Calcium ion binding to δ- and to β-crystallins. The presence of the "EF-hand" motif in δ-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to δ- and β-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one δ-crystallin tetramer with an affinity of 4.3 × 103 M-1. Analysis of the amino acid sequence of δ-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. β-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 × 103 M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure

Constitutive and Inducible Resistance to Atherigona soccata (Diptera: Muscidae) in Sorghum bicolor

Host plant resistance is one of the important components for minimizing the losses because of sorghum shoot fly, Atherigona soccata (Diptera: Muscidae) attack. Therefore, we studied the constitutive and inducible biochemical mechanisms of resistance to A. soccata in a diverse array of sorghum genotypes to identify lines with diverse mechanisms of resistance to this insect. Fifteen sorghum genotypes with different levels of resistance to A. soccata were evaluated. Methanol extracts of 10-d old damaged and undamaged sorghum seedlings were subjected to high-performance liquid chromatography analysis. Association between peak areas of the identified and unidentified compounds with parameters measuring A. soccata resistance was determined through correlation analysis. Amounts of p-hydroxy benzaldehyde and the unidentified compounds at RTs 24.38 and 3.70 min were associated with susceptibility to A. soccata. Genotypes exhibiting resistance to A. soccata were placed in four groups, and the lines showing constitutive and/ or induced resistance to A. soccata with different combinations of biochemical factors potentially could be used for increasing the levels of resistance to A. soccata in sorghum

Transgenic approaches for improving fungal disease resistance in groundnut

Fungal diseases in groundnut are the most significant limiting factor causing more than 50% yield losses throughout the world. Genetic enhancement in groundnut through conventional breeding and chemical control has yielded only limited success. More recently, genetic transformation has led to possibility of transforming crops for increased resistance to fungal diseases. This review summarizes the advances of genetic engineering applied for improvement of groundnut disease resistance against fungal pathogens. Fungal resistant transgene of plant, bacterial or fungal origin can be introduced into groundnut for enhanced disease resistance. Progress in engineering fungal disease resistance in transgenic ground nut has been accomplished through expression of PR proteins, antifungal proteins, antimicrobial proteins, ribosome-inactivating proteins (RIP) and phytoalexins

Characterization of disease resistance gene homologues isolated from finger millet (Eleusine coracana L. Gaertn)

Resistance gene homologues were isolated from finger millet (Eleusine coracana L.) using degenerate oligonucleotide primers designed to the conserved regions of the nucleotide binding site (NBS) of previously cloned plant disease resistance genes (R-genes) using polymerase chain reaction (PCR). Of the eleven primer combinations tested, only five showed amplification of resistance gene homologues in finger millet. BLAST search of cloned finger millet DNA fragments showed strong homology to NBSLRR- type R-genes of other crop species. Of the 107 clones sequenced, 41 showed homology to known R-genes, and are denoted as EcRGHs (Eleusine coracana resistance gene homologues), while 11 showed homology to pollen signalling proteins (PSiPs), and are denoted as EcPSiPs (Eleusine coracana pollen signalling proteins). The cloned EcRGH sequences were classified into four clusters, and EcPSiPs formed two separate clusters based on sequence homology at the amino acid level. The amino acid sequences of the cloned EcRGHs showed characteristic features of non-TIR-type R-genes, which have been identified in all the monocot species studied so far. Six EcRGHs-specific primers were designed based on the sequences obtained in finger millet; reverse transcription PCR was performed on the cDNA and revealed the expression of EcRGHs in finger millet. The ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) in the NBS domains of finger millet RGHs varied from 0.3 to 0.7 for the different classes, which suggests a purifying selection, though the LRR region also needs to be considered to make predictions. This is the first report on RGHs in finger millet, which will serve as a valuable resource for finger millet improvement using molecular tools

Physico-chemical mechanisms of resistance to shoot fly, Atherigona soccata in sorghum, Sorghum bicolor

Sorghum shoot fly, Atherigona soccata is an important pest of sorghum, and host plant resistance is one of the important components for minimizing the losses due to this pest. Therefore, we evaluated a diverse array of sorghum genotypes to identify physico-chemical characteristics conferring resistance to A. soccata. Susceptibility to shoot fly was associated with high amounts of soluble sugars, fats, leaf surface wetness and seedling vigour; while leaf glossiness, plumule and leaf sheath pigmentation, trichome density and high tannin, Mg and Zn showed resistance to shoot fly. Stepwise regression indicated that Mg, Zn, soluble sugars, tannins, fats, leaf glossiness, leaf sheath and plumule pigmentation and trichome density explained 99.8% of the variation in shoot fly damage. Path coefficient analysis suggested that leaf glossiness, trichome density, Mg and fat content and plant plumule pigmentation can be used as markers traits to select for shoot fly resistance in sorghu

An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciensmediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of markerfree transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies

Better root:shoot ratio conferred enhanced harvest index in transgenic groundnut overexpressing the rd29A:DREB1A gene under intermittent drought stress in an outdoor lysimetric dry-down trial

An outdoor confined trial was conducted during the postrainy season of 2009 for physiological evaluation of induced drought tolerance in transgenic plants of groundnut variety JL 24 overexpressing a transcription factor, DREB1A driven by the stress-inducible promoter of the rd29A gene, both from Arabidopsis thaliana. Lysimetric system was used for growing the plants, where intermittent drought stress was imposed at mid-flowering and peak pod-filling stages of the crop, by subjecting plants to a cycle of drying and re-watering. The lysimetric system facilitated complete recovery of roots, thereby, facilitating studies on variations in the root:shoot ratio induced across the genotypes under controlled wellwatered (WW) and imposed drought stress (DS) conditions. Under DS the root:shoot ratio showed a significant (P <0.005) positive correlation with pod yield and harvest index (HI), reflecting clearly the better performance of two transgenic events GNRD11 and GNRD33 than the untransformed variety JL 24. The transgenic event GNRD11, in particular, showed enhanced HI along with significantly higher (P <0.05) seed yield that was 22% and 25% higher than JL 24 and the elite breeding groundnut cultivar ICGV 86031, respectively. Better HI in these transgenic events, when compared to the untransformed control, was mainly due to the effective partitioning of the accumulated biomass, more towards roots and pods while relatively less towards shoot biomass, leading to higher root:shoot ratio and better yield, also suggesting better water use efficiency in the former compared to the latter

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T1 plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt delta-endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer

Development of a molecular linkage map of pearl millet integrating DArT and SSR markers

Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F7 Recombinant Inbred Lines (RILs) from cross H 77/833-2 9 PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available

Quantitative trait locus analysis and construction of consensus genetic map for drought tolerance traits based on three recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.)

Groundnut (Arachis hypogaea L.) is an important food and cash crop grown mainly in semi-arid tropics (SAT) regions of the world where drought is the major constraint on productivity. With the aim of understanding the genetic basis and identification of quantitative trait loci (QTL) for drought tolerance, two new recombinant inbred line (RIL) mapping populations, namely ICGS 76 × CSMG 84-1 (RIL-2) and ICGS 44 × ICGS 76 (RIL-3), were used. After screening of 3,215 simple sequence repeat (SSR) markers on the parental genotypes of these populations, two new genetic maps were developed with 119 (RIL-2) and 82 (RIL-3) SSR loci. Together with these maps and the reference map with 191 SSR loci based on TAG 24 × ICGV 86031 (RIL-1), a consensus map was constructed with 293 SSR loci distributed over 20 linkage groups, spanning 2,840.8 cM. As all these three populations segregate for drought-tolerance-related traits, a comprehensive QTL analysis identified 153 main effect QTL (M-QTL) and 25 epistatic QTL (E-QTL) for drought-tolerance-related traits. Localization of these QTL on the consensus map provided 16 genomic regions that contained 125 QTL. A few key genomic regions were selected on the basis of the QTL identified in each region, and their expected role in drought adaptation is also discussed. Given that no major QTL for drought adaptation were identified, novel breeding approaches such as marker-assisted recurrent selection (MARS) and genomic selection (GS) approaches are likely to be the preferred approaches for introgression of a larger number of QTL in order to breed drought-tolerant groundnut genotypes