Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

Active extrusion of protons and exudation of carboxylic acids in response to iron deficiency by roots of chickpea (Cicer arietinum L.)

A chickpea cultivar, K-850, acidified the nutrient solution in response to iron deficiency, with subsequent re-greening of chlorotic leaves. No recovery of chlorosis was observed when the nutrient solution was buffered at a pH 6.3. During the period of acidification induced by iron deficiency, the roots of K-850 exuded more carboxylic acids than when supplied with sufficient iron. However, the rate of extrusion of protons was much higher than the rate of exudation of carboxylic acids during the acidification period. The extrusion of protons was inhibited by the addition of vanadate at the beginning of the decrease in pH. It appeared that acidification of the solution in response to iron deficiency was mediated by a proton-pumping ATPase, located at the plasma membrane. The presence of cations in the solution was essential for the extrusion of protons under iron deficiency, but the species of cation made no significant difference to the rate of extrusion of protons from roots. Therefore, we concluded that non-specific H+/cation antiport was involved in the acidification process.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

Cobalt stress affects nitrogen metabolism,photosynthesis and antioxidant system in chickpea (Cicer arietinum L.)

Abstract

Plants of chickpea were exposed to varied levels of cobalt (Co) and sampled at the 60-day stage. Cobalt at concentration <100 µM significantly increased the number of nodules, their dry mass, leghemoglobin concentration and the activity of nitrogenase. Similarly, the activities of glutamate dehydrogenase, glutamine synthetase and glutamate synthase also exhibited an increase in the presence of Co <100 µM, in nodules and leaves, respectively. The various photosynthetic attributes in leaves and the activity of antioxidative enzymes both in nodules and leaves were inhibited by Co in a concentration-dependent manner. However, the lipid peroxidation and the content of proline exhibited a significant increase in response to Co and were at a maximum in the plants exposed to 250 µM concentration of cobalt. Since most of the parameters showed a significant increase in response to 50 µM cobalt, this concentration may be regarded as a threshold concentration.     

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.     

In vitro regeneration and propagation of chickpea (Cicer arietinum L.) from meristem tips and cotyledonary nodes

Summary Two representative cultivars ofCicer arietinum, the desi-type cv.Annigeri and the kabuli-type cv.ICCV6, were regenerated in vitro and clonally propagated from cotyledonary nodes and meristem tips. The explants were dissected from 1-wk-old seedlings aseptically germinated on WH medium. In both cultivars, all nodes cultured on B5 medium supplemented with 4.4μM 6-benzylaminopurine developed up to seven shoots per node within 3 wk. Meristem tips were much better suited for multiple shoot formation. Cultured on DKW-C-a medium supplemented with 4.4μM 6-benzylaminopurine and 0.05μM indole-3-butyric acid, 96% of the meristem tips produced up to 10 shoots per explant. A new method in improving clonal propagation was subdividing the meristem tips. Doing so, multiple shoot formation was considerably enhanced: up to 90 shoots per original explant could be obtained with cv.Annigeri, and up to 50 with cv.ICCV6. Indole-3-butyric acid proved to be the best rooting factor. From several media tested, the best root induction and development was achieved on WH medium supplemented with 2.5μ M indole-3-butyric acid: 72% rooting with cv.Annigeri and 68% rooting with cv.ICCV6. With both cultivars there were no differences in rooting capacity between shoots of nodal origin and those derived from meristem tips. The plantlets obtained were transferred into soil and kept under greenhouse conditions. The survival frequency was 28% with cv.Annigeri and 23% with cv.ICCV6. R₀ plants remained smaller than seed-grown controls and produced only a few fertile seeds. There was no difference between R₁ plants and controls in growth, development, and seed set.     

Molecular structure and chromosomal localization of major repetitive DNA families in the chickpea (Cicer arietinum L.) genome

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162–168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

The effects of salinity and sodicity upon nodulation and nitrogen fixation in chickpea (Cicer arietinum)

Production of grain legumes is severely reduced in salt-affected soils because their ability to form and maintain nitrogen-fixing nodules is impaired by both salinity and sodicity (alkalinity). Genotypes of chickpea, Cicer arietinum, with high nodulation capacity under stress were identified by field screening in a sodic soil in India and subsequently evaluated quantitatively for nitrogen fixation in a glasshouse study in a saline but neutral soil in the UK. In the field, pH 8.9 was the critical upper limit for most genotypes studied but genotypes with high nodulation outperformed all others at pH 9.0-9.2. The threshold limit of soil salinity for shoot growth was at ECe 3 dS m(-1), except for the high-nodulation selection for which it was ECe 6. Nodulation was reduced in all genotypes at salinities above 3 dS m(-1) but to a lesser extent in the high-nodulation selection, which proved inherently superior under both non-saline and stress conditions. Nitrogen fixation was also much more tolerant of salinity in this selection than in the other genotypes studied. The results show that chickpea genotypes tolerant of salt-affected soil have better nodulation and support higher rates of symbiotic nitrogen fixation than sensitive genotypes.     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

Osmotic adjustment as a mechanism of dehydration postponement in chickpea (Cicer arietinum L.) leaves

