Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

Sinapis genomes provide insights into whole-genome triplication and divergence patterns within tribe Brassiceae.

Sinapis alba and Sinapis arvensis are mustard crops within the Brassiceae tribe of the Brassicaceae family, and represent an important genetic resource for crop improvement. We performed the de novo assembly of Brassica nigra, S. alba, and S. arvensis, and conducted comparative genomics to investigate the pattern of genomic evolution since an ancient whole-genome triplication event. Both Sinapis species retained evidence of the Brassiceae whole-genome triplication approximately 20.5 million years ago (Mya), with subgenome dominance observed in gene density, gene expression, and selective constraint. While S. alba diverged from the ancestor of Brassica and Raphanus at approximately 12.5 Mya, the divergence time of S. arvensis and B. nigra was approximately 6.5 Mya. S. arvensis and B. nigra had greater collinearity compared with their relationship to either Brassica rapa or Brassica oleracea. Two chromosomes of S. alba (Sal03 and Sal08) were completely collinear with two ancestral chromosomes proposed in the Ancestral Crucifer Karyotype (ACK) genomic block model, the first time this has been observed in the Brassiceae. These results are consistent with S. alba representing a relatively ancient lineage of the species evolved from the common ancestor of tribe Brassiceae, and suggest that the phylogeny of the Brassica and Sinapis genera requires some revision. Our study provides new insights into the genome evolution and phylogenetic relationships of Brassiceae and provides genomic information for genetic improvement of these plants.

Introgression and QTL mapping conferring resistance for Alternaria brassicae in the backcross progeny of Sinapis alba + Brassica juncea somatic hybrids.

KEY MESSAGE: A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC2F3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02 cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae.

Lifetimes of the Aglycone Substrates of Specifier Proteins, the Autonomous Iron Enzymes That Dictate the Products of the Glucosinolate-Myrosinase Defense System in Brassica Plants.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(ß-d-glucopyranosyl)thiohydroximate-O-sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: epithionitrile (ESP), nitrile (NSP), or thiocyanate (TFP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives (t1/2) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3-epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.

Effects of sewage sludge supplementation on heavy metal accumulation and the expression of ABC transporters in Sinapis alba L. during assisted phytoremediation of contaminated sites.

ATP binding cassette (ABC) transporters, types C, G, and B were monitored via qPCR in order to investigate the influence of heavy metal (HM) contamination of post-industrial and post-agricultural soils and the effects of its supplementation with sewage sludge, on Sinapis alba plants. Five house-keeping genes were selected and validated to ensure the best reference points. The relative expression of ABC types C and G genes was profoundly affected by experimental conditions and included their upregulation after plants exposure to heavy metals and downregulation after supplementation with sewage sludge. However, ABC type C was more responsive then type G. The experimental conditions altered the expression of ABC type C gene faster than ABC type G and thus, the expression of ABC type C can therefore potentially be used as a bioindicator during assisted phytoremediation of degraded sites. In clean soil, supplementation with sewage sludge with a slight content of heavy metals still caused an upregulation in the expression of ABC types C and G, which showed that proper toxicity assessments are necessary to ensure safe application of sewage sludge into soils. Results showed that the analysed genes take a significant part in plants metal detoxification and that their expression is regulated at transcriptional level after exposure to soil contaminated with heavy metals by both, industrial activities and by sewage sludge supplementation. Thus, their expression can potentially be used as an early-warning biomarker when soil supplementation with sewage sludge is incorporated into the soil-management process.

Draft genome of multiple resistance donor plant Sinapis alba: An insight into SSRs, annotations and phylogenetics.

