Genomic diversity and distribution of Mesorhizobium nodulating chickpea (Cicer arietinum L.) from low pH soils of Ethiopia

Chickpea is the third most important grain legume worldwide. This is due in part to its high protein content that results from its ability to acquire bioavailable nitrogen when colonized by diverse, nitrogen fixing Mesorhizobium species. However, the diversity and distribution of mesorhizobia communities may depend on their adaptation to soil conditions. Therefore, this study was initiated in order to isolate and investigate the diversity and taxonomic identities of chickpea-nodulating Mesorhizobium species from low pH soils of Ethiopia. A total of 81 rhizobia strains were isolated from chickpea nodules harvested from low pH soils throughout Ethiopia, and their genomes were sequenced and assembled. Considering a representative set of the best-sequenced 81 genomes, the average sequence depth was 30X, with estimated average genome sizes of approximately 7 Mbp. Annotation of the assembled genome predicted an average of 7,453 protein-coding genes. Concatenation of 400 universal PhyloPhlAn conserved genes present in the genomes of all 81 strains allowed detailed phylogenetic analysis, from which eight well-supported species were identified, including M.opportunistum, M.australicum, Mesorhizobium sp. LSJC280BOO, M.wenxiniae, M.amorphae, M.loti and M.plurifarium, as well as a novel species. Phylogenetic reconstructions based on the symbiosis-related (nodC and nifH) genes were different from the core genes and consistent with horizontal transfer of the symbiotic island. The two major genomic groups, M.plurifarium and M.loti, were widely distributed in almost all the sites. The geographic pattern of genomic diversity indicated there was no relationship between geographic and genetic distance (r = 0.01, p > 0.01). In conclusion, low pH soils in Ethiopia harbored a diverse group of Mesorhizobium species, several of which were not previously known to nodulate chickpea.     

Agrobacterium mediated genetic transformation of chickpea, Cicer arietinum L.

In leaf and stem explants of chickpea, wild type strains of Agrobacteria were able to induce tumors. These tumors were capable of phytohormone independent growth. A supervirulent strain A281 was found to be most effective. Thus, using an agrobacterium R1601, which carries genes conferring supervirulent phenotype along with a plant selectable marker gene (npt II), transformed calli of chickpea were selected in the presence of 100 micrograms/ml level of kanamycin. Molecular analyses of genomic DNA from transformed calli confirmed the integration of the marker gene into chickpea genome.     

Quantitative inheritance and barriers to crossability in Cicer arietinum L.

Summary Three different diallel crosses were studied in Cicer arietinum; two of size 6×6, one within each of the two botanical groups macrosperma and microsperma of the cultivated subspecies, and one of 9×9 involving lines covering most of the morphological variation of chickpea. Barriers to crossability present neither a botanical nor a geographical pattern, being probably a direct consequence of interactions between genotypes. The genetic systems of twelve quantitative characters were analysed. Full dominance in a negative sense (small values dominant) is shown by leaflet length, width and shape index, rachis length, leaflet density on the rachis and pod length. Full dominance in a positive sense is shown by seeds per pod. Overdominance (in a positive sense) is evident for pods, seeds and yield per plant. Weak reciprocal differences were manifested by pod length, and pods, seeds and yield per plant. The system controlling number of leaflets per leaf is not clear. Dominance of primitive over selected characters seems to be the rule. As far as the environmental effects have permitted the analysis, no differences in genetic systems were observed between botanical groups.     

Genetic diversity among Rhizobium strains from Cicer arietinum L.

Following bacteriological cloning and determination of their symbiotic performance, 15 representative, diverse strains of chickpea rhizobia were genetically analysed for restriction fragment length polymorphisms. Analyses of genomic DNAs showed several different groups. Almost half (7) of the strains examined were very similar and clearly represented a single species. There was a related group of four strains which could be a subspecies. There was also one distinct group of four strains which were apparently unrelated to the reference strain 3377. This latter group may constitute a separate species. Phenotypic differences should be investigated further.     

Influence of salt stress on biochemical processes in chickpea,Cicer arietinum L.

