SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity.

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.

The cowpea trypsin inhibitor promoter drives expression in response to cellular maturation in Arabidopsis thaliana.

The bowman-birk type trypsin inhibitors accumulate in high concentration in legume and cereal seeds, especially during seed maturation and are considered to be involved in insect tolerance. The 5' flanking sequences of the trypsin inhibitor was isolated from cowpea genomic DNA using anchor PCR. Analysis of sequences showed presence of seed specific RY elements and also other elements associated with seed development such as abscisic acid responsive elements (ABA responsive elements; ABRE) and dehydration responsive elements (DRE). Spatial and temporal control of the promoter driven expression pattern was analyzed using gus as reporter. Expression was found to occur both in embryo and endosperm; starting from torpedo stage of embryogenesis and continuing till the stage of final maturation i.e. bent cotyledon stage. Additional expression analyses showed that the promoter actually drives expression in tissues like leaves, roots, stipules, etc., but followed a specific pattern. Comparative analysis of expression in seeds and other organs indicated that the promoter driven expression is in response to cellular maturation.

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.

Transgenic Indian mustard (Brassica juncea) expressing tomato glucanase leads to arrested growth of Alternaria brassicae.

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.).

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.

Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR.

Forty isolates of Macrophomina phaseolina, a pathogen causing charcoal dry root rot of soybean, cotton, and chickpea, were genetically characterized with universal rice primers (URP; primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of 12 URPs used in this study, 5 primers were effective in producing polymorphic fingerprint patterns from the DNA of M. phaseolina isolates. Three primers (URP-2F, URP-6R, and URP-30F) were quite informative and produced high levels of polymorphism among the isolates of M. phaseolina. Analysis of the entire fingerprint profiles using unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated M. phaseolina isolates obtained from soybean, cotton, and chickpea hosts into specific groups. In this study, we found for the first time transferability and use of PCR primers derived from plant genomes to generate host-specific fingerprint profiles of M. phaseolina, a broad host range plant pathogenic fungus. These results demonstrate that URPs are sensitive and technically simple to use for assaying genetic variability in M. phaseolina populations.

Isolation and characterisation of legumin promoter sequence from chickpea (Cicer arietinum L.).

Seed specific promoters are useful for expression of foreign genes in the seeds. We have isolated a Cicer arietinum legumin promoter from lambda EMBL genomic library and subcloned in pBluescript II KS (-) in Eco RV and Pst I site. The 2.762 kb Hind II Pst I fragment was sequenced completely by dideoxy chain termination method by creating a set of unidirectional deletions of the inserts in pAKKIII. The insert contains mainly upstream sequence (2240 bps) and only a part of structural gene (522 bps) sequence. The 522 bps of the structural gene shows approximately 80% homology with pea legumin A and this is almost the same as chickpea legumin in its sequence. The amino acid sequence derived from the part of the structural gene was similar to the chickpea 5' part of the legumin structural gene with a few variations. A 21 amino acid signal peptide was also deduced like many other legumes. Transcription start site (CAT) was located at 55 bp upstream of the initiation codon ATG. One codon downstream to ATG codon Hind III site was present. TATA box was observed at -30 position, with a consensus of CCTATAAATAACC. The consensus CATGCAAG, a part of legumin box was noticed at -110 bp position. At -295 to -265 bp upstream AGGA box like sequences were observed and a 56 bp perfect repeat was located between -913 bp and -972 bp. Strong homology with pea promoter sequence near the CAT sequence was noticed which gradually decreased towards the upstream region. Thus the cloned fragment contains a full length promoter which can be utilised for expression of foreign genes in seeds of chickpea.

Use of polymerase chain reaction in analysing recombinant cDNA clones encoding storage proteins of chickpea Cicer arietinum L.

A simple and reliable method was undertaken for the use of polymerase chain reaction in analyzing cDNA clones. Amplification was done of the inserts from positive legumin clones isolated from a cDNA library constructed from developing chickpea cotyledons in the expression vector, gt11. Amplification was made simple by using oligonucleotide primers which allowed convenient sizing, subcloning and sequencing of inserts by di-deoxy chain termination method. This simple method may provide opportunity to isolate large number of agronomically important genes from gene libraries.

Construction and screening of subgenomic library of chickpea (Cicer arietinum L.) for legumin genes and their analysis.

Legumin storage protein genes of chickpea were identified by hybridizing restricted chickpea genomic DNA transferred on Southern blot with heterologous pea legumin cDNA probes. Southern hybridization of restricted chickpea genomic DNA with pea legumin pDUB6 and pDUB8 cDNA probes indicated presence of pea legumin homologous sequences in chickpea. About 4-5 kb EcoRI fragment was hybridized with both the probes used. DNA corresponding to this size was isolated from the gel and used for construction of subgenomic library in lambda ZAPII vector. After amplification, library was screened for presence of legumin genes in chickpea. Out of clones screened (0.1 x 10(6)), three positive clones were obtained. Restriction analysis and hybridization studies showed presence of complete structural gene in each of the identified positive clones.

Isolation and subcloning of chitinase clone from chickpea genomic library.

Chickpea genomic library constructed earlier in phage lambda (EMBL-3) was screened for the presence of chitinase clone using tobacco chitinase cDNA as a probe. Positive clones obtained by primary screening of plaques (2 x 10(6)) were ascertained by secondary and tertiary screening. Presence of chitinase insert in the positive clones obtained, was further confirmed by restricting phage DNA with Sal I and then doing southern with tobacco chitinase. The insert band was eluted out and subcloned in puc 19 plasmid.

Construction of chickpea genomic library.

For construction of chickpea genomic library, DNA was isolated, purified on CsCl gradient and size fractionated into 15-20 Kb fragments after restriction with Sau 3A. These fragments were ligated to phage lambda (EMBL-3) vector and the recombinant molecules packaged in vitro into viable phage particles. The recombinant phages were obtained as phages on a P2 lysogen of E. coli (Spi- selection) and amplified to establish a permanent library. This is the first report of the construction of chickpea genomic library.

A Simple Technique of Studying Water Deficit Effects on Nitrogen Fixation in Nodules without Influencing the Whole Plant.

Cowpea (Vigna unguiculata L. Walp cv C-152) plants were grown in a system in which watering was withheld from the soil zone containing nodules, while the plants were able to maintain normal water status. The system was developed in a pot by making two soil zones, an upper and a lower separated by a gravel column between these two zones. Plants extended their roots into the lower layer of soil and were able to absorb water. The dry matter accumulation, photosynthesis rate, and leaf area development of the plant were not affected when the upper soil zone was dried, but the water potential of the nodules was lower than in the nodules in fully irrigated pots. Nitrogenase activity in the nodules obtained from plants stressed in the upper zone only was lower than in nodules obtained from fully irrigated plants. The present technique is helpful in distinguishing the direct water stress effects on nitrogen fixation compared to those mediated via photosynthate availability.