BACKGROUND: Sinapis alba is a wild member of the Brassicaceae family reported to possess genetic resistance against major biotic and abiotic stresses of oilseed brassicas. However, the resistance nature of S. alba was not exploited generously due to the unavailability of usable genome sequences in public databases. Therefore, the present study was conducted to assemble the first draft genome from raw whole genome shotgun sequences with annotation and develop simple sequence repeat markers for molecular genetics and marker-assisted breeding. RESULTS: The raw genome sequences had 96x coverage on the Illumina platform with 170 Gbp data. The developed assembly by SOAPdenovo2 has ~459 Mbp genome size covered in 403,423 contigs with an average size of 1138.04 bp. The assembly was BLASTX with Arabidopsis thaliana which showed 32.9% positive hits between both plants. The top hit species distribution analysis showed the highest similarity with A. thaliana. A total of 809,597 GO level annotations were recorded after BLASTX results, and 34,012 sequences were annotated with different enzyme codes grouped under seven classes. The gene prediction tool AUGUSTUS identified 113,107 probable genes with an average size of 684 bp. The biochemical pathway annotation assigned 16,119 potential genes to 152 KEGG maps and 1751 enzyme codes. The development of potential SSRs from the de-novo assembly yielded 70731 unique primer pairs. Out of 159 randomly selected SSR markers for validation, 149 successfully amplified in S. alba. However, 10 SSR markers did not amplify during the validation experiment. CONCLUSION: The annotated genome assembly with a large number of SSRs was developed in the present study. To the best of our knowledge, this is the first report of S. alba genome assembly development, annotation, and SSRs mining to date. The data presented here will be a very important resource for future crop improvement programs, especially for resistant breeding.

The complete chloroplast genome sequence of yellow mustard (Sinapis alba L.) and its phylogenetic relationship to other Brassicaceae species.

As a member of the large Brassicaceae family, yellow mustard (Sinapis alba L.) has been used as an important gene pool for the genetic improvement of cash crops in Brassicaceae. Understanding the phylogenetic relationship between Sinapis alba (S. alba) and other Brassicaceae crops can provide guidance on the introgression of its favorable alleles into related species. The chloroplast (cp) genome is an ideal model for assessing genome evolution and the phylogenetic relationships of complex angiosperm families. Herein, we de novo assembled the complete cp genome of S. alba by integrating the PacBio and Illumina sequencing platforms. A 153,760 bp quadripartite cycle without any gap was obtained, including a pair of inverted repeats (IRa and IRb) of 26,221 bp, separated by a large single copy (LSC) region of 83,506 bp and a small single copy (SSC) region of 17,821 bp. A total of 78 protein-coding genes, 30 tRNA genes, and four rRNA genes were identified in this cp genome, as were 89 simple sequence repeat (SSR) loci of 18 types. The codon usage analysis revealed a preferential use of the Leu codon with the A/U ending. The phylogenetic analysis using 82 Brassicaceae species demonstrated that S. alba had a close relationship with important Brassica and Raphanus species; moreover, it likely originated from a separate evolutionary pathway compared with the congeneric Sinapis arvensis. The synonymous (Ks) and non-synonymous (Ks) substitution rate analysis showed that genes encoding "Subunits of cytochrome b/f complex" were under the lowest purifying selection pressure, whereas those associated with "Maturase", "Subunit of acetyl-CoA", and "Subunits of NADH-dehydrogenase" underwent relatively higher purifying selection pressures. Our results provide valuable information for fully utilizing the S. alba cp genome as a potential genetic resource for the genetic improvement of Brassica and Raphanus species.

A combined transcriptional, biochemical and histopathological study unravels the complexity of Alternaria resistance and susceptibility in Brassica coenospecies.

Alternaria blight is one of the most devastating diseases of rapeseed-mustard caused by a necrotrophic fungus Alternaria brassicae. Lack of satisfactory resistance resource in Brassica is still a main obstruction for developing resistance against Alternaria. In this study, we have selected Brassica juncea, Sinapis alba and Camelina sativa to understand and unravel the mechanism of disease resistance against Alternaria. Histopathological studies showed early onset of necrosis in B. juncea (1 dpi) and delayed in S. alba (2 dpi) and C. sativa (3 dpi) respectively. Early and enhanced production of hydrogen peroxide (H2O2) was observed in C. sativa and S. alba (6 hpi) when compared to B. juncea (12 hpi). An increase in catalase activity was observed in both C. sativa (36 % at 6 hpi) and S. alba (15 % at 12 hpi), whereas it significantly decreased in B. juncea at 6 hpi (23 %), 12 hpi (30 %) and 24 hpi (8 %). Gene expression analysis showed induction of PR-3 and PR-12 genes only in C. sativa and S. alba when compared to B. juncea suggesting their vital role for Alternaria resistance. In contrast, SA marker genes were significantly expressed in B. juncea only which provides evidence of hormonal cross talk in B. juncea during Alternaria infection thereby increasing its susceptibility.

PCR-based identification of point mutation mediating acetolactate synthase-inhibiting herbicide resistance in weed wild mustard (Sinapis arvensis).

