Genetic dissection of drought tolerance in chickpea (Cicer arietinum L.)

Key message

Analysis of phenotypic data for 20 drought tolerance traits in 1–7 seasons at 1–5 locations together with genetic mapping data for two mapping populations provided 9 QTL clusters of which one present on CaLG04 has a high potential to enhance drought tolerance in chickpea improvement.

Abstract

Chickpea (Cicer arietinum L.) is the second most important grain legume cultivated by resource poor farmers in the arid and semi-arid regions of the world. Drought is one of the major constraints leading up to 50 % production losses in chickpea. In order to dissect the complex nature of drought tolerance and to use genomics tools for enhancing yield of chickpea under drought conditions, two mapping populations—ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261) segregating for drought tolerance-related root traits were phenotyped for a total of 20 drought component traits in 1–7 seasons at 1–5 locations in India. Individual genetic maps comprising 241 loci and 168 loci for ICCRIL03 and ICCRIL04, respectively, and a consensus genetic map comprising 352 loci were constructed (http://cmap.icrisat.ac.in/cmap/sm/cp/varshney/). Analysis of extensive genotypic and precise phenotypic data revealed 45 robust main-effect QTLs (M-QTLs) explaining up to 58.20 % phenotypic variation and 973 epistatic QTLs (E-QTLs) explaining up to 92.19 % phenotypic variation for several target traits. Nine QTL clusters containing QTLs for several drought tolerance traits have been identified that can be targeted for molecular breeding. Among these clusters, one cluster harboring 48 % robust M-QTLs for 12 traits and explaining about 58.20 % phenotypic variation present on CaLG04 has been referred as “QTL-hotspot”. This genomic region contains seven SSR markers (ICCM0249, NCPGR127, TAA170, NCPGR21, TR11, GA24 and STMS11). Introgression of this region into elite cultivars is expected to enhance drought tolerance in chickpea.     

Immunohistochemical localization of β-glucosidases in lignin and isoflavone metabolism in Cicer arietinum L. seedlings

Coniferin specific- and isoflavone 7-glucoside specific -glucosidases have been localized in stem and root sections of chick pea (Cicer arietinum L.) seedlings by the indirect immunofluorometrical method. The coniferin specific -glucosidase has been found in the cell walls of the tracheary elements and of the endo-, epi-, and exodermis. All these tissues are known to contain either lignin or polymers, like suberin and cutin, which consist partially of phenylpropanoid elements. The localization of this -glucosidase is therefore in agreement with its postulated relationship to the phenylpropanoid metabolism. The isoflavone 7-glucoside specific -glucosidase, on the other hand, is predominantly located in the parenchymatic cortex cells, and obviously in the cytoplasm. These cells are known to contain the isoflavone formononetin, which has been shown to undergo turnover in chick pea seedlings. We therefore have good reason to assume that this -glucosidase is involved in the metabolism of the 7-glucoside of this isoflavone.Abbreviations SDS sodium dodecylsulfate - PBS physiological phosphate saline The results are part of the thesis of Gerd Burmeister, 1980, University of Münster     

Cloning of genes involved in the production of bacteriocin in Cicer–Rhizobium PR 2109a

The molecular basis of bacteriocin production by a Cicer–Rhizobium strain PR2109a was studied. The bacterial strain showed in vitro growth inhibition of non-bacteriocin producing strain of Cicer–Rhizobium PR2005b. Tn5 mutagenesis of the wild-type strain helped in the isolation of the bacteriocin-defective mutant JN365. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR1 and maintained in Escherchia coli background. Complementation analysis with the cosmid library resulted in the isolation of a cosmid clone which complemented the defective character in the mutant JN365. The size of the complementary DNA fragment was found to be 23 kb.     

