Juncea Lines Research Articles

Evaluation of Indian Mustard Genotypes for White Rust Resistance Using BjuWRR1Gene and Their Phenotypic Performance

The present investigation was carried out to identify the potential donors of resistant gene(s)/the source of white rust disease in B. juncea using 30 genotypes, including locally adapted accessions and advanced breeding lines. Out of 30 genotypes, ten lines viz. Bio-YSR, CAULC-1, CAULC-2, CAULC-3, CAULC-4, CAURM-2, CAULR-7, CAURM-4, CAURM 4-1, and CAURM 4-2 exhibited a lower PDI value (lesser than mean 10.83) with a superior agronomic performance related with the disease. The evaluation of these ten genotypes for the presence of the BjuWRR1 gene using a gene-based marker depicted the presence of the functional allele of the BjuWRR1 gene in the five genotypes viz., Bio-YSR, CAULC-1, CAULC-3, CAURM 4-1 and CAURM 4-2. When compared with the sequenced amplicon of these genotypes, it is found to be identical with that of an east European Brassica juncea line, Donskaja-IV, the completely resistant genotype against various isolates of Albugo candida. The findings from the present study suggested that besides Bio-YSR, the local lines of Manipur CAULC-1 (Local Yella of Potshangbam) and CAULC-3 (Local Yella of Kakching Lamjao) can be used as the potential white rust resistance sources/donors in disease resistance breeding programmes for the development of elite B. juncea cultivars in the future. In addition to the local lines, two improved advanced lines, viz. CAURM 4-1 and CAURM 4-2, obtained from a hybridization programme, may be further evaluated for releasing resistant varieties against white rust.

Characterization and Mapping of retr04, retr05 and retr06 Broad-Spectrum Resistances to Turnip Mosaic Virus in Brassica juncea, and the Development of Robust Methods for Utilizing Recalcitrant Genotyping Data.

Turnip mosaic virus (TuMV) induces disease in susceptible hosts, notably impacting cultivation of important crop species of the Brassica genus. Few effective plant viral disease management strategies exist with the majority of current approaches aiming to mitigate the virus indirectly through control of aphid vector species. Multiple sources of genetic resistance to TuMV have been identified previously, although the majority are strain-specific and have not been exploited commercially. Here, two Brassica juncea lines (TWBJ14 and TWBJ20) with resistance against important TuMV isolates (UK 1, vVIR24, CDN 1, and GBR 6) representing the most prevalent pathotypes of TuMV (1, 3, 4, and 4, respectively) and known to overcome other sources of resistance, have been identified and characterized. Genetic inheritance of both resistances was determined to be based on a recessive two-gene model. Using both single nucleotide polymorphism (SNP) array and genotyping by sequencing (GBS) methods, quantitative trait loci (QTL) analyses were performed using first backcross (BC1) genetic mapping populations segregating for TuMV resistance. Pairs of statistically significant TuMV resistance-associated QTLs with additive interactive effects were identified on chromosomes A03 and A06 for both TWBJ14 and TWBJ20 material. Complementation testing between these B. juncea lines indicated that one resistance-linked locus was shared. Following established resistance gene nomenclature for recessive TuMV resistance genes, these new resistance-associated loci have been termed retr04 (chromosome A06, TWBJ14, and TWBJ20), retr05 (A03, TWBJ14), and retr06 (A03, TWBJ20). Genotyping by sequencing data investigated in parallel to robust SNP array data was highly suboptimal, with informative data not established for key BC1 parental samples. This necessitated careful consideration and the development of new methods for processing compromised data. Using reductive screening of potential markers according to allelic variation and the recombination observed across BC1 samples genotyped, compromised GBS data was rendered functional with near-equivalent QTL outputs to the SNP array data. The reductive screening strategy employed here offers an alternative to methods relying upon imputation or artificial correction of genotypic data and may prove effective for similar biparental QTL mapping studies.

Pyramiding of γ-TMT and gly I transgenes in Brassica juncea enhances salinity and drought stress tolerance.

