A nematode-inducible promoter can effectively drives RNAi construct to confer Meloidogyne incognita resistance in tomato.

KEY MESSAGE: Heterologous expression of a nematode-responsive promoter in tomato successfully driven the RNAi constructs to impart root-knot nematode resistance. The root-knot nematode Meloidogyne incognita seriously afflicts the global productivity of tomatoes. Nematode management options are extremely reliant on chemical methods, however, only a handful of nematicides are commercially available. Additionally, nematodes have developed resistance-breaking phenotypes against the commercially available Mi gene-expressing tomatoes. Nematode resistance in crop plants can be enhanced using the bio-safe RNAi technology, in which plants are genetically modified to express nematode gene-specific dsRNA/siRNA molecules. However, the majority of the RNAi crops conferring nematode tolerance have used constitutive promoters, which have many limitations. In the present study, using promoter-GUS fusion, we functionally validated two nematode-inducible root-specific promoters (pAt1g74770 and pAt2g18140, identified from Arabidopsis thaliana) in the Solanum lycopersicum-M. incognita pathosystem. pAt2g18140 was found to be nematode-responsive during 10-21 days post-inoculation (dpi) and became non-responsive during the late infection stage (28 dpi). In contrast, pAt1g74770 remained nematode-responsive for a longer duration (10-28 dpi). Next, a number of transgenic lines were developed that expressed RNAi constructs (independently targeting the M. incognita integrase and splicing factor genes) driven by the pAt1g74770 promoter. M. incognita parasitic success (measured by multiplication factor ratio) in pAt1g74770:integrase and pAt1g74770:splicing factor RNAi lines were significantly reduced by 60.83-74.93% and 69.34-75.31%, respectively, compared to the control. These data were comparable with the RNAi lines having CaMV35S as the promoter. Further, a long-term RNAi effect was evident, because females extracted from transgenic lines were of deformed shape with depleted transcripts of integrase and splicing factor genes. We conclude that pAt1g74770 can be an attractive alternative to drive localized expression of RNAi constructs rather than using a constitutive promoter. The pAt1g74770-driven gene silencing system can be expanded into different plant-nematode interaction models.

The SPL transcription factor genes are potential targets for epigenetic regulation in response to drought stress in chickpea (C. arietinum L.).

BACKGROUND: Crop improvement for tolerance to various biotic and abiotic stress factors necessitates understanding the key gene regulatory mechanisms. One such mechanism of gene regulation involves changes in cytosine methylation at the gene body and flanking regulatory sequences. The present study was undertaken to identify genes which might be potential targets of drought-induced DNA methylation in chickpea. METHODS AND RESULTS: Two chickpea genotypes, which contrast for drought tolerance, were subjected to drought stress conditions and their differential response was studied by analysing different morpho-physiological traits. Utilizing the in-house, high throughput sequencing data, the SQUAMOSA promoter-binding (SBP) protein-like (SPL) transcription factor genes were identified to be differentially methylated and expressed amongst the two genotypes, in response to drought stress. The methylation status of one of these genes was examined and validated through bisulfite PCR (BS-PCR). The identified genes could be possible homologs to known epialleles and can therefore serve as potential epialleles which can be utilized for crop improvement in chickpea. CONCLUSION: The SPL TF genes are potential targets of epigenetic regulation in response to drought stress in chickpea. Since these are TFs, they might play important roles in controlling the expression of other genes, thus contributing to differential drought response of the two genotypes.

Gall-specific promoter, an alternative to the constitutive CaMV35S promoter, drives host-derived RNA interference targeting Mi-msp2 gene to confer effective nematode resistance.

One of the major obligate plant parasites causing massive economic crop losses belongs to the class of root-knot nematodes (RKNs). Targeting of major nematode parasitism genes via Host Delivered-RNAi (HD-RNAi) to confer silencing is established as one of the most effective approaches to curb nematode infection. Utilizing nematode-responsive root-specific (NRRS) promoters to design a dsRNA molecule targeting approach to hamper nematode parasitism. Here, a previously validated peroxidase gall specific promoter, pAt2g18140, from Arabidopsis was employed to express the dsRNA construct of the nematode effector gene Mi-msp2 from Meloidogyne incognita. Arabidopsis RNAi lines of CaMV35S::Mi-msp2-RNAi and pAt2g18140::Mi-msp2-RNAi were compared with control plants to assess the decrease in plant nematode infection. When subjected to infection, the maximum reductions in the numbers of galls, females and egg masses in the CaMV35S::Mi-msp2-RNAi lines were 61%, 66% and 95%, respectively, whereas for the pAt2g18140::Mi-msp2-RNAi lines, they were 63%, 68% and 100%, respectively. The reduction in transcript level ranged from 79%-82% for CaMV35S::Mi-msp2-RNAi and 72%-79% for the pAt2g18140::Mi-msp2-RNAi lines. Additionally, a reduction in female size and a subsequent reduction in next-generation fecundity demonstrate the efficacy and potential of the gall specific promoter pAt2g18140 for utilization in the development of HD-RNAi constructs against RKN, as an excellent alternative to the CaMV35S promoter.

Host-delivered RNAi-mediated silencing of the root-knot nematode (Meloidogyne incognita) effector genes, Mi-msp10 and Mi-msp23, confers resistance in Arabidopsis and impairs reproductive ability of the root-knot nematode.

MAIN CONCLUSION: Mi-msp10 and Mi-msp23 effector genes play a significant role during Meloidogyne incognita parasitism on Arabidopsis roots. The role of these genes was confirmed by demonstrating the decrease of the level of susceptibility of Arabidopsis by the  silencing of Mi-msp10 and Mi-msp23 genes using HD-RNAi technology. Root-knot nematodes (RKNs) are the most damaging pathogens severely affecting global food production. The sustainable options to minimize menace of nematode populations through economically feasible measures are limited. Thus, the development of innovative and target-specific strategies that aid in their management is imperative. RNAi technology has emerged as a sustainable and target-specific alternative to control phytonematodes. Here, we characterized two novel subventral gland and dorsal gland-specific effectors, Mi-msp10 and Mi-msp23, to determine their potential effectiveness in controlling M. incognita. Comparative developmental profiling using qRT-PCR revealed higher expression of both effectors in the adult nematode female. Furthermore, functional evaluation of Mi-msp10 and Mi-msp23 dsRNA cassettes was performed using host-delivered RNAi (HD-RNAi) in Arabidopsis. The transgenic lines were examined against M. incognita, and the phenotypic effect of HD-RNAi was evident with a 61% and 51% reduction in gall formation in the Mi-msp10 and Mi-msp23 RNAi lines, respectively. A significant drop in the nematode adult females by 59% for Mi-msp10 and 49% for Mi-msp23-RNAi lines was observed. Similarly, production in egg masses decreased significantly by 76% (Mi-msp10) and 60% (Mi-msp23) for the RNAi lines, which eventually decreased the reproductive factor by 92% and 75%, respectively. The gene expression analysis showed a significant decrease in the transcript level by up to 72% (Mi-msp10) and 66% (Mi-msp23) in M. incognita females feeding on RNAi lines, providing further evidence of effective gene silencing. Overall, our findings provide useful information and support further development of RNAi-based strategies to control M. incognita.

Conferring root-knot nematode resistance via host-delivered RNAi-mediated silencing of four Mi-msp genes in Arabidopsis.

The root-knot nematode (RKN) Meloidogyne incognita is considered one of the most damaging pests among phytonematodes. The majority of nematode oesophageal gland effector genes are indispensable in facilitating M. incognita parasitization of host plants. We report the effect of host-delivered RNAi (HD-RNAi) silencing of four selected M. incognita effector genes, namely, Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24, in Arabidopsis thaliana. Mi-msp5, Mi-msp18 and Mi-msp24, which are dorsal gland genes, were found to be maximally expressed in the adult female stage, whereas Mi-msp3, which is a sub-ventral gland gene, was maximally expressed in an earlier stage. In transgenic plants expressing dsRNA, the reduction in the number of galls on roots was 89 %, 78 %, 86 % and 89 % for the Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24 RNAi events, respectively. Moreover, gene transcript abundance was significantly reduced in RKN females feeding on dsRNA-expressing lines by up to 60 %, 84 %, 31 % and 61 % for Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24, respectively. Furthermore, the M. incognita reproduction factor was reduced up to 71-, 344-, 107- and 114-fold in Arabidopsis plants expressing Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24 dsRNA constructs, respectively. This study provides a set of potential target genes to curb nematode infestation in economically important crops via the HD-RNAi approach.

