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'Polima' cytoplasmic male sterility (CMS) was transferred to three different genotypes of B. napus i.e. GSL-1, ISN706 and HNS-8 by repeated backcrossing and was found to be completely stable in the nuclear background of synthetic B. napus genotype, ISN706, derived from a cross B. campestris ssp. oleifera var. brown sarson × B. oleracea var. botrytis cv. 'Pusa Katki'. The BC[SUB5] and BC[SUB6] generation lines of ISN706, GSL-1 and HNS-8 with 'Polima' CMS were grown under a range of temperature and photo-period conditions to test their stability. No breakdown in sterility was observed in 'Polima' ISN706 and, therefore, this genotype can be used as a maintainer for hybrid seed production. [ABSTRACT FROM AUTHOR]

Chloroplast (cp) and mitochondrial (mt) DNA restriction profiles of a cytoplasmic male sterile (CMS) line of Brassica juncea and its maintainer line were compared and found to be markedly different. Comparison of cpDNA restriction profiles of fifty different species of genus Brassica and some allied genera showed that the cpDNA profiles of CMS lines were similar to that of B. tournefortii for twenty different restriction endonucleases. This CMS system is thus not of spontaneous origin as reported earlier, but is alloplasmic in nature. Comparison of restriction profiles of mtDNA of B. tournefortii and CMS lines revealed some differences which might either be due to changes in DNA pattern during the transfer, or, due to the cytoplasm coming from a B. tournefortii line different from the one used in this study. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea. [ABSTRACT FROM AUTHOR]

Brassica species (tribe Brassiceae) belonging to U's triangle—B. rapa (AA), B. nigra (BB), B. oleracea (CC), B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC)—originated via two polyploidization rounds: a U event producing the three allopolyploids, and a more ancient b genome-triplication event giving rise to the A-, B-, and C-genome diploid species. Molecular mapping studies, in situ hybridization, and genome sequencing of B. rapa support the genome triplication origin of tribe Brassiceae, and suggest that these three diploid species diversified from a common hexaploid ancestor. Analysis of plastid DNA has revealed two distinct lineages—Rapa/Oleracea and Nigra—that conflict with hexaploidization as a single event defining the tribe Brassiceae. We analysed an R-block region of A. thaliana present in six copies in B. juncea (AABB), three copies each on A- and B-genomes to study gene fractionation pattern and synonymous base substitution rates (Ks values). Divergence time of paralogues within the A and B genomes and homoeologues between the A and B genomes was estimated. Homoeologous R blocks of the A and B genomes exhibited high gene collinearity and a conserved gene fractionation pattern. The three progenitors of diploid Brassicas were estimated to have diverged approximately 12 mya. Divergence of B. rapa and B. nigra, calculated from plastid gene sequences, was estimated to have occurred approximately 12 mya, coinciding with the divergence of the three genomes participating in the b event. Divergence of B. juncea A and B genome homoeologues was estimated to have taken place around 7 mya. Based on divergence time estimates and the presence of distinct plastid lineages in tribe Brassiceae, it is concluded that at least two independent triplication events involving reciprocal crosses at the time of the b event have given rise to Rapa/Oleracea and Nigra lineages. [ABSTRACT FROM AUTHOR]

Background: Brassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra. Results: RNA-seq was carried out on the RNA isolated from young inflorescences containing unopened floral buds, floral axis and small leaves, using Illumina paired-end sequencing technology. Sequence assembly was carried out using the Velvet de-novo programme and the assembled contigs were organised against Chiifu gene models, available in the BRAD-CDS database. RNA-seq confirmed the presence of more than 17,000 single-copy gene models described in the BRAD database. The assembled contigs and the BRAD gene models were analyzed for the presence of SSRs and SNPs. While the number of SSRs was limited, more than 0.2 million SNPs were observed between Chiifu and the three oleiferous lines. Assays for SNPs were designed using KASPar technology and tested on a F7-RIL population derived from a Chiifu x Tetra cross. The design of the SNP assays were based on three considerations - the 50 bp flanking region of the SNPs should be strictly similar, the SNP should have a read-depth of ≥7 and no exon/intron junction should be present within the 101 bp target region. Using these criteria, a total of 640 markers (580 for genome-wide mapping and 60 for specific-region mapping) marking as many genes were tested for mapping. Out of 640 markers that were tested, 594 markers could be mapped unambiguously which included 542 markers for genome-wide mapping and 42 markers for fine mapping of the tet-o locus that is involved with the trait tetralocular ovary in the line Tetra. Conclusion: A large number of SNPs and PSVs are present in the transcriptome of B. rapa lines for genome-wide linkage mapping and specific-region fine mapping. Criteria used for SNP identification delivered markers, more than 93% of which could be successfully mapped to the F7-RIL population of Chiifu x Tetra cross. [ABSTRACT FROM AUTHOR]