Acetolactate synthase (ALS)-inhibiting herbicides have been widely used for effective management and control of wild mustard (Sinapis arvensis) biotypes in Iran. The resistance of the ALS inhibitor to weeds is attributed to either target site alteration or enhanced herbicide degradation. Molecular and genetic characterization of the resistance mechanism is relevant to the evolution and management of herbicide resistance. The aims of this research were (a) to characterize the mechanism molecular suspected to Granstar (tribenuron methyl) and Atlantis (Mesosulfuron + Iodosulfuron) resistance in S. arvensis biotypes in the greenhouse and laboratory (b) to investigate the organization of the target-site loci in field selected S. arvensis populations and (c) instantly recognize the mutations that cause resistance to ALS inhibitors. Eighty resistant populations of S. arvensis were carefully collected from fields repeatedly treated with Granstar and Atlantis. The resistance level and pattern of the population were determined through a greenhouse dose-response experiment by applying the above-mentioned herbicides. Extraction of genomic DNA was carried out for PCR and ALS gene analysis. Our results showed that by greenhouse experiment across 80 biotypes suspected to resistance collected in the fields of whole Kermanshah Province, 30 biotypes (37.5%) conferred S. arvensis resistance species reported in the farm. Among 30 biotypes screened in a greenhouse experiment, six biotypes (20%), No. 9, 14, 17, 19, 23 and 28 revealed a mutation in the ALS gene that was detected by PCR-based method. Biotype No. 9 in the position 376 (Asp376-Gly, GAC to GGC), biotypes 14 and 19 in the position 197 (Pro197-Ala, CCT to GCT), biotypes 17, 23 and 28 in the position 574 (Trp574-Leu, TGG to TTG) and biotype No. 23 in the position 122 (Thr-122-Ala, ACA to GCA) showed herbicide resistance. The specific mutation in the position of 122 of the ALS gene in S. arvensis is the first report. Other biotypes showed resistance in the greenhouse but didn't indicate any mutation by PCR-based method. Most of the resistance to Granstar and Atlantis are genetic and are induced by mutations in the ALS gene. The resistance to herbicides may contain a non-mutagenic and non-genetic origin. The reason of herbicide resistance as non-target-site in some of the biotypes may relate to the activity of the herbicide-metabolizing enzyme(s) or transporter proteins that will naturally lead to an increase in herbicide degradation or compartmentation away from its active site.

Gene expression, DNA damage and other stress markers in Sinapis alba L. exposed to heavy metals with special reference to sewage sludge application on contaminated sites.

Bioindicators are promising tools used to detect the long-term effects of selected biosolids on plants development and should be implemented before large-scale supplementation of sewage sludge into the soil. The presented study shows the impact of sewage sludge application on metal-sensitive toxicity biological parameters (biomarkers) in Sinapis alba including: germination, root length, the activity of guaiacol peroxidase, the chlorophyll content, the level of DNA damage and the expression level of Ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and metallothionein (mt). We evaluated data from selected biomarkers in order to broaden our understanding of plants defense mechanisms against heavy metal contamination and the application of sewage sludge into soils. Overall, in contaminated soil after supplementation with both municipal sewage sludges, an increase in toxicity was noticed in DNA damage, mt and rbcl expression and total chlorophyll content. The supplementation of both soils with municipal sewage sludge caused a two-time induction in the mt expression. Moreover, clean soil supplemented with sewage sludge caused an increase in DNA damage shown as the tail moment from approximately 12 µm on control to 40 µm after supplementation. Even if those biosolids increased the initial germination, roots length, and biomass in comparison to the unamended soil, the toxicity was evidenced with other stress markers. Results showed, that in order to accurately assess the influence of sewage sludge application on plants the use of several specific biomarkers is required for safe land restoration. The conducted study also confirmed, both under biochemical and genotoxic tests, that iron enrichment for biosolids or contaminated soil can significantly reduce the bioavailability and toxicity of other metals.

Trp574 substitution in the acetolactate synthase of Sinapis arvensis confers cross-resistance to tribenuron and imazamox.

Rate-response experiments with nine putative resistant wild mustard (Sinapis arvensis) populations from Greece showed cross-resistance to tribenuron and imazamox. The calculated GR50 values [herbicide rate (gaiha-1) required for 50% reduction of fresh weight] of the nine resistant (R) populations ranged from 51.8 to 555.6gaitribenuronha-1 and from 66.3 to 900.4gaiimazamoxha-1. Regarding the susceptible population, GR50 value was not estimated for tribenuron as its lower treatment reduced fresh weight by >95%, whereas the respective value for imazamox was 0.5gaiha-1. Gene sequencing of als revealed that a point mutation at Trp574 position, leading to amino acid substitution by Leu in the ALS enzyme was present and the likely cause of resistance. The in vitro activity of the ALS enzyme indicated I50 values (herbicide concentration required for 50% reduction of the ALS activity) ranging from 19.11 to 217.45µM for tribenuron, whereas the respective value for the S population was 1.17µM. All populations were susceptible to MCPA at the recommended rate. These results strongly support that cross-resistance of 9 S. arvensis populations was due a point mutation of the als gene, which resulted in a less sensitive ALS enzyme.

Overlapping toxic effect of long term thallium exposure on white mustard (Sinapis alba L.) photosynthetic activity.

BACKGROUND: Heavy metal exposure affect plant productivity by interfering, directly and indirectly, with photosynthetic reactions. The toxic effect of heavy metals on photosynthetic reactions has been reported in wide-ranging studies, however there is paucity of data in the literature concerning thallium (Tl) toxicity. Thallium is ubiquitous natural trace element and is considered the most toxic of heavy metals; however, some plant species, such as white mustard (Sinapis alba L.) are able to accumulate thallium at very high concentrations. In this study we identified the main sites of the photosynthetic process inhibited either directly or indirectly by thallium, and elucidated possible detoxification mechanisms in S. alba. RESULTS: We studied the toxicity of thallium in white mustard (S. alba) growing plants and demonstrated that tolerance of plants to thallium (the root test) decreased with the increasing Tl(I) ions concentration in culture media. The root growth of plants exposed to Tl at 100 µg L(-1) for 4 weeks was similar to that in control plants, while in plants grown with Tl at 1,000 µg L(-1) root growth was strongly inhibited. In leaves, toxic effect became gradually visible in response to increasing concentration of Tl (100 - 1,000 µg L(-1)) with discoloration spreading around main vascular bundles of the leaf blade; whereas leaf margins remained green. Subsequent structural analyses using chlorophyll fluorescence, microscopy, and pigment and protein analysis have revealed different effects of varying Tl concentrations on leaf tissue. At lower concentration partial rearrangement of the photosynthetic complexes was observed without significant changes in the chloroplast structure and the pigment and protein levels. At higher concentrations, the decrease of PSI and PSII quantum yields and massive oxidation of pigments was observed in discolored leaf areas, which contained high amount of Tl. Substantial decline of the photosystem core proteins and disorder of the photosynthetic complexes were responsible for disappearance of the chloroplast grana. CONCLUSIONS: Based on the presented results we postulate two phases of thallium toxicity on photosynthesis: the non-destructive phase at early stages of toxicant accumulation and the destructive phase that is restricted to the discolored leaf areas containing high toxicant content. There was no distinct border between the two phases of thallium toxicity in leaves and the degree of toxicity was proportional to the migration rate of the toxicant outside the vascular bundles. The three-fold (nearly linear) increase of Tl(I) concentration was observed in damaged tissue and the damage appears to be associated with the presence of the oxidized form of thallium - Tl(III).

Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids.

Unreduced gametes, sperm or egg cells with the somatic chromosome number, are an important mechanism of polyploid formation and gene flow between heteroploid plants. The meiotic processes leading to unreduced gamete formation are well documented, but the relative influence of environmental and genetic factors on the frequency of unreduced gametes remain largely untested. Furthermore, direct estimates of unreduced gametes based on DNA content are technically challenging and, hence, uncommon. Here, we use flow cytometry to measure the contribution of genetic (hybridization) and environmental (nutrient limitation, wounding) changes to unreduced male gamete production in Brassica napus, Sinapis arvensis and two hybrid lines. Treatments were applied to greenhouse grown plants in a random factorial design, with pollen sampled at two time intervals. Overall, the frequency of unreduced gametes averaged 0.59% (range 0.06-2.17%), plus a single outlier with 27%. Backcrossed hybrids had 39 to 75% higher unreduced gamete production than parental genotypes, averaged across all treatments, although the statistical significance of these differences depended on sampling period and wounding treatment. Unreduced gamete frequencies were higher for the second sampling period than the first. There were no direct effects of wounding or nutrient regime. Our results indicate that both genetic and environmental factors can induce increased unreduced gametes, highlighting the potential importance of environmental heterogeneity and genetic composition of populations in driving polyploid evolution.

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding.

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.