We previously generated Brassica juncea lines overexpressing either glyoxalase I (gly I) or γ-tocopherol methyltransferase (γ-TMT) involved in the glyoxalase system and tocopherol biosynthesis, respectively. These transgenic plants showed tolerance to multiple abiotic stresses. As tolerance is a complex trait that can be improved by pyramiding of several characteristics in a single genotype, we generated in this study B. juncea plants coexpressing gly I and γ-TMT by crossing the previously generated stable transgenic lines. The performance of the newly generated B. juncea lines coexpressing gly I and γ-TMT was compared with that of wild-type and the single transgenic lines under non-stressed and NaCl and mannitol stress conditions. Our results show a more robust antioxidant response of B. juncea plants coexpressing gly I and γ-TMT compared to the other lines in terms of higher chlorophyll retention, relative water content, antioxidant enzyme and proline levels, and photosynthetic efficiency and lower oxidative damage. The differences in response to the stress of the different lines were reflected in their yield parameters. Overall, we demonstrate that the pyramiding of multiple genes involved in antioxidant pathways could be a viable and useful approach for achieving higher abiotic stress tolerance in crop plants.

Deployment of Brassica carinata A. Braun Derived Brassica juncea (L.) Czern. Lines for Improving Heterosis and Water Use Efficiency Under Water Deficit Stress Conditions.

Among Brassica species, Ethiopian mustard (Brassica carinata A. Braun) is known to tolerate most abiotic stresses, including drought. Drought caused by low and erratic rainfall in semi-arid regions consistently challenges rapeseed mustard productivity. Development of B. carinata-derived lines (CDLs) in Brassica juncea (L.) Czern. nuclear background, carrying genomic segments from B. carinata, are expected to tolerate moisture deficit stress conditions. The present study was, thus, aimed to establish the phenomenon “heterosis” for drought tolerance and water use efficiency by evaluating 105 hybrids developed from intermating 15 CDLs in half diallel fashion. Data on 17 seed yield and yield contributing traits were recorded under two different environments, viz., irrigated and rainfed conditions. Traits under study were found to be governed by both additive and non-additive types of gene action. Average degree of dominance was higher (>2) for yield and yield contributing traits, viz., secondary branches/plant, point to first siliqua on main shoot, total siliquae/plant, 1,000-seed weight, seed yield/plant, biological yield, harvest index, and seed yield/hectare under rainfed conditions, clearly indicating that higher productivity under drought conditions can be realised through the development of hybrids. Out of 15, highly significant general combining ability (GCA) effects for seven CDLs were observed under rainfed condition. Furthermore, nine and six hybrids expressed highly significant specific combining ability (SCA) effects and > 50% heterobeltiosis for yield contributing traits under rainfed and irrigated conditions, respectively. Water use efficiency (WUE) of parental CDLs and hybrids varied from 2.05 to 2.57 kg m–3 under rainfed, while 1.10 to 1.28 kg m–3 under irrigated conditions. Hybrids expressed higher WUE than parental lines under both water regimes. Furthermore, selection indices such as drought tolerance index (DTI) and mean relative performance (MRP) were identified to be efficient in the selection of productive CDLs and hybrids under drought conditions. Nine hybrids, identified as highly productive in the present study, can further be exploited for improving the yield of Indian mustard in drought-prone areas. Usefulness of interspecific hybridisation in the development of B. carinata-derived B. juncea lines for improving heterosis and WUE is, thus, well demonstrated through the present study.

Candidate Rlm6 resistance genes against Leptosphaeria. maculans identified through a genome-wide association study in Brassica juncea (L.) Czern.