Characterization of STP4 promoter in Indian mustard Brassica juncea for use as an aphid responsive promoter.

OBJECTIVE: Brassica juncea, a major oilseed crop, suffers substantial yield losses due to infestation by mustard aphids (Lipaphis erysimi). Unavailability of resistance genes within the accessible gene pool underpins significance of the transgenic strategy in developing aphid resistance. In this study, we aimed for the identification of an aphid-responsive promoter from B. juncea, based on the available genomic resources. RESULTS: A monosaccharide transporter gene, STP4 in B. juncea was activated by aphids and sustained increased expression as the aphids colonized the plants. We cloned the upstream intergenic region of STP4 and validated its stand-alone aphid-responsive promoter activity. Further, deletion analysis identified the putative cis-elements important for the aphid responsive promoter activity. CONCLUSION: The identified STP4 promoter can potentially be used for driving high level aphid-inducible expression of transgenes in plants. Use of aphid-responsive promoter instead of constitutive promoters can potentially reduce the metabolic burden of transgene-expression on the host plant.

Identification and characterization of PEBP family genes reveal CcFT8 a probable candidate for photoperiod insensitivity in C. cajan.

Understanding the molecular mechanism underlying photoperiod sensitivity will play a crucial role in extending the cropping area of Cajanus cajan, a photoperiod sensitive major grain legume of India and Africa. In flowering plants, Flowering locus T (FT) gene is involved in the production of florigen molecule which is essential for induction of flowering, influenced largely by the duration of photoperiod. To understand the structural and regulatory nature of this gene, a genome-wide survey was carried out, revealing the presence of 13 PEBP (FT) family genes in C. cajan. Based on the gene expression profiling of 13 PEBP genes across the 30 tissues of C. cajan, CcFT6 and CcFT8 were found to be probable Flowering locus T genes responsible for the production of florigen as both of them showed expression in reproductive leaf. Expression analysis in photoperiod sensitive, MAL3 genotype revealed that CcFT6 is upregulated under SD. However, in photoperiod insensitive genotype (ICP20338) CcFT6 and CcFT8 were upregulated in SD and LD, respectively. Hence, in ICP20338 under SD, flowering induction occurs with the involvement of CcFT6 while under LD, flowering induction seems to be associated with the expression of CcFT8. CcFT6 was found to be expressed only under favourable photoperiodic condition (SD) in both MAL3 and ICP20338 and may be regulated through a photoperiod dependent pathway. The presence of additional florigen producing gene, CcFT8 in ICP20338 which has the ability to flower in a photoperiod independent manner under LD conditions might provide some clues on its photoperiod insensitive nature. This study will provide a detailed characterization of the genes involved in photoperiodic regulation of flowering in C. cajan.

Development of nematode resistance in Arabidopsis by HD-RNAi-mediated silencing of the effector gene Mi-msp2.

Root-knot nematodes (RKNs) are devastating parasites that infect thousands of plants. As RKN infection is facilitated by oesophageal gland effector genes, one such effector gene, Mi-msp2, was selected for a detailed characterization. Based on domain analysis, the Mi-MSP2 protein contains an ShKT domain, which is likely involved in blocking K+ channels and may help in evading the plant defence response. Expression of the Mi-msp2 gene was higher in juveniles (parasitic stage of RKNs) than in eggs and adults. Stable homozygous transgenic Arabidopsis lines expressing Mi-msp2 dsRNA were generated, and the numbers of galls, females and egg masses were reduced by 52-54%, 60-66% and 84-95%, respectively, in two independent RNAi lines compared with control plants. Furthermore, expression analysis revealed a significant reduction in Mi-msp2 mRNA abundance (up to 88%) in female nematodes feeding on transgenic plants expressing dsRNA, and northern blot analysis confirmed expression of the Mi-msp2 siRNA in the transgenic plants. Interestingly, a significant reduction in the reproduction factor was observed (nearly 40-fold). These data suggest that the Mi-msp2 gene can be used as a potential target for RKN management in crops of economic importance.