Structural stability and Sin a 1 anti-epitope antibody binding ability of yellow mustard (Sinapis alba L.) napin during industrial-scale myrosinase inactivation process.

This study investigated the structural stability of yellow mustard (YM, Sinapis alba L.) napin and the changes of its Sin a 1 anti-epitope antibody-binding ability during myrosinase enzyme inactivation process. The food industry uses myrosinase-inactive non-pungent YM for uses beyond spice applications. Napin was isolated from seeds received from an industrial processor before (YM + M) and after (YM - M) myrosinase inactivation. Secondary and tertiary structural features and surface hydrophobicity parameters of napin were analyzed. The Sin a 1 content in YM seeds and the stability of Sin a 1-containing napin during simulated in vitro gastrointestinal (GI) digestion were determined by a non-competitive indirect enzyme-linked immunosorbent assay using the Sin a 1 anti-epitope antibody (AE-Ab) as the primary Ab. YM napin retained the dominant alpha-helical components of secondary and tertiary structure folds during this process. YM - M napin showed changes in hydrophobicity parameters of the molecules and binding ability of AE-Ab: 2.19 ± 0.48 g per 100 g of YM - M seeds vs. 1.49 ± 0.16 g per 100 g YM + M seeds. YM - M proteins were more susceptible for in vitro GI digestion and also showed a 30% reduction in AE-Ab binding ability upon digestion of napins. This suggests that the myrosinase inactivation process has induced the surface modification of napin, exposing Sin a 1 epitope, leading to an increase in AE-Ab binding. However, the epitope region of YM - M napin showed improved susceptibility for hydrolysis during GI digestion resulting in fewer available epitope regions, suggesting a possible reduction in napin immune reactivity.

Bidirectional but asymmetrical sexual hybridization between Brassica carinata and Sinapis arvensis (Brassicaceae).

With transgenic crop development it is important to evaluate the potential for transgenes to escape into populations of wild, weedy relatives. Ethiopian mustard (Brassica carinata, BBCC) is easily transformed and is being investigated for uses from biodiesel fuels to biopharmaceuticals. However, little work has been done evaluating its ability to cross with relatives such as wild mustard (Sinapsis arvensis, SrSr), an abundant, cosmopolitan weedy relative. Here we conducted bidirectional crosses with Ethiopian mustard as a maternal parent in 997 crosses and paternal parent in 1,109 crosses. Hybrids were confirmed using flow cytometry and species-specific ITS molecular markers and indicate a high hybridization rate of 6.43 % between Ethiopian mustard (♀) and wild mustard (♂) and a lower, but not insignificant, hybridization rate of 0.01 % in the reverse direction. The majority of the hybrids were homoploid (BCSr) with less than 1 % of pollen production of their parents and low seed production (0.26 seeds/pollination) in crosses and backcrosses indicating a potential for advanced generation hybrids. The accession used had a significant effect on hybrid seed production with different accessions of Ethopian mustard varying in their production of hybrid offspring from 2.69 to 16.34 % and one accession of wild mustard siring almost twice as many hybrid offspring per flower as the other. One pentaploid (BBCCSr) and one hexaploid (BBCCSrSr) hybrid were produced and had higher pollen viability, though no and low seed production, respectively. As wild mustard is self-incompatible and the outcrossing rate of Ethiopian mustard has been estimated as 30 % potential for hybrid production in the wild appears to be high, though the hybridization rate found here represents a worst case scenario as it does not incorporate pre-pollination barriers. Hybridization in the wild needs to be directly evaluated as does the propensity of Ethiopian mustard to volunteer.

Validation and comparison of two commercial ELISA kits and three in-house developed real-time PCR assays for the detection of potentially allergenic mustard in food.

The study compares the applicability of two commercial mustard ELISA kits (Mustard ELISA Kit-specific and Mustard ELISA Kit-total) and three in-house developed real-time PCR assays (singleplex assay for white mustard, singleplex assay for black/brown mustard and duplex assay for the detection of white, black and brown mustard). Analyses of raw and brewed model sausages containing white and black/brown mustard in the range from 1 to 50 ppm indicate that both ELISAs and the three real-time PCR assays allow the detection of traces of mustard in raw and in brewed sausages. The ELISAs were found to be more sensitive than the real-time PCR assays. When the ELISAs and real-time PCR assays were applied to the analysis of 15 commercial foodstuffs differing in their labelling concerning mustard, in one sample mustard was detected with both ELISAs and the three real-time PCR assays although mustard was not indicated on the food ingredient list.