The objectives of this study were to test the existence of osmotic adjustment in a field-grown chickpea (Cicer arietinum L.) and to reproduce it in controlled conditions for a more complete study. In a first experiment, carried out in the field with the cultivar Casoar, we described two types of drought stress that a field-grown chickpea could experience during flowering in our conditions. They were characterized with soil and plant water status. Osmotic adjustment was taking place when the stress increased progressively. This evidence was obtained with the measurement of plant water potential and relative water content during a drying-rewatering cycle. In a second experiment, carried out in pots with rain shelter, with cultivars Casoar and Sombrero, we reproduced this particular type of drought stress, on the basis of soil water potential. Measurement of plant water status was based on water, osmotic, and turgor potentials, and relative water content. It showed that chickpea is able to realize osmotic adjustment during a controlled drying-rewatering cycle limited in intensity and duration. The analysis of a broad range of solutes (nitrate, sucrose, glucose, proline, malic acid and six other organic acids) gave a good explanation of the measured reduction of osmotic potential. Organic acids accounted for most of this reduction: 97% for Casoar and 96% for Sombrero. Malic acid, which represented about half of these acids, and malonic acid significantly accumulated during the drought stress. They explained 78.2% (for Casoar) and 75.8% (for Sombrero) of the reduction of osmotic potential. Cultivar Sombrero was the only one able to accumulate some sucrose.     

Isolation and characterization of a lectin from the seeds of Erythrina Edulis

A galactose-specific lectin was isolated from the seeds of Erythrina edulis. The protein was purified by affinity chromatography of the globulin fraction on an allyl-galactoside polyacrylamide gel. The hemagglutination properties, amino acid composition, A₂₈₀, MW of the protein and of its subunits, carbohydrate content, electrophoretic pattern and isoelectric point were determined. Comparison of its properties with those of other Erythrina lectins shows that the protein is a distinct member of this group of lectins.     

Response of field-grown chickpea (Cicer arietinum L.) to phosphorus fertilization for yield and nitrogen fixation

Application of phosphorus at 40, 60, 80 and 100 kg P₂O₅ ha^–1 in the presence of a uniform dressing of nitrogen (N) and potash (K₂O) each applied at 20 and 24 kg ha^–1 to chickpea (CM-88) grown in sandy loam soil in a replicated field experiment improved the nodulation response of the crop, increased its grain yield (ka ha^–1) by 18, 59, 40 and 14 percent, biomass yield (ka ha^–1) by 32, 32, 54 and 14 percent, biomass N (kg ha^–1) by 31, 48, 49, 19 percent, and biomass P (kg ha^–1) by 26, 40, 41 and 11 percent, respectively. The effect of phosphorus on the nitrogenase activity of the excised roots of chickpea was, however, inconsistent.     

Effect of an endogenous factor on arginase activity in germinating Bengal gram (Cicer arietinum) seeds

Arginase (EC 3.5.3.1) activity increased in the cotyledon, while it declined rapidly in the embryonic axis of Bengal gram ( Cicer arietinum L. cv. Desi type) seeds during germination. The decrease in enzyme activity in the embryonic axis was accompanied by changes in the properties of arginase in vitro such as decreased stability, increased heat lability and failure to bind to DEAE-cellulose. These alterations were due to the presence of a low-molecular-weight factor in the extract, which was purified and identified as a hexose derivative. During germination the concentration of the factor increased in the embryonic axis, while no detectable level of the factor was present in the cotyledons. We postulate that the factor may have a role in the regulation of arginase activity in vivo.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Coverage-based consensus calling (CbCC) of short sequence reads and comparison of CbCC results to identify SNPs in chickpea (Cicer arietinum; Fabaceae), a crop species without a reference genome

? Premise of the study: Next-generation sequencing (NGS) technologies are frequently used for resequencing and mining of single nucleotide polymorphisms (SNPs) by comparison to a reference genome. In crop species such as chickpea (Cicer arietinum) that lack a reference genome sequence, NGS-based SNP discovery is a challenge. Therefore, unlike probability-based statistical approaches for consensus calling and by comparison with a reference sequence, a coverage-based consensus calling (CbCC) approach was applied and two genotypes were compared for SNP identification. ? Methods: A CbCC approach is used in this study with four commonly used short read alignment tools (Maq, Bowtie, Novoalign, and SOAP2) and 15.7 and 22.1 million Illumina reads for chickpea genotypes ICC4958 and ICC1882, together with the chickpea trancriptome assembly (CaTA). ? Key results: A nonredundant set of 4543 SNPs was identified between two chickpea genotypes. Experimental validation of 224 randomly selected SNPs showed superiority of Maq among individual tools, as 50.0% of SNPs predicted by Maq were true SNPs. For combinations of two tools, greatest accuracy (55.7%) was reported for Maq and Bowtie, with a combination of Bowtie, Maq, and Novoalign identifying 61.5% true SNPs. SNP prediction accuracy generally increased with increasing reads depth. ? Conclusions: This study provides a benchmark comparison of tools as well as read depths for four commonly used tools for NGS SNP discovery in a crop species without a reference genome sequence. In addition, a large number of SNPs have been identified in chickpea that would be useful for molecular breeding.     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine