Water deficit is the major abiotic constraint affecting crop productivity in peanut (Arachis hypogaea L.). Water use efficiency under drought conditions is thought to be one of the most promising traits to improve and stabilize crop yields under intermittent water deficit. A transcription factor DREB1A from Arabidopsis thaliana, driven by the stress inducible promoter from the rd29A gene, was introduced in a drought-sensitive peanut cultivar JL 24 through Agrobacterium tumefaciens-mediated gene transfer. The stress inducible expression of DREB1A in these transgenic plants did not result in growth retardation or visible phenotypic alterations. T3 progeny of fourteen transgenic events were exposed to progressive soil drying in pot culture. The soil moisture threshold where their transpiration rate begins to decline relative to control well-watered (WW) plants and the number of days needed to deplete the soil water was used to rank the genotypes using the average linkage cluster analysis. Five diverse events were selected from the different clusters and further tested. All the selected transgenic events were able to maintain a transpiration rate equivalent to the WW control in soils dry enough to reduce transpiration rate in wild type JL 24. All transgenic events except one achieved higher transpiration efficiency (TE) under WW conditions and this appeared to be explained by a lower stomatal conductance. Under water limiting conditions, one of the selected transgenic events showed 40% higher TE than the untransformed control.

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.

Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR, (2015), AbstractTo facilitate gene expression studies in pearl millet (Pennisetum glaucum (L.) R. Br.), the key reference genes including ACP, ACT, TUB, CYP, EIF4A, GAPDH, MDH, PP2C, UBC and S24 were selected based on the available literature, and their expression stabilities were studied to determine their suitability for normalizing gene expression in pearl millet. Sequence information of the reference genes were obtained from the closely related species and cloned from pearl millet using homology based cloning strategy. Further, expression stabilities were validated for their accurate expression in different tissues, genotypes and abiotic stress treatments using three statistical algorithms including geNorm, NormFinder and RefFinder.Analysis showed that while the expression of EF-1α and EIF4A were most stable in different plant tissues, MDH and EIF4A were stable under different abiotic stress conditions. Amongst the different genotypes of pearl millet tested, while UBC and MDH genes exhibited most stable expression, MDH and ACP showed greater stability in all samples set. Interestingly, the widely used reference genes S24 and TUB were found to be least stable across all the tested samples.Pair-wise analysis showed that two reference genes were sufficient for proper normalization, except when analyzing the gene expression studies in all samples set. Results of this study can help in the selection of reference genes for quantitative real time PCR (qRT-PCR) normalization in pearl millet that will contribute towards more accurate and reliable quantification of transcripts in this important crop of the drylands.

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

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 NBS-LRR-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.

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Background Entomopathogens are frequent natural enemies of arthropods worldwide, and they are capable of alternative control agents against the important pests. The optimally selected botanical product can minimize their harmful effect on these entomopathogens, and it becomes essential to know the influence of combinations of botanicals and biopesticides (botanical biopesticide combination (BBC)) in comparison to their sole action. Main body Botanicals, especially neem products, are highly efficient to be combined with the entomopathogens (with some exceptions). There are many possible reasons for the synergistic action of these botanicals, attacking the immune system of the insect being one of the important ones. These botanicals when applied in combination with microbial pesticides showed maximum sublethal effects rather than complete mortality, making them the best alternatives for combating resistance development in insects. To work effectively, biological control agents must be used within a compatible program combined with botanicals. It is highly difficult for such products to compete with chemical controls in high-value crops, so where they can become a commercially viable option in organic cultivation. The increasing acreage is under organic production for high-value export crops, where pesticide residues are undesirable for the environment, and biopesticides and botanicals are good choices for crop protection. Concerning the effect of these products used in pest control, a significant reduction in dosage in relation to the individuals is noticed. Conclusion By combining the performance and safety, biopesticides and botanicals are efficacious. This knowledge should facilitate the choice of biopesticides compatible with less harmful or naturally occurring botanicals. And if these have to be incorporated into a pest management program through an organic approach, it is necessary to determine the effects of botanicals on the beneficial microbes, on the behavior of pest, the importance of application technique, and the role of application timing for these botanical biopesticide combinations.