Deep RNA-Seq to unlock the gene bank of floral development in Sinapis arvensis.

Sinapis arvensis is a weed with strong biological activity. Despite being a problematic annual weed that contaminates agricultural crop yield, it is a valuable alien germplasm resource. It can be utilized for broadening the genetic background of Brassica crops with desirable agricultural traits like resistance to blackleg (Leptosphaeria maculans), stem rot (Sclerotinia sclerotium) and pod shatter (caused by FRUITFULL gene). However, few genetic studies of S. arvensis were reported because of the lack of genomic resources. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive dataset for S. arvensis for the first time. We used Illumina paired-end sequencing technology to sequence the S. arvensis flower transcriptome and generated 40,981,443 reads that were assembled into 131,278 transcripts. We de novo assembled 96,562 high quality unigenes with an average length of 832 bp. A total of 33,662 full-length ORF complete sequences were identified, and 41,415 unigenes were mapped onto 128 pathways using the KEGG Pathway database. The annotated unigenes were compared against Brassica rapa, B. oleracea, B. napus and Arabidopsis thaliana. Among these unigenes, 76,324 were identified as putative homologs of annotated sequences in the public protein databases, of which 1194 were associated with plant hormone signal transduction and 113 were related to gibberellin homeostasis/signaling. Unigenes that did not match any of those sequence datasets were considered to be unique to S. arvensis. Furthermore, 21,321 simple sequence repeats were found. Our study will enhance the currently available resources for Brassicaceae and will provide a platform for future genomic studies for genetic improvement of Brassica crops.

Development and validation of a duplex real-time PCR assay for the simultaneous detection of three mustard species (Sinapis alba, Brassica nigra and Brassica juncea) in food.

The paper presents a duplex real-time PCR assay for the simultaneous detection of three potentially allergenic mustard species commonly used in food: white mustard (Sinapis alba), black mustard (Brassica nigra) and brown mustard (Brassica juncea). White mustard is detected in the "green" and black/brown mustard in the "yellow" channel. The duplex real-time PCR assay does not show cross-reactivity with other Brassicaceae species including broccoli, cauliflower, radish and rapeseed. Low cross-reactivities (difference in the Ct value ⩾ 11.91 compared with the positive control) were obtained with cumin, fenugreek, ginger, rye and turmeric. When applying 500 ng DNA per PCR tube, the duplex real-time PCR assay allowed the detection of white, black and brown mustard in brewed model sausages down to a concentration of 5mg/kg in 10 out of 10 replicates. The duplex real-time PCR assay was applied to verify correct labelling of commercial foodstuffs.

Construction of a genetic linkage map and QTL analysis of erucic acid content and glucosinolate components in yellow mustard (Sinapis alba L.).

BACKGROUND: Yellow mustard (Sinapis alba L.) is an important condiment crop for the spice trade in the world. It has lagged behind oilseed Brassica species in molecular marker development and application. Intron length polymorphism (ILP) markers are highly polymorphic, co-dominant and cost-effective. The cross-species applicability of ILP markers from Brassica species and Arabidopsis makes them possible to be used for genetic linkage mapping and further QTL analysis of agronomic traits in yellow mustard. RESULTS: A total of 250 ILP and 14 SSR markers were mapped on 12 linkage groups and designated as Sal01-12 in yellow mustard. The constructed map covered a total genetic length of 890.4 cM with an average marker interval of 3.3 cM. The QTL for erucic content co-localized with the fatty acid elongase 1 (FAE1) gene on Sal03. The self-(in)compatibility gene was assigned to Sal08. The 4-hydroxybenzyl, 3-indolylmethyl and 4-hydroxy-3-indolylmethyl glucosinolate contents were each controlled by one major QTL, all of which were located on Sal02. Two QTLs, accounting for the respective 20.4% and 19.2% of the total variation of 2-hydroxy-3-butenyl glucosinolate content, were identified and mapped to Sal02 and Sal11. Comparative synteny analysis revealed that yellow mustard was phylogenetically related to Arabidopsis thaliana and had undergone extensive chromosomal rearrangements during speciation. CONCLUSION: The linkage map based on ILP and SSR markers was constructed and used for QTL analysis of seed quality traits in yellow mustard. The markers tightly linked with the genes for different glucosinolate components will be used for marker-assisted selection and map-based cloning. The ILP markers and linkage map provide useful molecular tools for yellow mustard breeding.