One hundred and sixty-seven B. juncea varieties were genotyped on the 90K Brassica assay (42,914 SNPs), which led to the identification of sixteen candidate genes for Rlm6. Brassica species are at high risk of severe crop loss due to pathogens, especially Leptosphaeria maculans (the causal agent of blackleg). Brassica juncea (L.) Czern is an important germplasm resource for canola improvement, due to its good agronomic traits, such as heat and drought tolerance and high blackleg resistance. The present study is the first using genome-wide association studies to identify candidate genes for blackleg resistance in B. juncea based on genome-wide SNPs obtained from the Illumina Infinium 90K Brassica SNP array. The verification of Rlm6 in B. juncea was performed through a cotyledon infection test. Genotyping 42,914 single nucleotide polymorphisms (SNPs) in a panel of 167 B. juncea lines revealed a total of seven SNPs significantly associated with Rlm6 on chromosomes A07 and B04 in B. juncea. Furthermore, 16 candidate Rlm6 genes were found in these regions, defined as nucleotide binding site leucine-rich-repeat(NLR), leucine-rich repeat RLK (LRR-RLK) and LRR-RLP genes. This study will give insights into the blackleg resistance in B. juncea and facilitate identification of functional blackleg resistance genes which can be used in Brassica breeding.

QTL identification for nine seed-related traits in Brassica juncea using a multiparent advanced generation intercross (MAGIC) population

Agronomic traits are usually determined by multiple quantitative trait loci (QTLs) that can have pleiotropic effects. A multiparent advanced generation intercross (MAGIC) population is well suited for genetically analysing the effects of multiple QTLs on the traits of interest because it contains more QTL alleles than a biparental population and can overcome the problem of confounding the population structure of the natural germplasm population. We previously developed the B. juncea MAGIC population, derived from eight B. juncea lines with great diversity in agronomic and quality traits. In this study, we show that the B. juncea MAGIC population is also effective for the evaluation of multiple QTLs for complex agronomic traits in B. juncea. A total of twenty-two QTLs for nine seed-related traits were identified, including one QTL for each oil content, seed number per silique and thousand-seed weight; two QTLs for each acid detergent lignin and neutral detergent fibre; three QTLs for each acid detergent fibre and protein content; four QTLs for the seed maturity time; and five QTLs for the white index. Some of these QTLs overlapped. These results should be helpful for further fine mapping, gene cloning, plant breeding and marker-assisted selection (MAS) in B. juncea.

Investigating genetic relationship of Brassica juncea with B. nigra via virtual allopolyploidy and hexaploidy strategy.

The online version contains supplementary material available at 10.1007/s11032-020-01197-7.

Haploid Bio-Induction in Plant through Mock Sexual Reproduction.

SummaryHaploidization is invaluable for basic genetic research and crop breeding. The haploid bio-induction principle is an important topic that remains largely unexplored. In this study, both CenH3 RNAi and in vitro inhibition were used to simulate and induce haploids in allopolyploid crop. Notably, in vitro CenH3 inhibition showed that the results were much the same to that of RNAi in phenotype, chromosome behavior, microspore production, and haploid induction. Cytological analyses of RNAi and inhibitor-treated progenies revealed elimination of chromosomes, defective microspores with empty nuclei, thereby giving rise to pseudo male gametes, and haploid parthenogenesis induction. We found distinct defective empty microspores that were positively correlated with the decrease of CenH3 during RNAi manipulation. Investigation through both in vivo and in vitro studies revealed that haploidization was induced through the pseudo male gamete-mediated mock sexual reproduction. The present results provide insights for the haploid parthenogenesis induction process.

Advancement in Sclerotinia rot management in Indian mustard (Brassica juncea L.)

Sclerotinia sclerotiorum is one of the important a fungal plant pathogens affecting all Brassica oilseed speciesgrown in India. Management of the disease by crop rotation has limited success due to the pathogen’s wide host range and long-term survival as resting bodies (sclerotia) in the soil. Nine B. juncea lines showing low levels of disease have been identified. Two of these lines were crossed with high yielding B. juncea varieties and further work on mapping quantitative trait loci (QTL) conferring resistance to Sclerotinia using populations of recombinant inbred lines (RIL) derived from the two crosses is under progress.

A Mapped Locus on LG A6 of Brassica juncea Line Tumida Conferring Resistance to White Rust Contains a CNL Type R Gene.

