Sub-cellular immunolocalization of the glucosinolate sinigrin in seedlings of Brassica juncea

Polyclonal rat antibodies were raised to a bovine serum albumin-sinigrin conjugate and used to immunolocalize sinigrin (2-propenylglucosinolate) in imbibed seeds and developing seedlings of Brassica juncea. (L.) Czern. Sinigrin was localized to protein bodies in aleurone-like cells but shown to be absent from myrosin cells. Double labelling techniques were used to co-localize both myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) and sinigrin. Myrosin grains were labelled only with the anti-myrosinase antibody, but aleurone cells were labelled with both anti-myrosinase and anti-sinigrin antibodies. High-performance liquid chromatographic analysis of conventionally fixed and dehydrated seed tissues (4 h post imbibition in water), indicated a high proportion of sinigrin was retained in fixed tissues. Over a time course of 100 h, protein bodies within aleurone-like cells degraded, fused to form the cell vacuole and lost all myrosinase labelling but retained residual sinigrin labelling. The degradation of protein bodies corresponded to a decrease in retention of sinigrin in the fixed tissues. The results describe for the first time the co-localization of a plant enzyme and its substrate, a secondary metabolite. Received: 8 January 1998 / Accepted: 27 February 1998     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.     

Amphidiploid Brassica juncea contains conserved progenitor genomes.

To perform a detailed study of genome evolution in the natural Brassica amphidiploid B. juncea, we have constructed two linkage maps based on RFLP (restriction fragment length polymorphism) markers; one generated from a cross between a resynthesized B. juncea (a chromosome doubled interspecific B. rapa x B. nigra hybrid) and a natural B. juncea cultivar, the other from a cross between two B. juncea cultivars. By using a common cultivar in both crosses, the two maps could be unambiguously integrated. All loci exhibited disomic inheritance of parental alleles in the natural x resynthesized cross, showing that B. rapa chromosomes paired exclusively with their A-genome homologues in B. juncea and that B. nigra chromosomes likewise paired with their B-genome homologues. The maps derived from the two crosses were also perfectly collinear. Furthermore, these maps were collinear with maps of the diploid progenitor species (B. nigra and B. rapa) produced using the same set of RFLP probes. These data indicate that the genome of B. juncea has remained essentially unchanged since polyploid formation. Our observations appear to refute the suggestion that the formation of polyploid genomes is accompanied by rapid change in genome structure.     

Brassica napus DNA markers linked to white rust resistance in Brassica juncea

White rust, caused by Albugo candida, is an economically important disease of Brassica juncea mustard. The most efficient and cost effective way of protecting mustard plants from white rust is through genetic resistance. The development of canola quality B. juncea through interspecific crosses of B. juncea with Brassica napus has lead to the introgression of white rust resistance from B. napus into B. juncea. The objective of this study was to identify DNA markers for white rust resistance, derived from the introgressed B. napus chromosome segment, in a BC(3)F(2) population of condiment B. juncea mustard. This segregating population was phenotyped for white rust reaction and used to screen for AFLP markers associated with white rust resistance using bulked segregant analysis. Segregation data indicated that a single dominant gene controlled resistance to white rust. Eight AFLP markers linked to white rust resistance were identified, all derived from B. napus. The B. napus chromosome segment, carrying the white rust resistance gene ( Ac2V(1)), appeared to have recombined with the B. juncea DNA since recombinant individuals were identified. Comparative mapping of the eight B. napus-derived AFLP markers in a typical B. napus mapping population was inconclusive; therefore, the size of the introgressed B. napus fragment could not be determined.     