Summary Soybean plants were grown in pots with or without vesicular-arbuscular myocorrhizal (VAM) fungi in three soils of low plant-available P content, different texture and different water-holding capacities. Mineral nutrients, except P, were provided in a complete nutrient solution. The biomass of non-VAM plants was positively and fungal colonization negatively correlated with increasingly coarse soil texture. There was no correlation of soil P with host or endophyte growth. Plant growth enhancement was positively correlated with soil water content at −1.5 MPa. These observations suggest soil water status and the mycorrhizal condition interact in influencing plant growth.     

Pectin Depolymerase Activities Associated with Cell Walls from Cicer arietinum L. Epicotyl

The hydrolytic activity of the proteins extracted with 3 M LiClfrom chick-pea (Cicer arietinum) cell walls to pec-tic fractionsextracted with 50 mM trans-l,2-diaminocy-clohexane-N,N,N,N,-tetraaceticacid (CDTA) and 50 mM sodium carbonate was studied. The pecticfractions contained acidic polysaccharides with high molecularmass (higher than 5 x 10³ kDa), mainly composed of uronic acids,galac-tose, arabinose and rhamnose. The extracted proteins depo-lymerizedthe pectic polysaccharides and also a commercial preparationof polygalacturonic acid from citrus, detected by a decreasein their viscosity and a shift of their molecular mass distribution.The extract was able to depolymerize a uronic acid-rich componentin all the cases, although in different extent. Also, with regardto the CDTA-soluble pectins, a degradation of polyuronide anda shift of the molecular mass distribution of arabinogalactanwas observed. (Received March 11, 1997; Accepted September 10, 1997)     

Cytochemical characterization of two types of nuclear bodies in Cicer arietinum L.

The present study was carried out to characterize two distinct types of nuclear bodies, the nucleolus-associated bodies (NABs) and the satellites (SATs) using some specific staining, enzyme and immunogold labeling techniques in Cicer arietinum L. These bodies are of interest as the functional components of plant nucleus. DNA-specific staining and labeling with anti-DNA, a monoclonal antibody, were employed to verify the presence of DNA in NABs as well as in SATs. The enzyme-gold labeling technique was used to compare the relative amounts of RNase-gold and protease-gold labeling over the NABs. In NABs, RNase-gold labeling was relatively low compared to the protease-gold labeling. Ag-NOR staining revealed a similar content of NOR-silver proteins in both NABs and granular zone of the nucleolus. The NABs do not contain any DNA as they show negative response to DNA-specific stains and also when incubated with anti-DNA, only few gold particles are found over these structures. The SATs, on the other hand, react positively with DNA-specific stains, and high labeling is recorded with anti-DNA along with the dense chromatin masses.     

Genetics of nodulation in Chickpea (Cicer arietinum L.)

Combining ability, components of genetic variance and graphic analysis revealed that nodulation in the cultivars of Chickpea (Cicer arietinum L.) under study, was predominantly under the control of non-additive gene action although substantial additive effect was also present. The crosses giving high specific combining ability effects also manifested highly significant positive heterosis. The parents F-61, Giza and Annegiri possessed mostly dominant alleles while Phule G-5, NEC-249 and N-31 possessed mostly recessive alleles having positive effect on nodule weight.     

Colchicine induced polyploidy in chickpeas (Cicer arietinum L.)

Summary The most successful induction of tetraploidy was obtained with 2 hour treatment by 0.25% aqueous colchicine solution of 18-hour watersoaked desi chickpeas material. However, kabuli types needed only 1 hour treatment under similar conditions. Gigantism accompanied induction of polyploidy in desi as well as kabuli types but yield and fertility were greatly reduced. The meiotic abnormalities accompanying polyploidy were multivalent association of chromosomes followed by unequal disjunction, chromosome bridges, laggards etc. The percentage of stainable pollen, however, was at par between diploids and tetraploids. Gene control of percentage seed setting was observed in both levels of ploidy. A striking feature of the studies was the high seed setting percentage in 4n F ₁ material resulting from diverse crosses, viz., desi×kabuli. A probable reduction in multivalent association coupled with yield increases in segregants from the later generation of tetraploids indicates the possibility of selection for higher yield and fertility from polyploids, particularly from some hybrid material.     