Medicarpin and maackiain 3-O-glucoside-6′-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures

Summary The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6-O-malonate and maackiain 3-O-glucoside-6-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.Abbreviations MeGM medicarpin 3-O-glucoside-6-O-malonate - MaGM maackiain 3-O-glucoside-6-O-malonate - MeG medicarpin 3-O-glucoside - MaG maackiain 3-O-glucoside - FGM formononetin 7-O-glucoside-6-O-malonate - BGM biochanin A 7-O-glucoside-6-O-malonate - IFR NADPH: 2-hydroxyisoflavone oxidoreductase - PTS pterocarpan synthase - IGT UDP-glucose: isoflavone 7-O-glucosyltransferase - IMT malonyl-coA: isoflavone 7-O-glucoside-6 -O-malonyltransferase - RT retention time - sh shoulder - d duplette - m multiplette - s singulette     

The βI-galactosidase of Cicer arietinum is located in thickened cell walls such as those of collenchyma, sclerenchyma and vascular tissue

We report localisation of the chickpea βI-Gal, a member of the chickpea β-galactosidase family, which contains at least four members. After generation of specific antibodies, the distribution and cellular immunolocalisation of the protein in different organs and developmental stages of the plant was studied. βI-Gal protein is much longer than the other chickpea β-galactosidases because of the presence of a lectin-like domain in the carboxyl terminus of the protein. Western blot experiments indicated that the active βI-Gal retains this lectin-like domain for its function in the plant. The βI-Gal protein was mainly detected in cell walls of elongating organs, such as seedling epicotyls and stem internodes. An immunolocation study indicated a very good correlation between the presence of this βΙ-galactosidase and cells whose walls are thickening, not only in aged epicotyls and mature internodes in the final phase of elongation, but mostly in cells with a support function, such as collenchyma cells, xylem and phloem fibres and a layer of sclerenchyma cells surrounding the vascular cylinder (perivascular fibres). These results could suggest a function for the βI-Gal in modification of cell wall polymers, leading to thicker walls than the primary cell walls.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Microsomal isoflavone 2′- and 3′-hydroxylases from chickpea (Cicer arietinum L.) cell suspensions induced for pterocarpan phytoalexin formation

Microsomal fractions derived from suspension-cultured chickpea (Cicer arietinum L.) cells induced for phytoalexin biosynthesis catalyzed the monohydroxylation of 4′-methoxyisoflavones (biochanin A and formononetin) in the 2′- and 3′-positions. The reactions depended on NADPH and molecular oxygen. Post-microsomal supernatants or microsomes from non-induced cells are without detectable activity in the hydroxylase assay. 4′-Hydroxyisoflavones (genistein and daidzein) were not hydroxylated to any significant extent. The occurrence of these hydroxylases proceeds concomitantly with the accumulation of two pterocarpan phytoalexins, medicarpin and maackiain, by induced cell cultures. The results are discussed with regard to the biosynthetic sequences in the conversion of isoflavones to pterocarpans.     

Differential Sensitivity of Macrocarpa and Microcarpa Types of Chickpea （Cicer arietinum L.） to Water Stress： Association of Contrasting Stress Response with Oxidative Injury

Chickpea (Cicer arietinum L.) is particularly sensitive to water stress at its reproductive phase and, under conditions of water stress, will abort flowers and pods, thus reducing yield potential. There are two types of chickpea: (i) Macrocarpa (“Kabuli”), which has large, rams head‐shaped, light brown seeds; and (ii) Microcarpa (“Desi”), which has small, angular and dark‐brown seeds. Relatively speaking, “Kabuli” has been reported to be more sensitive to water stress than “Desi”. The underlying mechanisms associated with contrasting sensitivity to water stress at the metabolic level are not well understood. We hypothesized that one of the reasons for contrasting water stress sensitivity in the two types of chickpea may be a variation in oxidative injury. In the present study, plants of both types were water stressed at the reproductive stage for 14 d. As a result of the stress, the “Kabuli” type exhibited an 80% reduction in seed yield over control compared with a 64% reduction observed for the “Desi” type. The decrease in leaf water potential (Ψ_w) was faster in the “Kabuli” compared with the “Desi” type. At the end of the water stress period, Ψ_w was reduced to ?2.9 and ?3.1 MPa in the “Desi” and “Kabuli” types, respectively, without any significant difference between them. On the last day of stress, “Kabuli” experienced 20% more membrane injury than “Desi”. The chlorophyll content and photosynthetic rate were significantly greater in “Desi” compared with “Kabuli”. The malondialdehyde and H₂O₂ content were markedly higher at the end of the water stress in “Kabuli” compared with “Desi”, indicating greater oxidative stress in the former. Levels of anti‐oxidants, such as ascorbic acid and glutathione, were significantly higher in “Desi” than “Kabuli”. Superoxide dismutase and catalase activity did not differ significantly between the two types of chickpea, whereas on the 10th day, the activities of ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase were higher in “Desi”. These findings indicate that the greater stress tolerance in the “Desi” type may be ascribed to its superior ability to maintain better water status, which results in less oxidative damage. In addition, laboratory studies conducted by subjecting both types of chickpea to similar levels of polyethylene glycol‐induced water stress and to 10 μ.mol/L abscisic acid indicated a greater capacity of the “Desi” type to deal with oxidative stress than the “Kabuli” type. (Managing editor: Ping He)     

