Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers.

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2-5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.

Arabidopsis thaliana MCM2 plays role(s) in mungbean yellow mosaic India virus (MYMIV) DNA replication.

Geminiviruses are plant pathogens with single-stranded (ss) DNA genomes of about 2.7 kb in size. They replicate primarily via rolling-circle replication (RCR) with the help of a few virally encoded factors and various host-cell machineries. The virally encoded replication initiator protein (Rep) is essential for geminivirus replication. In this study, by interaction screening of an Arabidopsis thaliana cDNA library, we have identified a host factor, MCM2, that interacts with the Rep protein of the geminivirus mungbean yellow mosaic India virus (MYMIV). Using yeast two-hybrid, ß-galactosidase and co-immunoprecipitation assays, we demonstrated an interaction between MYMIV-Rep and the host factor AtMCM2. We investigated the possible role of AtMCM2 in geminiviral replication using a yeast-based geminivirus DNA replication restoration assay and observed that the AtMCM2 protein complemented the mcm2∆ mutation of S. cerevisiae. Our data suggest the involvement of AtMCM2 in the replication of MYMIV ex vivo. The role of MCM2 in replication was confirmed in planta by a transient replication assay in both wild-type and mutant Arabidopsis plants through agroinoculation. Our data provide evidence for the involvement of AtMCM2 in geminiviral DNA replication, presumably in conjunction with other host factors, and suggest its importance in MYMIV DNA replication.

NAC proteins: regulation and role in stress tolerance.

The plant-specific NAC (NAM, ATAF1,2 and CUC2) proteins constitute a major transcription factor family renowned for their roles in several developmental programs. Despite their highly conserved DNA-binding domains, their remarkable diversification across plants reflects their numerous functions. Lately, they have received much attention as regulators in various stress signaling pathways which may include interplay of phytohormones. This review summarizes the recent progress in research on NACs highlighting the proteins' potential for engineering stress tolerance against various abiotic and biotic challenges. We discuss regulatory components and targets of NAC proteins in the context of their prospective role for crop improvement strategies via biotechnological intervention.

Suramin ameliorates collagen induced arthritis.

Suramin, a polysulfonated polyaromatic symmetrical urea is known for multiple therapeutic effects including antineoplastic activity. It is known as an antagonist of ATP at P2X purinergic receptors. Suramin is also found to inhibit protein synthesis affecting both initiation and elongation of the polypeptide chain. As a growth factor blocker, it is reported to suppress experimental myocardial inflammation. Here, we describe the anti-arthritic property of suramin in the collagen induced arthritic (CIA) rat, a model of human rheumatoid arthritis (RA). Intraperitoneal (i.p) injection of suramin (10 mg/kg/day) for 3 weeks was found to reduce inflammation and repair joint destruction in CIA rats. Recovery of body weight (p<0.0001), reduction in splenic (p<0.05) and arthritic indices (p<0.0001) and reappearance of smooth synovial lining after suramin treatment to CIA rats were found to be significant. Levels of pro-inflammatory cytokines such as TNF-α, IL-1ß and IL-6 in plasma and joint extracts were reduced (p<0.0001) significantly in response to suramin treatment. Several acute phase proteins were normalized after suramin administration.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

Estimation of genetic diversity and evaluation of relatedness through molecular markers among medicinally important trees: Terminalia arjuna, T. chebula and T. bellerica.

Terminalia trees are being over-exploited because of their medicinal and economical importance leading to loss of valuable genetic resources. For sustainable utilization and conservation, assessment of genetic diversity therefore becomes imperative. We report a comprehensive first study on estimation and analysis of genetic variation through Amplified fragment length polymorphism (AFLP), inter simple sequence repeat polymorphism (ISSR) and random amplification of polymorphic DNA (RAPD) across three species of Terminalia. The study included (i) characterization of genetic diversity at interspecific level, and (ii) comparison of efficiency of the marker systems. That the three species are genetically distinct was revealed by all the three marker systems as unique DNA fingerprints were obtained. This led to identification of several species-specific amplification products. Further analysis helped in species-wise clustering. The species specific bands obtained from the present investigation can be used as diagnostic markers to identify the raw materials for herbal drug preparations for authentication purposes.