Glycanases Associated with Cell Walls of Cicer arietinum L: Arabinogalactan Degradation

The proteins extracted from Cicer arietinum cell walls showedendo-glycanase activity against the water-soluble hemicellulosicpolysaccharides extracted with 4% KOH. Such endo-glycanase activitywas able to shift the molecular mass distribution of the arabinogalactancausing a decrease in its average molecular mass, as well asto release small oligosaccharides constituted exclusively ofarabinose. Exo-glycanase activities such as glucosidase andgalactosidase were also present. Key words: Arabinogalactan, cell wall, Cicer arietinum, glycanase, hemicellulose     

Narrow Genetic Variability in Cicer arietinum L. as Revealed by RFLP Analysis

A subgenomic library constructed from small Pstl restriction fragments (0.4 to 2.0 kb) yielded 83.18% low-copy clones. Using 17 random genomic and 5 heterologous probes in 65 probe-enzyme combinations, Restriction Fragment Length Polymorphism (RFLP) for nuclear DNA was studied in five desi and five kabuli type chickpea cultivars. Only two clones revealed polymorphism in the cultivars tested. No polymorphism in chickpea varieties was detected with four Random Amplified Polymorphic DNA (RAPD) markers studied. However, some degree of polymorphism between C. arietinum and its wild relative C. reticulatum was detected. The RFLP analysis of chloroplast and mitochondrial genomes showed no polymorphism.     

Genetic transformation in the grain legume Cicer arietinum L. (chickpea)

In the grain legume Cicer arietinum L. (chickpea), the seed-derived embryo axes deprived of the apical meristem were able to regenerate adventitious shoots on Murashige and Skoog (1962) medium supplemented with kinetin. This protocol was suitable for Agrobacterium-mediated gene transfer by the co-cultivation technique. Chickpea transgenic plants showed neomycin phosphotransferase II and ß-glucuronidase activities and the presence in their genome of integrated bacterial DNA.Abbreviations 6-BAP 6-benzyl-aminopurine - CaMV cauliflower mosaic virus - GUS ß-glucuronidase - IAA indole-3-acetic acid - Kn kanamycin - MU methyl umbelliferone - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase II     

Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L

Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m⁷ GTP and m⁷ GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA.     

Sensitivity to Low Temperature in Pollen Germination and Fruit-Set in Cicer arietinum L.

Cicer arietinum L. cv. G 62404 was seen to have a very low fruit-setpercentage at low temperatures and as temperatures increased,either in nature or in culture conditions, the fruit-set alsoincreased. This effect of temperature could also be seen inpercent pollen germination and pollen tube growth. M 450, amutant of G 62404, was seen to have a better fruit-set percentageat low temperature and better pollen germination. This may bedue to differences in malic acid concentration, which increasedpollen germination, and pollen tube growth under in vitro cultureconditions.     

Abscisic acid concentration,root pH and anatomy do not explain growth differences of chickpea (Cicer arietinum L.) and lupin (Lupinus angustifolius L.) on acid and alkaline soils

The ABA concentrations of leaves, roots, soils and transport fluids of chickpea and lupin plants growing in acid (pH=4.8) and alkaline (pH=8.0) soils and an acid soil with an alkaline subsoil and an alkaline soil with an acid subsoil were measured with the aim of explaining the poor growth of narrow-leafed lupins in alkaline soil. The ABA concentration in the leaves was higher in lupin than chickpea, but did not differ when the plants were grown in alkaline compared to acid soil. The ABA concentration of the roots and xylem sap of lupin did not differ significantly when grown in acid or alkaline soil. Chickpea roots and xylem sap had, however, lower ABA concentrations in acid soil. The ABA concentration in the soil solution was higher in the acid than in the alkaline soil. Roots of lupin and chickpea showed no suberization of the hypodermis or exodermis whether grown aeroponically or hydroponically and the pH of the cytoplasm did not change significantly when root cells of lupin and chickpea were exposed to external pHs of 4.8 or 8.0. The chickpea roots had greater suberization of the endodermal cells adjacent to radial xylem rays and maintained a slightly higher vacuolar pH than lupin in both acid and alkaline external media, but these small differences are insufficient to explain the reductions in lupin growth in alkaline soil.