Sequence analysis of Maturase K (matK): A chloroplast-encoding gene in some selected pulses

The application and utilization of sequence data has been found very informative in the characterization and phylogenetic relationship of different crops species. This study aimed to use bioinformatics tools to characterize the matK gene in some  selected legumes with special reference to pigeon pea [cajanus cajan (L.)Millsp]  matK sequence as a quarry sequence. Nucleotide and amino acid sequence of matK gene of 10 legumes were retrieved from NCBI database and analysed for homology, physiochemical properties, motifs, GC content as well as phylogenetic relationships. Results showed that the nucleotide and amino acid sequence lengths of this gene among the selected legumes differs. Its nucleotide length varied between  631-1580bp, while the amino acids sequence varied between 21 and 509 residues. P. tetragonolobus matK and C. cajan matK sequences had percentage identity of 88% while V. sativa had the lowest percentage identity of 70%. G.tomentella and P. tetragonolobus matK sequence shared the same percentage similarity of 91% with C.cajan while V. sativa had the least (78%) with C.cajan. The motif predicted were tyrosine kinase phosphorylation site, N-myristoylation site, N-glycosylation site, protein kinase phosphorylation site, casein kinase II phosphorylation site and cAMP- and –cGMP dependent protein kinase phosphorylation site. However,  microbodies C-terminal targeting site was only predicted in the amino acid  sequence of matK gene of P. sativum and C.cajan. Phylogenetically, two major clades were revealed with P.sativum, V.sativa, and C. arientinum matK gene sequence in clade A and matK gene sequence of P.tetragonolobus, C. cajan, G. tomentella,  P.vulgaris, V.unguiculata, V. angularis and V. radiate in clade B. It showed that clade A diverged from the ancestry legume approximately 39MYA while legume  sequences in clade B diverged from the ancestor about 57MYA. GC content of the nucleotide sequence of matK gene of V. sativa was highest (31.37%) with the range in the selected legume varying between 7.29%-31.37%. The secondary structure of amino acids sequence of matK gene in the selected legume revealed the alpha helix (34.14%-41.27%), extended strand (11.56%-20.99%) and random coil (39.48%- 51.76%). The major domain architecture found in the amino acid  sequence were single and double types. Implicitly, though maturase K gene  sequences in the selected legumes differ in lengths physiochemical properties, GC content and motif. The result of this study revealed that C.cajan matK gene sequences is closely related to that of P. tetragonolobus but distant to V.  unguiculata as well as P. vulgaris.Keywords: Maturase K (matK) gene, bioinformatics, phylogenetics, selected legumes, breedin

Flow cytometry determination of ploidy level in winged bean [Psophocarpus tetragonolobus (L.) DC] and its response to colchicine-induced mutagenesis

Ploidy determination and mutation breeding of crop plants are inseparable twins given that mutation breeding is hinged majorly on polyploidization of crop’s chromosome number. The present research was aimed at determining the ploidy level of 20 accessions of winged bean (Psophoscarpus tetragonolobus) using known diploid standard as control and also subjecting two accessions to colchicine mutagenesis. For flow cytometric analysis, approximately 50mm2 of young fresh intact leaves of 8 weeks post-seedling emergence from the 20 accessions were used. Crude isolation of nuclei was done by chopping the leaf in 1ml of ice cold buffer from DNA kit (cysteine UV Partec GmbH Germany) using a razor blade. The crude suspension was then filtered through a 30μm pore diameter nylon mesh and stained with 4,6- diaminodino-2-phenylindole (DAPI). Fluorescence intensity of isolated nuclei was measured using a partec PAII flow cytometry equipped with an argon ion laser at 488nm.For mutation induction, 20 seeds from accessionsTPt26 and TPt154 were soaked in three different concentrations of colchicine (5,10, 15mg/l) for 24, 48, and 72 hours, while the control group was soaked in water. A plot of land measuring 10X10 meters in the University of Calabar Experimental farm was used. Four beds were made and three seeds were sown in per hole using a Randomized Complete Block Design (RCBD) in a 2X3X4 factorial experiment layout with 5 replications. At the end of the field experiment, phenological, yield and yield-related traits were collected and subjected to analysis of variance using Predictive Analytical Software (PASW) version 20. Results obtained showed that there were significant effect of colchicine on phenological traits except on germination. However, there were significant increasing effects on yield and yield-related traits, which was colchicine concentration, exposure time as well as accession-specific. Exposing winged bean seeds to 15mg/l of colchicine for 24 and 48 hour duration gave encouraging results and should be considered for further tests and exploitation.Keywords: Ploidy determination, flow cytometry, mutation breeding, colchicine, Psophoscarpus tetragonolobu