Isolation and characterization of Ty1-copia group of LTRs in genome of three species of Datura: D. innoxia, D. stramonium and D. metel.

Retrotransposons (RT) constitute a major fraction of plant genome. They are implicated in evolution and sequence organization. These elements have been proposed to have major role in evolution and variation in genome size. The sequence information of these RT regions in terms of divergence and conservation could be utilized for determining the interrelationship among various copia retrotransposons within the genome. In order to assess the diversity of Ty1-copia group of retroelements, reverse transcriptase (RT) sequence was amplified from genomes of three medicinally important Datura species: D. innoxia, D. stramonium and D. metel using the primers derived from two conserved domains of RT region. A total of twenty one independent amplicons from RT regions were cloned, sequenced and compared. The intra-family divergence at amino acid level ranged from 4 to 52 %. Though intra-family RT sequences are conserved, no two sequences are identical. Southern blot hybridization suggested that Ty1-copia-like retrotransposons are dispersed throughout the Datura genome. The results indicate a high degree of heterogeneity among the Ty1-copia group of retroelements in Datura species.

Comparative transcriptome analysis of contrasting foxtail millet cultivars in response to short-term salinity stress.

Soil salinity represents a major abiotic stress that adversely affects crop growth and productivity. In this study, 21-day-old seedlings of two foxtail millet (Setaria italica) cultivars differing in salt tolerance were found to also differ in lipid peroxidation, ion balance and activity of antioxidative enzymes (glutathione reductase and catalase) under short-term salinity stress (250 mM NaCl for 1-48 h). With the aim of better understanding the molecular mechanisms underlying plant responses to short-term salinity stress, two suppression subtractive hybridization cDNA libraries (forward and reverse) were constructed of these cultivars. A total of 249 non-redundant ESTs was identified by random EST sequencing and grouped into 11 functional categories. Macroarray analysis of these clones showed that 159 (63.9%) were differentially expressed (≥ 2-fold) in response to salinity stress, with 115 (72.3%) up and 44 (27.7%) down-regulated. A data search of transcriptional profiling under salinity stress in other species revealed that 81 (51%) of the 159 differentially expressed transcripts found in foxtail millet have not been reported in previous studies. Hence, these new transcripts may represent untapped gene sources allowing specific responses to short-term salt-stress in an orphan crop known to possess a natural adaptation capacity to abiotic stress. Quantitative real-time PCR of 21 highly up-regulated (≥ 2.5-fold) transcripts showed temporal variation in expression in both cultivars under salinity. Among them, several transcription factors and signalling genes were preferentially expressed in the tolerant cultivar. These results suggest that the tolerant cultivar possesses more effective signal-perception mechanisms for metabolic adjustments in plants under harsh saline conditions. Our findings provide evidence that the unknown genes identified in this study, in addition to several known genes, may play important roles in stress tolerance mechanisms present in foxtail millet.

Physical mapping, expression analysis and polymorphism survey of resistance gene analogues on chromosome 11 of rice.

Rice is the first cereal genome with a finished sequence and a model crop that has important syntenic relationships with other cereal species. The objectives of our study were to identify resistance gene analogue (RGA) sequences from chromosome 11 of rice, understand their expression in other cereals and dicots by in silico analysis, determine their presence on other rice chromosomes, and evaluate the extent of polymorphism and actual expression in a set of rice genotypes. A total of 195 RGAs were predicted and physically localised. Of these, 91.79% expressed in rice, and 51.28% expressed in wheat, which was the highest among other cereals. Among monocots, sugarcane showed the highest (78.92%) expression, while among dicots, RGAs were maximally expressed in Arabidopsis (11.79%). Interestingly, two of the chromosome 11-specific RGAs were found to be expressing in all the organisms studied. Eighty RGAs of chromosome 11 had significant homology with chromosome 12, which was the maximum among all the rice chromosomes. Thirty-one per cent of the RGAs used in polymerase chain reaction (PCR) amplification showed polymorphism in a set of rice genotypes. Actual gene expression analysis revealed post-inoculation induction of one RGA in the rice line IRBB-4 carrying the bacterial blight resistance gene Xa-4. Our results have implications for the development of sequence-based markers and functional validation of specific RGAs in rice.

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.