White rust caused by Albugo candida (Pers.) Kuntze is a major disease of the oilseed mustard Brassica juncea. Almost all the released varieties of B. juncea in India are highly susceptible to the disease. This causes major yield losses. Hence, there is an urgent need to identify genes for resistance to white rust and transfer these to the existing commercial varieties through marker-assisted breeding. While the germplasm belonging to the Indian gene pool is highly susceptible to the disease, the east European germplasm of B. juncea is highly resistant. In the present study, we have tagged two independent loci governing resistance to A. candida race 2V in two east European lines, Heera and Donskaja-IV. Two doubled haploid populations were used; the first population was derived from a cross between Varuna (susceptible Indian type) and Heera (partially resistant east European line) and the second from a cross between TM-4 (susceptible Indian type) and Donskaja-IV (fully resistant east European line). In both the resistant lines, a single major locus was identified to confer resistance to white rust. In Heera, the resistance locus AcB1-A4.1 was mapped to linkage group A4, while in Donskaja-IV, the resistant locus AcB1-A5.1 was mapped to linkage group A5. In both the cases, closely linked flanking markers were developed based on synteny between Arabidopsis and B. juncea. These flanking markers will assist introgression of resistance-conferring loci in the susceptible varieties. [ABSTRACT FROM AUTHOR]

Fine mapping of six seed glucosinolate QTL ( J2Gsl1, J3Gsl2, J9Gsl3, J16Gsl4, J17Gsl5 and J3Gsl6) (Ramchiary et al. in Theor Appl Genet 116:77–85, ) 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. [ABSTRACT FROM AUTHOR]

• Seed-glucosinolate chemotypes of 641 Brassica juncea genotypes were analyzed. • PCA of glucosinolates data showed the distinctiveness of four populations used. • A comprehensive Vis-NIRS model developed using MPLS regression analysis. • The model has high predictability for 8 glucosinolates of B. juncea. • The Vis-NIRS model also facilitates cross-species glucosinolates quantification. The globally cultivated Brassica crops contain high deliverable concentrations of health-promoting glucosinolates. Development of a Visible-Near InfraRed Spectroscopy (Vis-NIRS) calibration to profile different glucosinolate components from 641 diverse Brassica juncea chemotypes was attempted in this study. Principal component analysis of HPLC-determined glucosinolates established the distinctiveness of four B. juncea populations used. Subsequently, modified partial least square regression based population-specific and combined Vis-NIRS models were developed, wherein the combined model exhibited higher coefficient of determination (R2; 0.81–0.97) for eight glucosinolates and higher ratio of prediction determination (RPD; 2.42–5.35) for seven glucosinolates in B. juncea populations. Furthermore, range error ratio (RER > 4) for twelve and RER > 10 for eight glucosinolates make the combined model acceptable for screening and quality control. The model also provided excellent prediction for aliphatic glucosinolates in four oilseed Brassica species. Overall, our work highlights the potential of Vis-NIR spectroscopy in estimating glucosinolate content in the economically important Brassica oilseeds. [ABSTRACT FROM AUTHOR]

Background: Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP) markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome) and A. thaliana and analyzed the arrangement of 24 (previously described) genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes. Results: IP markers were highly efficient and generated easily discernable polymorphisms on agarose gels. Comparative analysis of the segmental organization of the A and B genomes of B. juncea (present study) with the A and B genomes of B. napus and B. nigra respectively (described earlier), revealed a high degree of colinearity suggesting minimal macro-level changes after polyploidization. The ancestral block arrangements that remained unaltered during evolution and the karyotype rearrangements that originated in the Oleracea lineage after its divergence from Rapa lineage were identified. Genomic rearrangements leading to the gain or loss of one chromosome each between the A-B and A-C lineages were deciphered. Complete homoeology in terms of block organization was found between three linkage groups (LG) each for the A-B and A-C genomes. Based on the homoeology shared between the A, B and C genomes, a new nomenclature for the B genome LGs was assigned to establish uniformity in the international Brassica LG nomenclature code. Conclusion: IP markers were highly effective in generating comparative relationships between Arabidopsis and various Brassica species. Comparative genomics between the three Brassica lineages established the major rearrangements, translocations and fusions pivotal to karyotype diversification between the A, B and C genomes of Brassica species. The inter-relationships established between the Brassica lineages vis-à-vis Arabidopsis would facilitate the identification and isolation of candidate genes contributing to traits of agronomic value in crop Brassicas and the development of unified tools for Brassica genomics. [ABSTRACT FROM AUTHOR]