Host-mediated RNAi of a Notch-like receptor gene in Meloidogyne incognita induces nematode resistance.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47-50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40-60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes.

Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi) to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

Identification, Validation and Utilization of Novel Nematode-Responsive Root-Specific Promoters in Arabidopsis for Inducing Host-Delivered RNAi Mediated Root-Knot Nematode Resistance.

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants.

Host Delivered RNAi of Two Cuticle Collagen Genes, Mi-col-1 and Lemmi-5 Hampers Structure and Fecundity in Meloidogyne incognita.

Root-knot nematodes have emerged as devastating parasites causing substantial losses to agricultural economy worldwide. Tomato is the most favored host for major species of root-knot nematodes. Control strategies like use of nematicides have proved to be harmful to the environment. Other control methods like development of resistant cultivars and crop rotation have serious limitations. This study deals with the application of host generated RNA interference toward development of resistance against root-knot nematode Meloidogyne incognita in tomato. Two cuticle collagen genes viz. Mi-col-1 and Lemmi-5 involved in the synthesis and maintenance of the cuticle in M. incognita were targeted through host generated RNA interference. Expression of both Mi-col-1 and Lemmi-5 was found to be higher in adult females followed by egg masses and J2s. Tomato var. Pusa Ruby was transformed with the RNAi constructs of these genes to develop transgenic lines expressing the target dsRNAs. 30.80-35.00% reduction in the number of adult females, 50.06-65.73% reduction in the number of egg mass per plant and 76.47-82.59% reduction in the number of eggs per egg mass were observed for the T1 events expressing Mi-col-1 dsRNA. Similarly, 34.14-38.54% reduction in the number of adult females, 62.34-66.71% reduction in number of egg mass per plant and 67.13-79.76% reduction in the number of eggs per egg mass were observed for the T1 generation expressing Lemmi-5 dsRNA. The multiplication factor of M. incognita reduced significantly in both the cases and the structure of adult females isolated from transgenic plants were heavily distorted. This study demonstrates the role of the cuticle collagen genes Mi-col-1 and Lemmi-5 in the structure and development of M. incognita cuticle inside the host and reinforces the potential of host generated RNA interference for management of plant parasitic nematodes (PPNs).

Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.).

Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Aneurysmal bone cyst of the lumbar spine.

An aneurysmal bone cyst (ABC) is a benign, locally proliferative vascular disorder of non-neoplastic osseous lesions in children and young adults. Seventy-five percent of ABCs occur before the age of 20 years. They comprise 1.4% of all primary bone tumors, and commonly occur in the long bones. Spinal ABCs are much rarer. We present to you one such rare case of ABC involving the lumbar spine which was successfully treated with surgery. The clinical pathological and radiological features are described. The treatment options available are discussed.

The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor.

The AP2/ERF family is one of the largest transcription factor gene families that are involved in various plant processes, especially in response to biotic and abiotic stresses. Complete genome sequences of one of the world's most important pulse crops chickpea (Cicer arietinum L.), has provided an important opportunity to identify and characterize genome-wide ERF genes. In this study, we identified 120 putative ERF genes from chickpea. The genomic organization of the chickpea ERF genes suggested that the gene family might have been expanded through the segmental duplications. The 120 member ERF family was classified into eleven distinct groups (I-X and VI-L). Transcriptional factor CarERF116, which is differentially expressed between drought tolerant and susceptible chickpea cultivar under terminal drought stress has been identified and functionally characterized. The CarERF116 encodes a putative protein of 241 amino acids and classified into group IX of ERF family. An in vitro CarERF116 protein-DNA binding assay demonstrated that CarERF116 protein specifically interacts with GCC box. We demonstrate that CarERF116 is capable of transactivation activity of and show that the functional transcriptional domain lies at the C-terminal region of the CarERF116. In transgenic Arabidopsis plants overexpressing CarERF116, significant up-regulation of several stress related genes were observed. These plants also exhibit resistance to osmotic stress and reduced sensitivity to ABA during seed germination. Based on these findings, we conclude that CarERF116 is an abiotic stress responsive gene, which plays an important role in stress tolerance. In addition, the present study leads to genome-wide identification and evolutionary analyses of chickpea ERF gene family, which will facilitate further research on this important group of genes and provides valuable resources for comparative genomics among the grain legumes.

