D. K. Yadava

Genome-wide analysis of drought induced gene expression changes in flax (Linum usitatissimum)

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.

Genetic and Pathogenic Variability of Indian Strains of Xanthomonas campestris pv. campestris Causing Black Rot Disease in Crucifers

Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) causing black rot of crucifers is a serious disease in India and causes >50% crop losses in favorable environmental conditions. Pathogenic variability of Xcc, X. oryzae pv. oryzae (Xoo), and X. axonopodis pv. citri (Xac) were tested on 19 cultivars of cruciferae including seven Brassica spp. viz., B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa, and Raphanus sativus for two consecutive years viz., 2007–2008 and 2008–2009 under field conditions at Indian Agricultural Research Institute, New Delhi. Xcc (22 strains) and other species of Xanthomonas (2 strains), they formed three distinct groups of pathogenic variability i.e., Group 1, 2, and 3 under 50% minimum similarity coefficient. All strains of Xcc clustered under Groupl except Xcc-C20. The strains of Xcc further clustered in 6 subgroups viz., A, B, C, D, E, and F based on diseases reaction on host. Genetic variability of 22 strains of Xcc was studied by using Rep-PCR (REP-, BOX- and ERIC-PCR) and 10 strains for hrp (hypersensitive reaction and pathogenecity) gene sequence analysis. Xcc strains comprised in cluster 1, Xac under cluster 2, while Xoo formed separate cluster 3 based on >50% similarity coefficient. Cluster 1 was further divided into 8 subgroups viz., A, B, C, D, E, F, G, and H at 75% similarity coefficient. The hrpF gene sequence analysis also showed distinctness of Xcc strains from other Xanthomonads. In this study, genetic and pathogenic variability in Indian strains of Xcc were established, which will be of immense use in the development of resistant genotypes against this bacterial pathogen.

Breeding Major Oil Crops: Present Status and Future Research Needs

Oils extracted from plants have been used predominantly as edible oil. Soybean, peanut, rapeseed mustard, sunflower, safflower, Sesamum, linseed, castor and cotton seed are predominant oil crops. Global status of nine major and minor oil crops has been discussed which includes their classification, contribution, major growing countries and objectives. Major objectives in oil crop improvement are enhancement of seed and oil yield, quality of oil according to its use, i.e. edible or industrial uses, breeding of varieties which fit in different cropping systems and breeding biotic and abiotic stress resistant/tolerant varieties. Achievements in varietal development programme of nine oil crops in India have also been discussed and future research needs to meet the increasing demand have also been highlighted. This review describes developments in use of biotechnological tools in seven edible oil crops, namely, Brassica, soybean, sunflower, groundnut, Sesamum, linseed and safflower and also highlights the prospects of using markers in genetic improvement of these crops. Molecular markers reported for genetic diversity assessment, mapping and tagging genes/QTLs for different qualitative and quantitative traits and their use in marker-assisted selection have been presented.

Cross-transferability and Polymorphic Potential of Genomic STMS Markers of Brassica Species

The present study was carried out with the objective of evaluating genomic STMS markers developed earlier in Brassica napus, B. oleracea, B. rapa and B. nigra for their use in Brassica juncea and B. carinata. Ninety-six of the 100 STMS markers used under standardized annealing temperatures and gel concentrations produced clear reproducible amplification pattern. For majority of the markers 60 °C annealing temperature and 3.5% metaphor agarose gel were found suitable. High cross-transferability of STMS markers to related Brassica species including B. carinata (91.6%) and B. juncea (87.5%) suggested the possibility of utilizing these markers for genome analysis in the species where no such markers are available. The ‘B’ genome derived markers showed lower level of transferability to the ‘A’ and ‘C’ genome Brassica species. The potential of STMS markers to detect polymorphism among Brassica species and genera was 98.9%. The level of inter-specific polymorphism was much higher than the intea-specific polymorphism. The markers capable of revealing polymorphism among Brassica species and genera would be useful in Brassica introgression breeding programme. The polymorphic markers were found efficient in establishing the expected evolutionary relationships among the six different Brassica species and two related genera. Low level of intra-specific polymorphism revealed by these markers suggested use of a large set of such markers for various applications in Brassica genetics, genomics and breeding.

