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.     

Sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.).

Two small-insert genomic libraries of chickpea (Cicer arietinum L.) were screened with a set of microsatellite-specific oligonucleotide probes. A total of 121 positive clones were identified among 13,000 plated colonies. Thirty-nine clones were recognized by (TAA)5, 26 by (GA)8, 18 by (GT)8, 27 by a pool of AT-rich trinucleotide repeats [(CAA)5, (CAT)5, and (GAA)5], and 11 by a pool of GC-rich trinucleotides [(TCC)5, (CAC)5, (CAG)5, and (CGA)5]. Of 53 clones selected for sequencing, 43 carried a microsatellite. Flanking primer pairs were designed for 28 loci, and used on a small test-set comprising one C. reticulatum and four C. arietinum accessions. Separation of the PCR products on agarose or polyacrylamide gels revealed single bands of the expected size with 22 of the primer pairs. Sixteen of these "Cicer arietinum sequence-tagged microsatellite site" (CaSTMS) markers were polymorphic at an intraspecific level, detecting 2-4 alleles within the four accessions examined. Primer pairs CaSTMS10 and CaSTMS15 revealed 25 and 16 alleles among 63 C. arietinum accessions from different geographic locations, reflecting gene diversity values of 0.937 and 0.922, respectively. Mendelian inheritance of CaSTMS markers was demonstrated using a set of recombinant inbred lines and their parents.     

Genetic variability and population structure of Mexican chickpea (Cicer arietinum L.) germplasm accessions revealed by microsatellite markers

Journal of Plant Biochemistry and Biotechnology - Globally, the chickpea is the second most important dried grain legume after bean. Mexico occupies eighth place as producer and fourth as exporter...     

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.     

Genetic variability of Fusarium wilt pathogen isolates of chickpea (Cicer arietinum L.) assessed by molecular markers

Genetic variability among 43 isolates of Fusarium oxysporum f.sp. ciceri, the chickpea wilt pathogen, collected from nine states of India including the four well-characterized races of the pathogen were assessed using the molecular markers, RAPDs and AFLP. Principal coordinate analysis of the similarity index data generated from the molecular marker studies mostly gave three different clusters: Of these two clusters represented race-1 and race-2, and the third cluster consisted of race-3 and race-4 pathogen isolates. In RAPDs a fourth cluster was seen which did not go with any of the four races of the pathogen. The molecular markers established the distinctness of race-1 and race-2 pathogen isolates and the close similarity of pathogen isolates of race-3 with that of race-4. AFLP was found to be more informative as it differentiated more number of the pathogen isolates with the known races with minimum of outliers. The high levels of DNA polymorphism observed with the molecular markers suggest the rapid evolution of new recombinants of the pathogen in the chickpea growing fields. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Integrated physical,genetic and genome map of chickpea (Cicer arietinum L.)

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.     

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.     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.

     

Molecular analysis of kabuli and desi type of Indian chickpea (Cicer arietinum L.) cultivars using STMS markers

Fifty one Sequence tagged microsatellite sites (STMS) primer pairs were employed to assess the genetic diversity and relationships with morphological characters among the sixty-eight chickpea (Cicer arietinum L.) cultivars of India. A total of 32 out of 51 STMS primers were found polymorphic and a total of 121 alleles were generated out of which 102 (83 %) were detected for the 32 polymorphic STMS markers with an average of 2.22 alleles per locus. The PIC values of all the polymorphic loci ranged from 0.15 (TS82) to 0.69 (TS29) with the mean value of 0.27. Three primers showed PIC value of more than 0.60. The highest PIC value was observed for the primer TS29 (0.69), succeeded by the primer GA 11 (0.61) and TS71 (0.60). Gene diversity (He) was observed in the range of 0.16 (TS82) to 0.74 (TS29) with an average value of 0.33. The heterozygosity (Ho) was observed to be 0.39 (average) with a range of 0.04 (TA18) to 1.00 (TA76, STMS 5, TA72 and TA122). Based on the above STMS marker analysis by considering the parameters of PIC value (≥0.55), gene diversity (≥0.62), and polymorphic alleles (≥4), six highly polymorphic STMS loci GA11, TA76S, TA89, TS29, TS43 and TS71 were observed which can effectively be used in further molecular studies. Dendrogram generated by the UPGMA analysis and POWER MARKER v3.0 showed similar results and there was no clear demarcation of Kabuli and Desi genotypes. The present study resulted in identification of highly distinct genotypes JG 130 and C 235 (57 %) followed by two pairs of genotypes B 108 and JG 11 (57.8 %) and, JG 315 and RSG 2 (59 %) which can be used effectively in a breeding programs in order to develop transgressive segregants with wider genetic base and better promising genotypes. Effective use of these three pairs of chickpea genotypes is expected to give better products for the development of higher yielding Kabuli and Desi genotypes with tolerance/resistance to biotic and abiotic stresses and quality traits.     