Seed glucosinolate content in Brassica juncea is a complex quantitative trait. A recurrent selection backcross (RSB) method with a doubled haploid (DH) generation interspersing backcross generations was used for the introgression of low glucosinolate alleles from an east European gene pool B. juncea line, Heera into an Indian gene pool variety, Varuna. Phenotypic comparisons among the DH populations derived from early to advanced backcrosses revealed a shift in the mean values for various glucosinolates with the advancement of backcrossing, indicating a change in the selective values of the alleles with change in the genetic background due to the existence of epistasis and context dependencies. QTL mapping for various seed glucosinolates from early (F1DH) and advanced generation (BC4DH) populations confirmed the presence of epistasis and context dependency. The common QTL detected in both F1DH and BC4DH changed their R 2 values from the former to the later generation. Some of the QTL detected in the F1DH became irrelevant in the BC4DH population. Further, new QTL were detected in the BC4DH population for various glucosinolates. A validation study on a population of low glucosinolate DH lines derived from all the backcross generations of the RSB breeding programme revealed that the QTL detected in BC4DH were the ‘true’ QTL. Using glucosinolate as an example, the study provides strong evidence for the importance of the RSB method for the identification of the ‘true’ QTL which would be significant for marker assisted introgression of a complex quantitative trait whose expression is influenced by epistatic interactions. [ABSTRACT FROM AUTHOR]

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes ( BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene ( BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm. [ABSTRACT FROM AUTHOR]

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high × low erucic acid F1 hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 (FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes (FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild (E1E2) and mutant (e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (<2%) and E1E1E2E2 identified individuals with highest erucic acid content (>40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica species. [ABSTRACT FROM AUTHOR]

Abstract Analysis of the glucosinolate content and composition by high-pressure liquid chromatography indicated that varieties of Brassica juncea bred and grown in India have a high glucosinolate content characterized by the presence of 2-propenyl (allyl) and 3-butenyl as the major and 4-pentenyl as the minor fractions. In contrast, the B. juncea germplasm from other countries is characterized by the presence of 2-propenyl as the major glucosinolate fraction, trace amounts of 3-butenyl and a total lack of the 4-pentenyl types. In order to transfer the low glucosinolate trait to Indian B. juncea , the inheritance of total glucosinolates was investigated using doubled haploid (DH) populations derived from F1 (DH1 ) and BC1 (BC1 DH) of a cross between ‘Varuna’ (the most widely cultivated high glucosinolate variety of India) and ‘Heera’ (a non-allyl type low glucosinolate line). A total of 752 DH1 and 1263 BC1 DH gave rise to seven and 11 low glucosinolate (containing less than 18 µmol/g seed) individuals, respectively. On the basis of the frequency of the low glucosinolate individuals, the total glucosinolate was found to be under the control of seven genes. There was presence of both allyl and non-allyl types in DH1 and BC1 DH low-glucosinolate individuals and absence of 3-butenyl glucosinolate in some of the BC1 DH low glucosinolate individuals, indicating segregation for these fractions in the population. The size of the segregating DH population proved to be crucial for precise determination of the number of genes controlling the trait. Because of the large number of genes involved, incorporation of low glucosinolate trait in Indian B. juncea should be approached through doubled haploid (DH) breeding. [ABSTRACT FROM AUTHOR]

The objective of this study was to broaden the genetic base in oleiferous Brassica juncea by resynthesis, using 10 diverse parental lines of oleiferous B. rapa and two lines of B. nigra of both Indian and exotic origin. Out of 14 crosses attempted using B. rapa as the female parent, eight were successful. Embryo rescue was necessary to obtain interspecific plants. A total of 29 fertile interspecific plants were obtained after colchicine treatment. In the S2 generation, the expression of component characters in the majority of the resynthesized plants showed a negative trend. The resynthesized B. juncea lines are being maintained through repeated selfing and selection at each generation for desirable plant types. This process will continue till the progeny lines of the desirable plants achieve uniformity. [ABSTRACT FROM AUTHOR]

Brassica carinata (BBCC), a potential oilseed crop for dry land agriculture, is sensitive to high temperatures during germination and early stages of growth, which thereby restricts the possibility of using the residual soil moisture available after the rainy season for its cultivation. To overcome this problem, a three-genome hybrid, RCBB, was synthesized using Raphanus sativus (RR) and Brassica oleracea (CC) as donor sources for the desired heat tolerance. Protoplasts of RC hybrids obtained through sexual crosses between R. sativus (female) and B. oleracea (male) were fused with protoplasts of Brassica nigra (BB) to produce RCBB somatic hybrids. The hybrid colonies regenerated with an average frequency of 7.6%. Twelve out of 36 hybrids grown to maturity were characterized for their nuclear and organelle genomes. While all the hybrids showed the presence of B. nigra chloroplasts, 10 of the hybrids showed B. nigra -specific mitochondria and two had Raphanus -specific mitochondria. The somatic hybrids could be backcrossed to B. carinata . [ABSTRACT FROM AUTHOR]