White rust, causal agent oomycete Albugo candida, is a significant disease of the cultivated Brassica species. The Indian gene pool lines of oilseed mustard, Brassica juncea, are highly susceptible to the pathogen. Resistance to A. candida has been reported in the east European gene pool lines of mustard and mapped to LG A4 in line Heera and LG A5 in line Donskaja-IV. A new resistance-conferring locus to A. candida isolate AcB1 has been mapped to LG A6 of B. juncea line Tumida—a Chinese vegetable type mustard using an F1DH mapping population that has been developed from a Tumida × Varuna (susceptible Indian gene pool line) cross. A molecular map containing 8,303 genic and GBS markers was used to map the resistance trait to an interval of 63.0 cM—70.8 cM on LG A6. Genome assemblies of Tumida and Varuna were used to find the genes present within the flanking markers discerned by genetic mapping. The most likely candidate gene in the mapped interval is BjuA046215, a CC-NBS-LRR (CNL) type R gene that encodes a protein with all the specific subdomains of the proteins encoded by such genes. Alleles of BjuA046215 in Varuna and other lines of the Indian and the east European gene pools encode proteins that have truncated LRR domains. Analysis of the syntenic regions in some of the Brassicaceae genomes and phylogenetic analysis of CNL type R genes showed BjuA046215 to be closely related to a recently described white rust resistance-conferring R gene BjuWRR1 in B. juncea Donskaja-IV, both belonging to the CNL-D group of R genes. Related R genes in Arabidopsis thaliana confer resistance to another oomycete, Peronospora parasitica.

Genome wide association studies for yield and its component traits under terminal heat stress in Indian mustard (Brassica juncea L.)

Breeding for terminal heat stress (THS) in Brassica juncea L. Czern & Coss is recognized as an imperative objective for sustained productivity in contemporary climatic changes. A fixed diversity stock of 491 genotypes was documented for wide range of variations for seed yield under natural terminal heat stress. A set of top 20 genotypes comprising introgression lines from wild species Erucastrum cardaminoides and B. tournefortii; derived B. juncea lines using B. carinata and B. napus; land races;, commercial cultivars and breeding lines, having the lowest heat susceptibility index and the least yield reduction under heat stress, have been identified as potential heat tolerant donors. A panel of 96 genotypes was constituted from this stock on the basis of their differential response to heat susceptibility index and seed yield reduction under natural THS. The constituted panel was evaluated for validation under controlled conditions for ten seed yield-related traits. Moderate to low correlations between SY and its related traits were observed in NS and THS conditions. Double digest restriction site associated DNA sequencing of 71 genotypes identified 18,258 SNPs after filtration. Least square means of all the traits under NS and THS conditions and the best linear unbiased predictors along with identified SNPs were used for genome-wide association study. A total of 34 SNPs under NS, 24SNPs under THS and 30SNPs using BLUP values were found to be associated with all seed yield-related traits. Chromosome B05 harbored the maximum number of SNPs (nine) followed by chromosomes A07 and A09 (eight SNPs each). SNPs under NS conditions could not be associated with THS. This is the first report on the identification of 24 marker-traits associations detected for SY and its component traits under THS conditions. It may be possible to develop the molecular markers for significant SNPs after due validation. The constituted panel may also serve as a source of allelic diversity for genes controlling various economic traits. The derived introgression lines as potential heat tolerant donors indicated the possibility of using wild species to breed for abiotic stress tolerance in Indian mustard.

Rorippa indica HSPRO2 expression in transgenic Brassica juncea induces tolerance against mustard aphid Lipaphis erysimi