Glycolytic enzyme activities and gene expression in Cicer arietinum exposed to water-deficit stress.

The specific activities and transcript levels of glycolytic enzymes were examined in shoots of chickpea (Cicer arietinum L.) cultivars, Pusa362 (drought tolerant) and SBD377 (drought sensitive), subjected to water-deficit stress 30 days after sowing. Water-deficit stress resulted in decrease in relative water content, chlorophyll content, plant dry weight, and NADP/NADPH ratio and increase in NAD/NADH ratio in both the cultivars. A successive decline in the specific activities of fructose-1,6-bisphosphate aldolase (aldolase), 3-phosphoglycerate kinase (PGK), and NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and elevation in the specific activities of phosphoglycerate mutase (PGM) and triosephosphate isomerase (TPI) was observed in both the cultivars under stress as compared to their respective control plants. The specific activities of hexokinase, fructose-6-phosphate kinase (PFK), and NAD-GAPDH were least affected. The transcript levels of PGK and NADP-GAPDH decreased and that of glucose-6-phosphate isomerase (GPI), PGM, and PFK increased in response to water-deficit stress while water-deficit stress had no effect on the steady-state transcript levels of hexokinase, aldolase, TPI, and NAD-GAPDH. The results suggest that under water-deficit stress, the activities and transcript levels of most of the glycolytic enzymes are not significantly affected, except the increased activity and transcript level of PGM and decreased activities and transcript levels of PGK and NADP-GAPDH. Further, the glycolytic enzymes do not show much variation between the tolerant and sensitive cultivars under water deficit.

Molecular characterization of primary gene pool of chickpea based on ISSR markers.

Genetic diversity and relationships within and among members of the primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR markers. The study revealed moderate diversity, detecting 141 fragments, of which 79 (56%) were polymorphic. Averages were 0.125 for polymorphic information content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between wild and cultivated accessions. The clustering pattern did not strictly follow the grouping of accessions by geographic origin but was in good agreement with the pedigree data and the seed type. The study demonstrates that ISSRs provide promising marker tools in revealing genetic diversity and relationships in chickpea and can contribute to efficient identification, conservation, and utilization of germplasm for plant improvement through conventional as well as molecular breeding approaches.

Upstream sequence of fatty acyl-CoA reductase (FAR6) of Arabidopsis thaliana drives wound-inducible and stem-specific expression.

An Arabidopsis mutant line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified from a population of Arabidopsis thaliana tagged with a promoterless ß-glucuronidase (GUS) in the T-DNA. Sequence flanking the insertion from the right border was amplified by TAIL PCR and cloned. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5' untranslated region (UTR) of the fatty acyl-CoA reductase 6 (FAR6) gene. RT-PCR analysis of the FAR6 gene revealed that the gene is expressed predominantly in stem tissue. Semi-quantitative RT-PCR showed that the expression is also induced by wounding in the epidermal layer of mature stem internodes. The transcription initiation site (TSS) was identified by 5' RACE PCR. Different 5' deletion fragments of the promoter sequences were developed and linked to the GUS reporter gene as transcriptional fusions and the expression patterns of GUS were histochemically analyzed in transgenic Arabidopsis plants. Sequences from -510 bp upstream to the transcriptional start site were sufficient to exhibit wound-inducible GUS expression in the stems. The addition of further upstream sequences (-510 to -958, -1,400 or -1,456) enhanced and extended the wound-inducible GUS expression throughout the mature stem.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.