Novel phosphate solubilizing bacteria ‘Pantoea cypripedii PS1’ along with Enterobacter aerogenes PS16 and Rhizobium ciceri enhance the growth of chickpea (Cicer arietinum L.)

Phosphate solubilizing bacteria (PSB) are known to convert the insoluble forms of phosphate to soluble one and make them available for plant uptake. The present study aimed to isolate PSB from the rhizosphere of chickpea (Cicer arietinum L. cv. GPF2) and examine their effect on the growth and seed number. The isolated PSB were analyzed for phosphate solubilization, indole acetic acid and siderophore production. PSB were characterized for phenotypic and biochemical properties, BIOLOG and whole-cell fatty acid methyl ester profile and found to be closely related to Pantoea cypripedii and Enterobacter aerogenes based on 16s rRNA gene sequencing. A high increase in growth of C. arietinum was observed when innoculated with PSB in tricalcium phosphate amended soils. A higher uptake in total P (53 %) of plants was observed when inoculated with mixture of P. cypripedii and E. aerogenes along with Rhizobium ciceri as compared to respective control plants which significantly increased the seed number (98.3 %) and seed weight (46.1 %). This study demonstrated the ability of novel PSB P. cypripedii along with E. aerogenes and R. ciceri to promote chickpea growth.     

A linkage map of chickpea (Cicer arietinum L.) based on populations from Kabuli × Desi crosses: location of genes for resistance to fusarium wilt race 0

Two recombinant inbred line (RIL) populations derived from intraspecific crosses with a common parental line (JG62) were employed to develop a chickpea genetic map. Molecular markers, flower colour, double podding, seed coat thickness and resistance to fusarium wilt race 0 (FOC-0) were included in the study. Joint segregation analysis involved a total of 160 markers and 159 RILs. Ten linkage groups (LGs) were obtained that included morphological markers and 134 molecular markers (3 ISSRs, 13 STMSs and 118 RAPDs). Flower colour (B/b) and seed coat thickness (Tt/tt) appeared to be linked to STMS (GAA47). The single-/double-podding locus was located on LG9 jointly with two RAPD markers and STMS TA80. LG3 included a gene for resistance to FOC-0 (Foc0₁/foc0₁) flanked by RAPD marker OPJ20₆₀₀ and STMS marker TR59. The association of this LG with FOC-0 resistance was confirmed by QTL analysis in the CA2139 × JG62 RIL population where two genes were involved in the resistance reaction. The STMS markers enabled comparison of LGs with preceding maps.     

Poly-β-hydroxybutyrate (PHB) biosynthesis,tricarboxylic acid activity and PHB content in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) root nodule

An experiment was conducted to assess the relationship between poly-β-hydroxybutyrate (PHB) biosynthesis and tricarboxylic acid (TCA) activity in desi and kabuli chickpea (Cicer arietinum L.) genotypes. The specific activities of enzymes of PHB metabolism viz., β-ketothiolase (PHB-A), acetoacetyl coenzyme A reductase (PHB-B) and PHB synthase (PHB-C), and those of tricarboxylic acid cycle (citrate synthase (CS) and malate dehydrogenase (MDH) under symbiosis were measured in bacteroids and compared with the PHB accumulation in the nodule and the root. The significant positive correlation was observed between shoot and nodule mass and PHB-A, PHB-B, and PHB-C activities. However, nodule and shoot weights were not significantly correlated with PHB content either in the roots or nodules. The same was true for PHB levels and citrate synthase activity. MDH activity showed a significant negative correlation with nodule PHB. A marked variation and an age dependant increase in malate dehydrogenase activity were measured. A higher capacity for malate oxidation by an increased MDH is likely alter the balance between malate decarboxylation and oxidation, resulting in a higher steady-state concentration of oxaloacetate and that may favor the utilization of acetyl-CoA in the TCA cycle rather than for the synthesis of PHB.     

Definition of the novel symbiovar canariense within Mesorhizobium neociceri sp. nov., a new species of genus Mesorhizobium nodulating Cicer canariense in the “Caldera de Taburiente” National Park (La Palma,Canary Islands)

Cicer canariense is a highly promiscuous wild chickpea nodulated by Mesorhizobium strains in La Palma Island located at Canary archipelago. Four of these strains, CCANP34, CCANP35^T, CCANP38 and CCANP95 belong to a group phylogenetically close to Mesorhizobium caraganae with 100% similarity values in the 16S rRNA gene. However, the genomes of the strains CCANP35^T and M. caraganae LMG 24397^T obtained in this work showed ANIb and dDDH values of 90.02% and 44.1%, respectively. These values are lower than those currently accepted for different bacterial species showing that the Canarian strains do not belong to the species M. caraganae. The Canarian strains also differ from M. caraganae in the amounts of several fatty acids and in several phenotypic traits. Based on the obtained results the Canarian strains belong to a novel species for which we propose the name Mesorhizobium neociceri sp. nov. and whose type strain is CCANP35^T. The results of the phylogenetic analyses of nodC and nifH symbiotic genes showed that the Canarian strains represent a novel symbiovar within genus Mesorhizobium phylogenetically divergent to that encompassing M. caraganae. We propose the names canariense and caraganae for the symbiovars encompassing the strains of M. neociceri and M. caraganae, respectively.     

A linkage map of the chickpea (Cicer arietinum L.) genome based on recombinant inbred lines from a C. arietinum×C. reticulatum cross: localization of resistance genes for fusarium wilt races 4 and 5

An integrated molecular marker map of the chickpea genome was established using 130 recombinant inbred lines from a wide cross between a cultivar resistant to fusarium wilt caused by Fusarium oxysporum Schlecht. emend. Snyd. &. Hans f. sp. ciceri (Padwick) Snyd & Hans, and an accession of Cicer reticulatum (PI 489777), the wild progenitor of chickpea. A total of 354 markers were mapped on the RILs including 118 STMSs, 96 DAFs, 70 AFLPs, 37 ISSRs, 17 RAPDs, eight isozymes, three cDNAs, two SCARs and three loci that confer resistance against different races of fusarium wilt. At a LOD-score of 4.0, 303 markers cover 2077.9 cM in eight large and eight small linkage groups at an average distance of 6.8 cM between markers. Fifty one markers (14.4%) were unlinked. A clustering of markers in central regions of linkage groups was observed. Markers of the same class, except for ISSR and RAPD markers, tended to generate subclusters. Also, genes for resistance to races 4 and 5 of fusarium wilt map to the same linkage group that includes an STMS and a SCAR marker previously shown to be linked to fusarium wilt race 1, indicating a clustering of several fusarium-wilt resistance genes around this locus. Significant deviation from the expected 1 : 1 segregation ratio was observed for 136 markers (38.4%, P<0.05). Segregation was biased towards the wild progenitor in 68% of the cases. Segregation distortion was similar for all marker types except for ISSRs that showed only 28.5% aberrant segregation. The map is the most extended genetic map of chickpea currently available. It may serve as a basis for marker-assisted selection and map-based cloning of fusarium wilt resistance genes and other agronomically important genes in future. Received: 17 November 1999 / Accepted: 4 June 2000