Vibha Gupta

Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers: homoeologous relationships, diversification and evolution of the A, B and C Brassica genomes

BackgroundExtensive 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.ResultsIP 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.ConclusionIP 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.

Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration.

SummaryProtocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. ‘Pusa Kalyani’. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and β-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450–625 μ from the cut surface. Such shoots were negative for GUS activity.

The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-specific promoters, TA29 and A9. Several transformation vectors based on different promoter and marker gene combinations were developed and tested for their efficacy in generating agronomically viable male-sterile lines. Use of strong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-sterile plants at an extremely low frequency with most plants showing abnormalities in vegetative morphology, poor female fertility, low seed germination frequencies and/or distortion in segregation ratios of transgenes. Such abnormalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fragment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exhibited normal morphology, growth and seed set on backcrossing as observed for wild-type plants. Around 75% of single-copy events tested further also showed proper segregation of the marker gene/male-sterile phenotype among backcross progeny. Constructs based on the use of Spacer DNA fragments as insulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of agronomically viable male-sterile lines in crop plants and for cell/tissue ablation studies in general.

Molecular tagging of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide polymorphisms in FAE1 gene

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.

AFLP-based genetic diversity assessment amongst agronomically important natural and some newly synthesized lines of Brassica juncea

Abstract AFLP markers were employed to assess the genetic diversity amongst 21 established natural and nine synthetic varietes and lines of Brassica juncea originating from Asia, Australia, Canada, Eastern Europe and Russia. Six of the synthetics used for diversity studies have been developed recently. Twenty one EcoRI/MseI-based AFLP primer pairs generated a total of 1251 scorable fragments among the 30 genotypes studied, of which 778 bands were polymorphic with an average of 37 polymorphic bands per primer pair. On the basis of the similarity coefficients (F value), cluster analysis was performed using the UPGMA method. The 30 B. juncea lines could be grouped into three distinct clusters. All the Indian, Chinese and previously developed synthetics formed one cluster (cluster A), the recently developed synthetics formed a separate cluster (cluster B) and the lines from Australia, Canada, Eastern Europe and Russia formed the third cluster (cluster C). A majority of the lines were uniquely identified by one or more primer pairs due to the presence or absence of variety specific band(s). Four primer pairs were found to be most informative, since these uniquely identified all the genotypes assayed. These four primer pairs, could therefore be used as fingerprinting primers for varietal identification.

Mapping of yield influencing QTL in Brassica juncea: implications for breeding of a major oilseed crop of dryland areas.

Quantitative trait loci (QTL) analysis of yield influencing traits was carried out in Brassica juncea (AABB) using a doubled haploid (DH) mapping population of 123 lines derived from a cross between Varuna (a line representing the Indian gene pool) and Heera (representing the east European gene pool) to identify potentially useful alleles from both the parents. The existing AFLP based map of B. juncea was further saturated with RFLP and SSR markers which led to the identification of the linkage groups belonging to the A (B. rapa) and B (B. nigra) genome components of B. juncea. For QTL dissection, the DH lines were evaluated at three different environments and phenotyped for 12 quantitative traits. A total of 65 QTL spread over 13 linkage groups (LG) were identified from the three environments. QTL analysis showed that the A genome has contributed more than the B genome to productivity (68% of the total QTL detected) suggesting a more prominent role of the A genome towards domestication of this crop. The east European line, Heera, carried favorable alleles for 42% of the detected QTL and the remaining 58% were in the Indian gene pool line, Varuna. We observed clustering of major QTL in a few linkage groups, particularly in J7 and J10 of the A genome, with QTL of different traits having agronomically antagonistic allelic effects co-mapping to the same genetic interval. QTL analysis also identified some well-separated QTL which could be readily transferred between the two pools. Based on the QTL analysis, we propose that improvement in yield could be achieved more readily by heterosis breeding rather than by pure line breeding.

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.

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5’ and 3’ RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (α-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.

Structure-Function Analysis of Tritrypticin, an Antibacterial Peptide of Innate Immune Origin

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity againstEscherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and β-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a β-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.

QTL analysis reveals context-dependent loci for seed glucosinolate trait in the oilseed Brassica juncea: importance of recurrent selection backcross scheme for the identification of ‘true’ QTL

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 R2 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.