiorum in comparison to untransformed controls. There was a concomitant reduction in expression of Ssoah1 and accumulation of oxalic acid in the necrotic regions of transgenic lines as compared to the non-transgenic controls. Specific Ssoah1-siRNA was highly expressed in HIGS Ssoah1 transgenic lines, as compared with WT and EV plants. The outcomes of oxalic acid estimation revealed that silencing of Ssoah1 results in decreased OA accumulation. The recovered mycelium plugs from HIGS Ssoah1 transgenic lines showed decreased Ssoah1 expression and pathogenesis. These results provide the possibility of using HIGS of Ssoah1 for engineering resistance against S. sclerotiorum.

Sclerotinia sclerotiorum Thioredoxin1 (SsTrx1) is required for pathogenicity and oxidative stress tolerance.

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host-induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference-induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1-based host-induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS-A) and Nicotiana benthamiana (HIGS-N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum.

Comparative analysis of draft genome assemblies developed from whole genome sequences of two Hyaloperonospora brassicae isolate samples differing in field virulence on Brassica napus.

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.

Anchoring alien chromosome segment substitutions bearing gene(s) for resistance to mustard aphid in Brassica juncea-B. fruticulosa introgression lines and their possible disruption through gamma irradiation.

KEY MESSAGE: 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 structural evolution in Brassica crops.

The cultivated Brassica species include numerous vegetable and oil crops of global importance. Three genomes (designated A, B and C) share mesohexapolyploid ancestry and occur both singly and in each pairwise combination to define the Brassica species. With organizational errors (such as misplaced genome segments) corrected, we showed that the fundamental structure of each of the genomes is the same, irrespective of the species in which it occurs. This enabled us to clarify genome evolutionary pathways, including updating the Ancestral Crucifer Karyotype (ACK) block organization and providing support for the Brassica mesohexaploidy having occurred via a two-step process. We then constructed genus-wide pan-genomes, drawing from genes present in any species in which the respective genome occurs, which enabled us to provide a global gene nomenclature system for the cultivated Brassica species and develop a methodology to cost-effectively elucidate the genomic impacts of alien introgressions. Our advances not only underpin knowledge-based approaches to the more efficient breeding of Brassica crops but also provide an exemplar for the study of other polyploids.

Genome wide association analyses to understand genetic basis of flowering and plant height under three levels of nitrogen application in Brassica juncea (L.) Czern & Coss.

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.

Development and Validation of Kompetitive Allele-Specific PCR Assays for Erucic Acid Content in Indian Mustard [Brassica juncea (L.) Czern and Coss.].

Brassica juncea L. is the most widely cultivated oilseed crop in Indian subcontinent. Its seeds contain oil with very high concentration of erucic acid (≈50%). Of late, there is increasing emphasis on the development of low erucic acid varieties because of reported association of the consumption of high erucic acid oil with cardiac lipidosis. Erucic acid is synthesized from oleic acid by an elongation process involving two cycles of four sequential steps. Of which, the first step is catalyzed by ß-ketoacyl-CoA synthase (KCS) encoded by the fatty acid elongase 1 (FAE1) gene in Brassica. Mutations in the coding region of the FAE1 lead to the loss of KCS activity and consequently a drastic reduction of erucic acid in the seeds. Molecular markers have been developed on the basis of variation available in the coding or promoter region(s) of the FAE1. However, majority of these markers are not breeder friendly and are rarely used in the breeding programs. Present studies were planned to develop robust kompetitive allele-specific PCR (KASPar) assays with high throughput and economics of scale. We first cloned and sequenced FAE1.1 and FAE1.2 from high and low erucic acid (<2%) genotypes of B. juncea (AABB) and its progenitor species, B. rapa (AA) and B. nigra (BB). Sequence comparisons of FAE1.1 and FAE1.2 genes for low and high erucic acid genotypes revealed single nucleotide polymorphisms (SNPs) at 8 and 3 positions. Of these, three SNPs for FAE1.1 and one SNPs for FAE1.2 produced missense mutations, leading to amino acid modifications and inactivation of KCS enzyme. We used SNPs at positions 735 and 1,476 for genes FAE1.1 and FAE1.2, respectively, to develop KASPar assays. These markers were validated on a collection of diverse genotypes and a segregating backcross progeny. KASPar assays developed in this study will be useful for marker-assisted breeding, as these can track recessive alleles in their heterozygous state with high reproducibility.

