Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome

A size-selected genomic library comprising 280,000 colonies and representing ≈18% of the chickpea genome, was screened for (GA)_n, (GAA)_n and (TAA)_n microsatellite-containing clones, of which 389 were sequenced. The majority (～75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%–9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613?cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P?≥?0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.     

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.     

Relationship between P and Mn in chickpea (Cicer arietinum L.)

Summary Phosphorus and Mn relationship was studied in chickpea at two stages of growth in pot culture using 0, 7.5, 15 and 30 ppm P and 0, 5, 10 and 15 ppm Mn. The dry matter yield increased with P at both stages of growth. Manganese improved the yield only in the first stage. Initial levels of Mn enhanced while higher levels had a depressing effect on tissue P. Addition of 7.5 ppm P enhanced Mn concentration at first stage and at higher levels a marked reduction in Mn content was observed at both the stages.     

Assembling a puzzle of dispersed retrotransposable sequences in the genome of chickpea (Cicer arietinum L.)

Several repetitive elements are known to be present in the genome of chickpea (Cicer arietinum L.) including satellite DNA and En/Spm transposons as well as two dispersed, highly repetitive elements, CaRep1 and CaRep2. PCR was used to prove that CaRep1, CaRep2, and previously isolated CaRep3 of C. arietinum represent different segments of a highly repetitive Ty3-gypsy-like retrotransposon (Metaviridae) designated CaRep that makes up large parts of the intercalary heterochromatin. The full sequence of this element including the LTRs and untranslated internal regions was isolated by selective amplification. The restriction pattern of CaRep was different within the annual species of the genus Cicer, suggesting its rearrangement during the evolution of the genus during the last 100 000 years. In addition to CaRep, another LTR and a non-LTR retrotransposon family were isolated, and their restriction patterns and physical localization in the chickpea genome were characterized. The LINE-like element CaLin is only of comparatively low abundance and reveals a considerable heterogeneity. The Ty1-copia-like element (Pseudoviridae) CaTy is located in the distal parts of the intercalary heterochromatin and adjacent euchromatic regions, but it is absent from the centromeric regions. These results together with earlier findings allow to depict the distribution of retroelements on chickpea chromosomes, which extensively resembles the retroelement landscape of the genome of the model legume Medicago truncatula Gaertn.     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

A gene for leaf necrosis in chickpea (Cicer arietinum L.)

Necrosis of leaves was observed in the glabrous mutant (ICC 15566) of desi chickpea (Cicer arietinum L.). It was characterized by drying of leaflet margins to drying of complete leaflets of older leaves. The oldest leaves were the most affected and the intensity of necrosis decreased toward the apical meristem. A single recessive gene, designated nec, was found to govern the necrotic characteristic. The nec locus was linked to gl (glabrous shoots) with a map distance of 16 +/- 3 cM. The loci slv (simple leaves), mlv (multipinnate leaves), nlv (narrow leaflets), hg (prostrate growth habit), P (pink corolla), and shp (round seed shape) segregated independently of nec.     

Pathotype-specific genetic factors in chickpea (Cicer arietinum L.) for quantitative resistance to ascochyta blight

Ascochyta blight in chickpea (Cicer arietinum L.) is a devastating fungal disease caused by the necrotrophic pathogen, Ascochyta rabiei (Pass.) Lab. To elucidate the genetic mechanism of pathotype-dependent blight resistance in chickpea, F₇-derived recombinant inbred lines (RILs) from the intraspecific cross of PI 359075(1) (blight susceptible) × FLIP84-92C(2) (blight resistant) were inoculated with pathotypes I and II of A. rabiei. The pattern of blight resistance in the RIL population varied depending on the pathotype of A. rabiei. Using the same RIL population, an intraspecific genetic linkage map comprising 53 sequence-tagged microsatellite site markers was constructed. A quantitative trait locus (QTL) for resistance to pathotype II of A. rabiei and two QTLs for resistance to pathotype I were identified on linkage group (LG)4A and LG2+6, respectively. A putative single gene designated as Ar19 (or Ar21d) could explain the majority of quantitative resistance to pathotype I. Ar19 (or Ar21d) appeared to be required for resistance to both pathotypes of A. rabiei, and the additional QTL on LG4A conferred resistance to pathotype II of A. rabiei. Further molecular genetic approach is needed to identify individual qualitative blight resistance genes and their interaction for pathotype-dependent blight resistance 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.     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

28-Homobrassinolide ameliorates the saline stress in chickpea (Cicer arietinum L.)

The response of chickpea (Cicer arietinum L.) cv. KPG-59 to pre-sowing seed treatment with 28-homobrassinolide (HBR) and/or sodium chloride (NaCl) was investigated. The seeds imbibed in aqueous solution of 10⁻¹⁰ or 10⁻⁸ M of HBR for 8 h, resulted in an increase in the values for most of the characteristics of shoot and root at 90-day stage and seed yield, at harvest. The plants resulting from the seeds soaked in HBR (10⁻⁸ M) possessed 23% and 31% higher leaf nitrate reductase (E.C. 1.6.6.1) and carbonic anhydrase (E.C. 4.2.2.1) activities, 34% more dry mass, 30% higher nodule number, 31% and 18% more nodule fresh and dry mass, compared with water soaked, control. Leghaemoglobin content and nitrogenase activity (E.C. 1.7.99.2) were 28% and 30% higher while nodule nitrogen and carbohydrate contents decreased by 5% and 6%, compared with the control. Moreover, seed yield increased by 26% over the control, at harvest. The values for all the above characteristics declined significantly, in the plants raised from the seeds soaked in NaCl. However, this ill effect was overcome, if NaCl treatment was given before or after HBR treatment.     

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.     

Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.     

Paenibacillus lentimorbus enhances growth of chickpea (Cicer arietinum L.) in chromium-amended soil

Chromium (Cr), with its great economic importance in industrial use, is a major metal pollutant of the environment. It affects soil microbial activity and soil fertility, resulting in losses in yield of plants. Paenibacillus lentimorbus B-30488^r (B-30488^r) tolerated 200 μg ml⁻¹ of Cr under in vitro conditions and produced the plant growth promoting substance indole acetic acid in the presence of Cr. Our in vitro study indicates enhancement in B-30488^r biofilm formation by sodium alginate (SA) and calcium chloride (CaCl₂) both in absence and presence of supplemented Cr(VI) as compared to unsupplemented control. The plant growth promoting effects caused by the B-30488^r biofilm in rhizosphere of chickpea under Cr(VI) stress suggests a phytoprotective role of B-30488^r biofilm. Our study reflects the multifarious role of strain B-30488^r and presents it as a potent plant growth promoting and bioremediation agent useful in Cr-contaminated rhizosphere soil, whereby the SA and CaCl₂ induced B-30488^r biofilm on plant root acts as a shield in preventing the direct access of toxic Cr to plant tissues, thus reducing its uptake in plants.     

Effects of selection criteria on yield stability in chickpea (Cicer arietinum L.)

Summary Selection in the F₃ generation for seed yield, fruiting branches/plant, effective pods/plant, and seed index (100-seed weight) was carried out in two chickpea crosses. Sixty F₅ lines (15 lines/selection criterion) along with check variety were evaluated for seed yield in three distinct environments. The effects of selection criteria on yield stability was examined using linear regression approach and genotype-grouping technique. There were no differences between selection criteria for linear yield responses of F₅ lines to different environments. Within all four selection criteria the lines showed similar linear responses. The non-linear component was relatively higher for lines selected for effective pods and seed index than lines selected for yield and fruiting branches. On the basis of mean yield and coefficient of variation across environments, the seed index was the least effective selection criterion for developing high yielding and stable lines. When the results of stability parameters and genotype-grouping technique were considered together, selection for yield and fruiting branches was highly effective for isolating stable and high yielding lines.     

Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.)

Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 μM 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyacetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 μM gibberellic acid and 1 μM indole-3-butyric acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics.     

Early generation yield testing versus visual selection in chickpea (Cicer arietinum L.)

Summary Four F₃ populations of chickpea (Cicer arietinum L.) were simultaneously evaluated for yield in an F₃ yield trial and in single plant progeny rows. Ten high yielding, 10 low yielding and 10 randomly sampled lines, along with 10 lines visually selected for yield from the progeny rows, were retained for further evaluation. The lines from each of the four selection groups in each population were bulked and evaluated in a replicated yield trial at three locations and four environments. The bulk of visually selected lines was not superior in yield to the bulk of randomly sampled lines at all locations. The present results indicate that an early generation yield testing selection procedure is more efficient than visual selection for yield improvements in chickpea.