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     

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.     

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     

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.     

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.     

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.     

Conservation and variability of sequence-tagged microsatellite sites (STMSs) from chickpea (Cicer aerietinum L.) within the genus Cicer

The conservation of 90 microsatellite-flanking sequences from chickpea in 39 accessions of eight annual and 1 accession of a perennial species of the genus Cicer was investigated. All of the primer sequences successfully amplified microsatellites in related species, indicating the conservation of microsatellite-flanking sequences in chickpea’s relatives. However, the degree of conservation of the primer sites varied between species depending on their known phylogenetic relationship to chickpea, ranging from 92.2% in C. reticulatum, chickpea’s closest relative and potential ancestor, down to 50% for C. cuneatum. A phylogenetic tree revealed that chickpea and the other members of its crossability group were more closely related to the perennial C. anatolicum than to other annual species of the genus. Considerable variation in size and number of amplification products between and within species was observed. Sequence analysis of highly divergent amplification products proved that variation is either due to large differences in the number of microsatellite repeats or to the amplification of a locus unrelated to the one amplified from chickpea. Sequence information and bootstrapping using PAUP suggested that STMSs derived from chickpea may be efficiently and reliably used for synteny studies in chickpea’s crossability group, including C. anatolicum. However, care should be taken when applying these markers to other species of the genus. Considering the data presented here and the known historical record, the age of section Monocicer, including chickpea, is estimated to be about 100,000 years. Received: 13 August 1999 / Accepted: 11 November 1999     

Genetic marker discovery,intraspecific linkage map construction and quantitative trait locus analysis of ascochyta blight resistance in chickpea (Cicer arietinum L.)

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr., is a highly destructive disease of chickpea (Cicer arietinum L.) on a global basis, and exhibits considerable natural variation for pathogenicity. Different sources of ascochyta blight resistance are available within the cultivated species, suitable for pyramiding to improve field performance. Robust and closely linked genetic markers are desirable to facilitate this approach. A total of 4,654 simple sequence repeat (SSR) and 1,430 single nucleotide polymorphism (SNP) markers were identified from a chickpea expressed sequence tag (EST) database. Subsets of 143 EST–SSRs and 768 SNPs were further used for validation and subsequent high-density genetic mapping of two intraspecific mapping populations (Lasseter × ICC3996 and S95362 × Howzat). Comparison of the linkage maps to the genome of Medicago truncatula revealed a high degree of conserved macrosynteny. Based on field evaluation of ascochyta blight incidence performed over 2 years, two genomic regions containing resistance determinants were identified in the Lasseter × ICC3996 family. In the S95362 × Howzat population, only one quantitative trait locus (QTL) region was identified for both phenotypic evaluation trials, which on the basis of bridging markers was deduced to coincide with one of the Lasseter × ICC3996 QTLs. Of the two QTL-containing regions identified in this study, one (ab_QTL1) was predicted to be in common with QTLs identified in prior studies, while the other (ab_QTL2) may be novel. Markers in close linkage to ascochyta blight resistance genes that have been identified in this study can be further validated and effectively implemented in chickpea breeding programs.