Genetics of days to flowering, maturity and plant height in natural and derived forms of Brassica rapa L.

KEY MESSAGE: 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.

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

KEY MESSAGE: 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.

Association Mapping of Seed Quality Traits Under Varying Conditions of Nitrogen Application in Brassica juncea L. Czern & Coss.

Indian mustard (Brassica juncea) is a major source of vegetable oil in the Indian subcontinent. The seed cake left after the oil extraction is used as livestock feed. We examined the genetic architecture of oil, protein, and glucosinolates by conducting a genome-wide association study (GWAS), using an association panel comprising 92 diverse genotypes. We conducted trait phenotyping over 2 years at two levels of nitrogen (N) application. Genotyping by sequencing was used to identify 66,835 loci, covering 18 chromosomes. Genetic diversity and phenotypic variations were high for the studied traits. Trait performances were stable when averaged over years and N levels. However, individual performances differed. General and mixed linear models were used to estimate the association between the SNP markers and the seed quality traits. Population structure, principal components (PCs) analysis, and discriminant analysis of principal components (DAPCs) were included as covariates to overcome the bias due to the population stratification. We identified 16, 23, and 27 loci associated with oil, protein, and glucosinolates, respectively. We also established LD patterns and haplotype structures for the candidate genes. The average block sizes were larger on A-genome chromosomes as compared to the B- genome chromosomes. Genetic associations differed over N levels. However, meta-analysis of GWAS datasets not only improved the power to recognize associations but also helped to identify common SNPs for oil and protein contents. Annotation of the genomic region around the identified SNPs led to the prediction of 21 orthologs of the functional candidate genes related to the biosynthesis of oil, protein, and glucosinolates. Notable among these are: LACS5 (A09), FAD6 (B05), ASN1 (A06), GTR2 (A06), CYP81G1 (B06), and MYB44 (B06). The identified loci will be very useful for marker-aided breeding for seed quality modifications in B. juncea.

Physical mapping of introgressed chromosome fragment carrying the fertility restoring (Rfo) gene for Ogura CMS in Brassica juncea L. Czern & Coss.

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

Identification of Chromosomes and Chromosome Rearrangements in Crop Brassicas and Raphanus sativus: A Cytogenetic Toolkit Using Synthesized Massive Oligonucleotide Libraries.

Crop brassicas include three diploid [Brassica rapa (AA; 2n = 2x = 16), B. nigra (BB; 2n = 2x = 18), and B. oleracea (CC; 2n = 2x = 20)] and three derived allotetraploid species. It is difficult to distinguish Brassica chromosomes as they are small and morphologically similar. We aimed to develop a genome-sequence based cytogenetic toolkit for reproducible identification of Brassica chromosomes and their structural variations. A bioinformatic pipeline was used to extract repeat-free sequences from the whole genome assembly of B. rapa. Identified sequences were subsequently used to develop four c. 47-mer oligonucleotide libraries comprising 27,100, 11,084, 9,291, and 16,312 oligonucleotides. We selected these oligonucleotides after removing repeats from 18 identified sites (500-1,000 kb) with 1,997-5,420 oligonucleotides localized at each site in B. rapa. For one set of probes, a new method for amplification or immortalization of the library is described. oligonucleotide probes produced specific and reproducible in situ hybridization patterns for all chromosomes belonging to A, B, C, and R (Raphanus sativus) genomes. The probes were able to identify structural changes between the genomes, including translocations, fusions, and deletions. Furthermore, the probes were able to identify a structural translocation between a pak choi and turnip cultivar of B. rapa. Overall, the comparative chromosomal mapping helps understand the role of chromosome structural changes during genome evolution and speciation in the family Brassicaceae. The probes can also be used to identify chromosomes in aneuploids such as addition lines used for gene mapping, and to track transfer of chromosomes in hybridization and breeding programs.

Molecular and genetic analysis of defensive responses of Brassica juncea - B. fruticulosa introgression lines to Sclerotinia infection.

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.

Detection of First Marker Trait Associations for Resistance Against Sclerotinia sclerotiorum in Brassica juncea-Erucastrum cardaminoides Introgression Lines.

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.

Near-infrared reflectance spectroscopy calibrations for assessment of oil, phenols, glucosinolates and fatty acid content in the intact seeds of oilseed Brassica species.

BACKGROUND: Very few near-infrared reflectance spectroscopy (NIRS) calibration models are available for non-destructive estimation of seed quality traits in Brassica juncea. Those that are available also fail to adequately discern variation for oleic acid (C18:1 ), linolenic (C18:3 ) fatty acids, meal glucosinolates and phenols. We report the development of a new NIRS calibration equation that is expected to fill the gaps in the existing NIRS equations. RESULTS: Calibrations were based on the reference values of important quality traits estimated from a purposely selected germplasm set comprising 240 genotypes of B. juncea and 193 of B. napus. We were able to develop optimal NIRS-based calibration models for oil, phenols, glucosinolates, oleic acid, linoleic acid and erucic acid for B. juncea and B. napus. Correlation coefficients (RSQ) of the external validations appeared greater than 0.7 for the majority of traits, such as oil (0.766, 0.865), phenols (0.821, 0.915), glucosinolates (0.951, 0.986), oleic acid (0.814. 0.810), linoleic acid (0.974, 0.781) and erucic acid (0.963, 0.943) for B. juncea and B. napus, respectively. CONCLUSION: The results demonstrate the robust predictive power of the developed calibration models for rapid estimation of many quality traits in intact rapeseed-mustard seeds which will assist plant breeders in effective screening and selection of lines in quality improvement breeding programmes. © 2018 Society of Chemical Industry.

Mapping resistance responses to Sclerotinia infestation in introgression lines of Brassica juncea carrying genomic segments from wild Brassicaceae B. fruticulosa.

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014-15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.

Segregation for fertility and meiotic stability in novel Brassica allohexaploids.

KEY MESSAGE: Allohexaploid Brassica populations reveal ongoing segregation for fertility, while genotype influences fertility and meiotic stability. Creation of a new Brassica allohexaploid species is of interest for the development of a crop type with increased heterosis and adaptability. At present, no naturally occurring, meiotically stable Brassica allohexaploid exists, with little data available on chromosome behaviour and meiotic control in allohexaploid germplasm. In this study, 100 plants from the cross B. carinata × B. rapa (A2 allohexaploid population) and 69 plants from the cross (B. napus × B. carinata) × B. juncea (H2 allohexaploid population) were assessed for fertility and meiotic behaviour. Estimated pollen viability, self-pollinated seed set, number of seeds on the main shoot, number of pods on the main shoot, seeds per ten pods and plant height were measured for both the A2 and H2 populations and for a set of reference control cultivars. The H2 population had high segregation for pollen viability and meiotic stability, while the A2 population was characterised by low pollen fertility and a high level of chromosome loss. Both populations were taller, but had lower average fertility trait values than the control cultivar samples. The study also characterises fertility and meiotic chromosome behaviour in genotypes and progeny sets in heterozygous allotetraploid Brassica derived lines, and indicates that genotypes of the parents and H1 hybrids are affecting chromosome pairing and fertility phenotypes in the H2 population. The identification and characterisation of factors influencing stability in novel allohexaploid Brassica populations will assist in the development of this as a new crop species for food and agricultural benefit.