Genetics and Molecular Mapping of Black Rot Resistance Locus Xca1bc on Chromosome B-7 in Ethiopian Mustard (Brassica carinata A. Braun).

Black rot caused by Xanthomonas campestris pv. campestris (Pam.) Dowson is the most destructive disease of cauliflower causing huge loss to the farmers throughout the world. Since there are limited sources of resistance to black rot in B. oleracea (C genome Brassica), exploration of A and B genomes of Brassica was planned as these were thought to be potential reservoirs of black rot resistance gene(s). In our search for new gene(s) for black rot resistance, F2 mapping population was developed in Brassica carinata (BBCC) by crossing NPC-17, a susceptible genotype with NPC-9, a resistant genotype. Out of 364 Intron length polymorphic markers and microsatellite primers used in this study, 41 distinguished the parental lines. However, resistant and susceptible bulks could be distinguished by three markers At1g70610, SSR Na14-G02 and At1g71865 which were used for genotyping of F2 mapping population. These markers were placed along the resistance gene, according to order, covering a distance of 36.30 cM. Intron length polymorphic markers At1g70610 and At1g71865 were found to be linked to black rot resistance locus (Xca1bc) at 6.2 and 12.8 cM distance, respectively. This is the first report of identification of markers linked to Xca1bc locus in Brassica carinata on B-7 linkage group. Intron length polymorphic markers provided a novel and attractive option for marker assisted selection due to high cross transferability and cost effectiveness for marker assisted alien gene introgression into cauliflower.

Evaluation of Microsatellite Markers for Genome Mapping in Indian Mustard (Brassica juncea L)

Microsatellites are robust markers for genome mapping, gene tagging and marker assisted selection. The genus Brassica, having a large and complex genome, requires such type of markers for various applications in genetics and breeding. A set of 202 microsatellite markers were used to screen two parental genotypes of Indian mustard (Brassica juncea) namely, ‘Varuna’, an indigenous cultivar and BEC144, an exotic collection from Poland, of which 36 (17.8%) were informative and usable for segregation analysis. The polymorphic markers detected heterozygosity in advanced generation recombinant inbred lines (RILs) developed earlier from the cross Varuna × BEC144 with a varying frequency that ranged from 0% to 23.5%. Normal Mendelian segregation for majority of microsatellite markers was observed. Eleven markers showed significant deviation from the expected 1:1 segregation ratio. Twelve markers were assigned to six different linkage groups of Indian mustard genome map. The level of polymorphism between the parents and the percentage of useful informative markers as observed in this study, suggested that many more markers are needed to achieve a reasonable coverage of mustard genome. This is the first report on the evaluation of microsatellite markers for genome mapping in B. juncea.

Development and validation of functional CAPS markers for the FAE genes in Brassica juncea and their use in marker-assisted selection

Low erucic acid is a major breeding target to improve the edible oil quality in Brassica juncea. The single nucleotide polymorphism (SNP) in fatty acid elongase 1 (FAE1.1 and FAE1.2) gene was exploited to expedite the breeding program. The paralogs of FAE1 gene were sequenced from low erucic acid genotype Pusa Mustard 30 and SNPs were identified through homologous alignment with sequence downloaded from NCBI GenBank. Two SNPs in FAE1.1 at position 591 and 1265 and one in FAE1.2 at 237 were found polymorphic among low and high erucic acid genotypes. These SNPs either create or change the recognition site of restriction enzymes. Transition of a single nucleotide at position 591 and 1265 in FAE1.1, and at position 237 in FAE1.2, leads to a change in the recognition site of Hpy99I, BglII and MnlI restriction enzymes, respectively. Two CAPS markers for FAE1.1 and one for FAE1.2 were developed to differentiate low and high erucic acid genotypes. The efficiency of these CAPS markers was found 100 per cent when validated in Brassica juncea, and B. nigra genotypes and used in back-cross breeding. These CAPS markers will facilitate in marker-assisted selection for improvement of oil quality in Brassica juncea.

