Characterization of Ty1/copia-like retrotransposon families from pigeonpea genome.

Retrotransposons contribute significantly to the size, organization, and genetic diversity of their host genomes. To characterize novel retrotransposon families in pigeonpea and develop retrotransposon-based sequence-specific amplification polymorphic markers, in silico homology sequence search was carried out against the whole genome shotgun sequence of pigeonpea variety Asha (ICPL87119). For homology searching, 5 copia-like retro elements belonging to soybean, common bean, mungbean, chickpea, and field pea were used as query sequences. Contigs with at least 80% query coverage and >70% similarity were searched for retroelements using the long terminal repeat finder. A total of 28 copia-like retroelements were identified using this method. Multiple sequence alignment for the reverse transcriptase domain indicated conserved reverse transcriptase domains in all 28 elements compared with other reported elements. Phylogenetic analysis based on reverse transcriptase domains revealed 11 families. The copy number per family ranged from 1 (for B, J, and K family) to 8 (I). The sequence-specific amplification polymorphic marker-based insertion site profiling for one of the retrotransposon families (G) confirmed multiple insertions of this element across the pigeonpea genome. This study showed that our in silico homology search strategy was efficient for identifying and characterizing the Ty1/copia-like retrotransposon. The results of this study are useful for developing retrotransposon-based sequence-specific amplification polymorphic markers for pigeonpea crop improvement.

A protocol for high-quality genomic DNA extraction from legumes.

Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/µL with an average of 1459 ng/µL was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US$2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species.

Antagonism by imidazoline-type drugs of muscarinic and other receptors in the guinea-pig ileum.

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.