We have previously established that RiHSPRO2, a nematode resistance protein-like homolog from wild crucifer Rorippa indica (L.) Hiern is a potent candidate to control mustard aphid Lipaphis erysimi. The present study further exploits this protein through structure prediction, biosafety assessment and transgenic study. The RiHSPRO2 protein showed an abundance of alpha helices intervened with loops in the homology-based three-dimensional model. No allergenic moiety was found in its amino acid sequence based on homology search in Protein Data Base. The secondary structure of RiHSPRO2 was unstable at temperatures above 50 °C. In vitro pepsin digestion assay revealed the protein to be digested in pepsin supplemented Simulated Gastric Fluid (SGF) within 2 minutes. The protein is proved to be biologically safe as per the FAO/WHO guidelines. An efficient Agrobacterium-mediated Brassica juncea transformation involving direct organogenesis with mean transformation frequency of 5.06 ± 0.28% is reported. RiHSPRO2, under the influence of constitutive promoter CaMV35S, was transformed into susceptible B. juncea cv. B85. Southern hybridization confirmed stable integration of the transgene and Western blotting confirmed consistent expression of RiHSPRO2 in the genetically modified Brassica lines. The transgene segregated following Mendelian 3:1 ratio in the successive generation. Detached leaf aphid bioassay and in planta aphid bioassay in transgenic B. juncea lines revealed a reduction in aphid survivability by 45% and a decrease in aphid fecundity by 45.6%.

Expression of Colocasia esculenta tuber agglutinin in Indian mustard provides resistance against Lipaphis erysimi and the expressed protein is non-allergenic.

Transgenic Brassica juncea plants expressing Colocasia esculenta tuber agglutinin (CEA) shows the non-allergenic nature of the expressed protein leading to enhanced mortality and reduced fecundity of mustard aphid-Lipaphis erysimi. Lipaphis erysimi (common name: mustard aphid) is the most devastating sucking insect pest of Indian mustard (Brassica juncea L.). Colocasia esculenta tuber agglutinin (CEA), a GNA (Galanthus nivalis agglutinin)-related lectin has previously been reported by the present group to be effective against a wide array of hemipteran insects in artificial diet-based bioassays. In the present study, efficacy of CEA in controlling L. erysimi has been established through the development of transgenic B. juncea expressing this novel lectin. Southern hybridization of the transgenic plants confirmed stable integration of cea gene. Expression of CEA in T0, T1 and T2 transgenic plants was confirmed through western blot analysis. Level of expression of CEA in the T2 transgenic B. juncea ranged from 0.2 to 0.47% of the total soluble protein. In the in planta insect bioassays, the CEA expressing B. juncea lines exhibited enhanced insect mortality of 70-81.67%, whereas fecundity of L. erysimi was reduced by 49.35-62.11% compared to the control plants. Biosafety assessment of the transgenic B. juncea protein containing CEA was carried out by weight of evidence approach following the recommendations by FAO/WHO (Evaluation of the allergenicity of genetically modified foods: report of a joint FAO/WHO expert consultation, 22-25 Jan, Rome, http://www.fao.org/docrep/007/y0820e/y0820e00.HTM , 2001), Codex (Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome; Codex, Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome, 2003) and ICMR (Indian Council of Medical Research, guidelines for safety assessment of food derived from genetically engineered plants, http://www.icmr.nic.in/guide/Guidelines%20for%20Genetically%20Engineered%20Plants.pdf , 2008). Bioinformatics analysis, pepsin digestibility, thermal stability assay, immuno-screening and allergenicity assessment in BALB/c mice model demonstrated that the expressed CEA protein from transgenic B. juncea does not incite any allergenic response. The present study establishes CEA as an efficient insecticidal and non-allergenic protein to be utilized for controlling mustard aphid and similar hemipteran insects through the development of genetically modified plants.

Retraction: Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

[This retracts the article on p. 2019 in vol. 7, PMID: 28111587.].

Resynthesis of <i>Brassica juncea</i> for resistance to <i>Plasmodiophora brassicae</i> pathotype 3

The oilseed crop Brassica juncea carries many desirable traits; however, resistance to clubroot disease, caused by Plasmodiophora brassicae, is not available in this species. We are the first to report the clubroot resistant resynthesized B. juncea lines, developed through interspecific crosses between a clubroot resistant B. rapa ssp. rapifera and two susceptible B. nigra lines, and the stability of the resistance in self-pollinated generations. The interspecific nature of the resynthesized B. juncea plants was confirmed by using A- and B-genome specific SSR markers, and flow cytometric analysis of nuclear DNA content. Self-pollinated progeny (S1 and S2) of the resynthesized B. juncea plants were evaluated for resistance to P. brassicae pathotype 3. The S1 and S2 progenies of one of the resynthesized B. juncea lines were resistant to this pathotype. However, resistance was lost in 6 to 13% plants of the S2 progenies derived from the second resynthesized B. juncea line; this apparently resulted from the loss of the genomic region carrying resistance due to meiotic anomalies.