Oil Improvement in Maize: Potential and Prospects

High-quality maize oil, having low level of saturated fatty acids, is highly suitable for human consumption. It is considered to be better than most of other edible oils due to its fatty acid composition and stability during storage and cooking. There is about 3–4 % oil content in maize kernel. However, more than 6–7 % oil is reported in high-oil corn genotypes. High-oil lines, in general, have reduced yields. Large numbers of genes/QTLs were reported to control this trait, thus, making it difficult to improve. A combination of conventional breeding methods, marker-assisted selection and transgenic approach would help in developing high-yielding genotypes with enhanced oil content in maize.

Cloning and characterization of drought stress-induced NAC transcription factors from Brassica juncea and Sinapis alba

The plant specific NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] TFs are one of the largest plant TF families that play important roles in plant development and stress tolerance. Suppression subtractive hybridization (SSH) analysis with using drought stressed plants of S. alba lead to the identification of several stress responsive ESTs. Two of them homologous to Arabidopsis NAC14 and NAC19 were selected for cloning of full length CDS and expression analysis in Brassica and related species with contrasting drought tolerance. NAC14 and NAC19 genes were cloned from drought tolerant Sinapis alba and Brassica juncea cvs. RGN73 and Varuna, and drought sensitive B. juncea cvs. RLM619, BEC144 and BioYSR. Sequencing of genomic region coding for these NACs revealed that both NAC14 and NAC19 contain 3 exons and 2 introns each. In silico analysis of protein structure led to development of 3D models of these stress responsive NAC TFs. Although both proteins have 7 strands of β sheets, the NAC14 had 5 β sheets of type A and 2 β sheets of type B, while NAC14 have all 7 β sheets of type A. These proteins also differed in helix content, β turns and g turns. This suggest their functional diversity under abiotic stresses. Real-time RT-PCR expression analysis revealed that both the genes were up-regulated under drought stress in the leaves of B. juncea genotypes Varuna and BioYSR. In addition, NAC14 was up-regulated in the leaves of RLM619, while NAC19 was up-regulated in the leaves of S. alba and BEC144 under drought stress as compared to control conditions. Interestingly, drought stress did not up-regulate these genes in RGN73. This study revealed genotypic variation in the drought regulation of NAC TFs in B. juncea and S. alba.

Validation of molecular markers linked to low glucosinolate QTLs for marker assisted selection in Indian mustard (Brassica juncea L. Czern & Coss)

Six earlier reported markers closely linked to low glucosinolate QTLs of Brassica juncea, spread across ‘A’ genome (A2, A3 and A9) were validated in a recombinant inbred line (RIL) population of a cross between Pusa Mustard-21 (low erucic acid) and EC-597325 (double low) genotypes, to utilize them in marker-assisted selection (MAS). Of them, four markers viz., GER 1 amplified alleles of 650 bp and of 950 bp, GER 5 amplified 310 bp and 350 bp, At5gAJ67 amplified 500 bp and 450 bp and Myb28 amplified alleles of size 900 bp and 920 bp in EC597325 and Pusa Mustard-21, respectively and therefore differentiated low and high glucosinolate parents. These four polymorphic markers were then used to genotype the phenotyped RIL population consisting 608 plants. Marker-trait association was tested for goodness of fit using 2 test. Of the four markers, GER1 and GER5 showed higher phenotypic variance (R2 value) compared to the others, indicating their significance in determination of glucosinolates and prospects for use in MAS for development of Indian mustard genotypes with low glucosinolates content.