Feeding behaviour of generalist pests on Brassica juncea: implication for manipulation of glucosinolate biosynthesis pathway for enhanced resistance

Differential accumulation of plant defence metabolites has been suggested to have important ecological consequence in the context of plant–insect interactions. Feeding of generalist pests on Brassica juncea showed a distinct pattern with selective exclusion of leaf margins which are high in glucosinolates. Molecular basis of this differential accumulation of glucosinolates could be explained based on differential expression profile of BjuMYB28 homologues, the major biosynthetic regulators of aliphatic glucosinolates, as evident from quantitative real‐time PCR and promoter:GUS fusion studies in allotetraploid B. juncea. Constitutive overexpression of selected BjuMYB28 homologues enhanced accumulation of aliphatic glucosinolates in B. juncea. Performance of two generalist pests, Helicoverpa armigera and Spodoptera litura larvae, on transgenic B. juncea plants were poor compared to wild‐type plants in a no‐choice experiment. Correlation coefficient analysis suggested that weight gain of H. armigera larvae was negatively correlated with gluconapin (GNA) and glucobrassicanapin (GBN), whereas that of S. litura larvae was negatively correlated with GNA, GBN and sinigrin (SIN). Our study explains the significance and possible molecular basis of differential distribution of glucosinolates in B. juncea leaves and shows the potential of overexpressing BjuMYB28 for enhanced resistance of Brassica crops against the tested generalist pests.     

Molecular markers for seed colour in Brassica juncea.

A detailed RFLP map was used to map QTLs associated with seed colour in Brassica juncea using a doubled-haploid population derived from a cross between a black/brown-seeded cultivar and a yellow-seeded breeding line. Segregation analysis suggested that seed colour was under control of 2 unlinked loci with duplicate gene action. However, QTL analysis revealed 3 QTLs, SC-B4, SC-A10 and SC-A6, affecting seed colour. The QTLs were consistent across environments, and individually explained 43%, 31%, and 16%, respectively, and collectively 62% of the phenotypic variation in the population. Digenic interaction analysis showed that closest flanking locus of QTL SC-B4, wg7b6cNM, had strong epistasis with the locus wg5a1a, which is tightly linked to QTL SC-A6. The interaction of these 2 loci explained 27% of the phenotypic variation in the population, while the whole model explained 84%. In a multiple regression model, the effects of QTL SC-A10, as well as its interaction with other loci, were non-significant, whereas the effects of loci wg7b6cNM and wg5a1a and their interaction were significant. Ninety-eight percent of the DH lines carried the expected alleles of loci wg7b6cNM and wg5a1a for seed colour, confirming that only these 2 loci were linked to seed colour in B. juncea. Four additional digenic interactions significantly affected seed colour, and all 5 digenic interactions were consistent across environments.     

Molecular marker-assisted breeding for improved Ogura cms restorer line (RfoRfo) and mapping of the restorer gene (Rfo) in Brassica juncea

Developing a fully functional hybrid system is a must for hybrid breeding in Brassica juncea. The B. napus Ogura cms hybrid system was transferred into B. juncea by the researchers at INRA, France. The B. juncea restorer (R) line (RfoRfo) exhibited poor vigor, low fertility and was black-seeded due to linkage drag. Our studies indicated that the Rfo gene in B. juncea R line was linked to the 5 C9 markers of B. napus (sN3553F, sS2285, sN3841, sN12905 and At5g58730) and 4 radish markers (At3g27100, At5g25080, At4g13720 and At5g06240) in addition to the 6 radish markers reported before (ScH03, ScA14, OPF3, BolJon, CAB and PGIint). These markers were used to screen for improved restorer plants in the three crosses of B. juncea restorer plant O39-16 (Rforf) × condiment var. Cutlass, O39-16 (Rforf) × canola B. juncea line C668 and O39-16 (Rforf) × resynthesized B. juncea line 15043. One improved homozygous R line VR441 (RfoRfo) with only 1 C9 marker sN12905 and 2 radish markers ScH03 and BolJon was successfully developed via marker-assisted selection in the cross O39-16 × 15043. VR441 had good seed-setting (average: 14.3 seeds/pod), strong growth vigor and was yellow-seeded. Linkage mapping revealed that the Rfo gene was introgressed into chromosome 9 of the A genome in B. juncea. The development of the improved R line VR441 has made the Ogura cms hybrid system fully functional in B. juncea. We are currently using the improved system for developing high yielding hybrid varieties in condiment and canola B. juncea.     

Fine mapping of loci involved with glucosinolate biosynthesis in oilseed mustard (Brassica juncea) using genomic information from allied species

Fine mapping of six seed glucosinolate QTL (J2Gsl1, J3Gsl2, J9Gsl3, J16Gsl4, J17Gsl5 and J3Gsl6) (Ramchiary et al. in Theor Appl Genet 116:77–85, 2007a) was undertaken by the candidate gene approach. Based on the DNA sequences from Arabidopsis and Brassica oleracea for the different genes involved in the aliphatic glucosinolate biosynthesis, candidate genes were amplified and sequenced from high to low glucosinolate Brassica juncea lines Varuna and Heera, respectively. Of the 20 paralogues identified, 17 paralogues belonging to six gene families were mapped to 12 of the 18 linkage groups of B. juncea genome. Co-mapping of candidate genes with glucosinolate QTL revealed that the candidate gene BjuA.GSL-ELONG.a mapped to the QTL interval of J2Gsl1, BjuA.GSL-ELONG.c, BjuA.GSL-ELONG.d and BjuA.Myb28.a mapped to the QTL interval of J3Gsl2, BjuA.GSL-ALK.a mapped to the QTL interval of J3Gsl6 and BjuB.Myb28.a mapped to the QTL interval of J17Gsl5. The QTL J9Gsl3 and J16Gsl4 did not correspond to any of the mapped candidate genes. The functionality and contribution of different candidate genes/QTL was assessed by allelic variation study using phenotypic data of 785 BC₄DH lines. It was observed that BjuA.Myb28.a and J9Gsl3 contributed significantly to the base level glucosinolate production while J16Gsl4, probably GSL-PRO, BjuA.GSL-ELONG.a and BjuA.GSL-ELONG.c contributed to the C3, C4 and C5 elongation pathways, respectively. Three A genome QTL: J2Gsl1harbouring BjuA.GSL-ELONG.a, J3Gsl2 harbouring both BjuA.GSL-ELONG.c and BjuA.Myb28.a and J9Gsl3, possibly the ‘Bronowski genes’, were identified as most important loci for breeding low glucosinolate B. juncea. We observed two-step genetic control of seed glucosinolate in B. juncea mainly effected by these three A genome QTL. This study, therefore, provides clues to the genetic mechanism of ‘Bronowski genes’ controlling the glucosinolate trait and also provides efficient markers for marker-assisted introgression of low glucosinolate trait in B. juncea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Loss of C-genome-specific markers during transgene introgression from Brassica napus to wild Brassica juncea

Transgene flow from engineered Brassica napus to wild weed relatives could potentially have an environmental effect. To evaluate the introgression of transgenic B. napus into wild Brassica juncea, the hybrid F₁ and backcross progenies derived from B. juncea (genome constitution AABB) and transgenic B. napus (AACC) crosses were investigated. C-genome-specific simple sequence repeat (SSR) markers corresponding to linkage groups N11–N19 in B. napus were screened and used to estimate the marker frequency in hybrid F₁ and backcross progenies. C-genome-specific markers could be stably detected in hybrid F₁ and backcross BC₁ plants, but were only rarely found in the BC₂–BC₅ generations. For example, a specific SSR marker for linkage group N12 segregated in BC₂ generation but were completely lost in BC₃–BC₅, while a specific SSR marker of linkage group N15 segregated in BC₁, BC₂ and BC₃ generations and was absent in more advanced backcrossed generations (BC₄ and BC₅). The results indicate that a certain gene regions in Brassica napus plants are transmitted at a relatively lower frequency to wild relatives, and more rapidly disappeared in subsequent backcross generations. We propose that a foreign gene or transgene that is integrated in the C-chromosome of Brassica napus could reduce the risk of introgression in nature.     

Transfer of auxinic herbicide resistance from Brassica kaber to Brassica juncea and Brassica rapa through embryo rescue

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops.     

Molecular mapping of seed aliphatic glucosinolates in Brassica juncea.

An RFLP genomic map with 316 loci was used to study the inheritance of aliphatic glucosinolates in Brassica juncea using doubled-haploid (DH) populations developed from a cross between RLM-514, an agronomically superior non-canola quality B. juncea (high erucic acid and high glucosinolates), and an agronomically poor canola quality B. juncea breeding line. Two QTLs (GSL-A2a and GSL-A2b) associated with 3-butenyl were consistent across years and locations, and explained 75% of the phenotypic variance in the population. Three QTLs (GSL-A2a, GSL-F, GSL-B3) affected 2-propenyl and explained 78% of the phenotypic variance in the population. For total aliphatic glucosinolates, five QTLs explained 30% to 45% of the total phenotypic variance in the population in different environments. Several QTLs (GSL-A7 and GSL-A3) were highly inconsistent in different environments. Major QTLs (GSL-A2a and GSL-A2b) associated with individual glucosinolates were non-significant for total aliphatic glucosinolates. A marker-assisted selection strategy based on QTLs associated with individual glucosinolates rather than total aliphatic glucosinolates is proposed for B. juncea.     

Molecular markers linked to white rust resistance in mustard Brassica juncea

 White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of Brassica juncea (L.) Czern. and Coss mustard, particularly in India. The most efficient and cost-effective way of protecting mustard plants from white rust disease is through genetic resistance. The objective of this study was to identify RAPD markers for white rust resistance in an F₁-derived doubled-haploid (DH) population originating from a cross between white rust-susceptible and white rust-resistant breeding lines of B. juncea from the canola-quality B. juncea breeding project of the Agriculture and Agri-Food Canada-Saskatoon Research Centre. The DH population was used to screen for RAPD markers associated with white rust resistance/susceptibility using bulked segregant analysis. Two markers, WR2 and WR3, linked to white rust resistance, flanked the resistance locus Ac2 ₁ and were highly effective in identifying the presence or absence of the resistance gene in the DH population. These two markers were shown to be specific to the Russian source of white rust resistance utilized in this project. It is concluded that the availability of these RAPD markers will enhance the breeding for white rust resistance in B. juncea. Received: 17 December 1997 / Accepted: 7 April 1998     

不同品种芥菜对Cu胁迫响应的差异

采用水培法研究了不同品种芥菜对Cu胁迫响应的差异。结果表明:4μmol·L-1和8μmol·L-1Cu处理均抑制根和地上部生物量的积累,根受到的抑制程度大于地上部,敏感品种受到的抑制程度大于抗性品种。根中Cu积累量大于地上部,敏感品种根中Cu积累量大于抗性品种。8μmol·L-1Cu处理能明显诱导芥菜根中的MDA、Pro含量的增加及AsA含量的减少,APX、POD活性上升和GR活性下降,而不改变SOD活性。     

Glyoxalase I from Brassica juncea is a calmodulin stimulated protein.

Brassica juncea glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1. 5) is a 56 kDa, heterodimeric protein. It requires magnesium (Mg2+) for its optimal activity. In this report we provide biochemical evidence for modulation of glyoxalase I activity by calcium/calmodulin (Ca2+/CaM). In the presence of Ca2+ glyoxalase I showed a significant (2.6-fold) increase in its activity. It also showed a Ca2+ dependent mobility shift on denaturing gels. Its Ca2+ binding was confirmed by Chelex-100 assay and gel overlays using 45CaCl2. Glyoxalase I was activated by over 7-fold in the presence of Ca2+ (25 microM) and CaM (145 nM) and this stimulation was blocked by the CaM antibodies and a CaM inhibitor, trifluroperazine (150 microM). Glyoxalase I binds to a CaM-Sepharose column and was eluted by EGTA. The eluted protein fractions also showed stimulation by CaM. The stimulation of glyoxalase I activity by CaM was maximum in the presence of Mg2+ and Ca2+; however, magnesium alone also showed glyoxalase I activation by CaM.