Enhancing food security in arid areas of Pakistan through newly developed drought tolerant and short duration mustard (Brassica juncea L.) Canola

Food security is the crucial global issue, especially in developing countries like Pakistan. Since edible oil is an essential food item, its persistent paucity in the country and huge import for meeting domestic requirements, has attained it second largest import item after petroleum products. The aim of present study is qualitative and quantitative evaluation of newly developed short duration and drought tolerant canola quality Brassica juncea lines ZBJ-06012 and ZBJ-08051 to overcome the unfavorable edible oil situation in the country. Thirteen lines were evaluated in randomized complete block design (RCBD) for seed yield, oil quality, maturity period and drought tolerance under different agro-climatic zones both in irrigated and arid areas across the Punjab province at eight locations in Micro Yield Trials during Rabi season 2012-13 and 2013-14. Presently, grown non-canola mustard varieties Khanpur Raya and Anmol Raya were used as check varieties. Brassica napus cultivars Punjab canola and Faisal canola were also included in the trials for comparison study of mustard and rapeseed genotypes. Data for all traits under observation was analyzed through Principle Component Analysis (PCA) to evaluate the best performing lines in irrigated as well as in rain fed areas. Principal Component Analysis showed first 2 PCs having Eigen value >1 explaining 76.4% and 72% of the total variation at irrigated areas and rain fed areas respectively. The mean seed yield was also compared by Least Significant Difference (LSD) test to study the significance at 5% probability level. Canola quality B. juncea lines ZBJ-06012 and ZBJ-08051 have shown good adaptability, early maturity, non-shattering, disease and drought tolerance traits with high yield potential in comparison with presently grown Brassica napus cultivars ?Punjab Canola? and ?Faisal Canola?. Due to these prominent features, these lines have a great scope for motivating farmers to grow canola quality B. juncea when compared with B. napus and non-canola B. juncea. Future challenges demand further development of high yielding, short duration and aphid tolerant mustard cultivars having high oil content and canola quality. There is a great potential of exploiting genetic variability in the existing B. juncea material to achieve the aforesaid goals by using conventional plant breeding techniques.

Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes.

Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes—SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.

Overexpression of biologically safe Rorippa indica defensin enhances aphid tolerance in Brassica juncea.

Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71-2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest-L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

Fine mapping the BjPl1 gene for purple leaf color in B2 of Brassica juncea L. through comparative mapping and whole-genome re-sequencing

Purple plants with higher anthocyanin content have attracted increasing attention in recent years due to their advantageous biological functions and nutritional value. A spontaneous mutant with purple leaves, designated 1280-1, was discovered in Brassica juncea line 1280. A previous genetic analysis indicated that the purple leaf trait in 1280-1 was controlled by a dominant gene (BjPl1). In the present study, an analysis of total anthocyanin content further indicated that the purple leaf trait was controlled by a complete dominance gene. According to a survey of 426 primers available from public resources, BjPl1 was assigned to linkage group B2 of B. juncea. In the early stage of this research, based on comparative mapping in Brassica, two simple sequence repeat (SSR) markers developed from A2 of B. rapa delimited the BjPl1 gene to a 0.7-cM genetic interval in the corresponding linkage map. According to information on the B. juncea genome released recently, the location of BjPl1 was further narrowed to a 225-kb interval (17.74–17.97 Mb). Within the target region, whole-genome re-sequencing identified two candidate regions (17.74–17.78 Mb and 17.93–17.96 Mb). Through Blast analysis of the two candidate intervals, four homologous anthocyanin biosynthetic genes were identified and localized to a 17.93–17.96 Mb interval of B2 (approximately 27 kb), which might include BjPl1. This work lays the foundation for the isolation of BjPl1 and will further improve our understanding of the molecular mechanisms of the anthocyanin metabolic pathway in Brassica.

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.