Annual Cicer Research Articles

Cicer super-pangenome provides insights into species evolution and agronomic trait loci for crop improvement in chickpea.

Chickpea (Cicer arietinum L.)-an important legume crop cultivated in arid and semiarid regions-has limited genetic diversity. Efforts are being undertaken to broaden its diversity by utilizing its wild relatives, which remain largely unexplored. Here, we present the Cicer super-pangenome based on the de novo genome assemblies of eight annual Cicer wild species. We identified 24,827 gene families, including 14,748 core, 2,958 softcore, 6,212 dispensable and 909 species-specific gene families. The dispensable genome was enriched for genes related to key agronomic traits. Structural variations between cultivated and wild genomes were used to construct a graph-based genome, revealing variations in genes affecting traits such as flowering time, vernalization and disease resistance. These variations will facilitate the transfer of valuable traits from wild Cicer species into elite chickpea varieties through marker-assisted selection or gene-editing. This study offers valuable insights into the genetic diversity and potential avenues for crop improvement in chickpea.

First report of Ascochyta rabiei infections on endemic Turkish populations of Cicer bijugum and C. turcicum.

Ascochyta rabiei causes Ascochyta blight disease on C. arietinum as well as on annual C. reticulatum, C. pinnatifidum and C. judaicum and perennial C. montbretii, C. isauricum, C. ervoides species (Can et al. 2007; Frenkel et al. 2007; Ozkilinc et al. 2019; Peever et al. 2007; Tekin et al. 2018). During field survey studies carried out on annual Cicer spp. in June 2022, concentric ring-shaped lesions were observed on the stems and leaves of C. bijugum in Mardin province and C. turcicum in Elazig province. Cicer reticulatum and C. arietinum plants were also found in the location where C. bijugum was found. No disease symptoms were observed in other Cicer species, while C. bijugum had 32% disease incidence. The disease incidence among the C. turcicum population was 37.3 %, and no chickpea cultivation area was found near it. Diseased plant parts were surface sterilized, placed on ½ potato dextrose agar (PDA) and incubated at 24±2 oC in 12 hours light/dark photoperiod. Each symptomatic plant was considered as one isolate. Monosporic isolates were obtained and the same colony morphology developed from all plant parts of C. turcicum and C. bijugum. Spores were oblong and spore sizes were 10.73±0.62 µm (n=15) in length and 3.60±0.25 (n=15) µm in width, 10.64±0.98 (n=15) µm in length and 3.00±0.26 (n=15) µm in width for isolates obtained from C. turcicum and C. bijugum, respectively. Amplicons for all 40 isolates were generated with mating type (MAT) primers, and the ITS region was amplified and sequenced by using the ITS4 and ITS5 primers (Peever et al. 2007). For the MAT primers, a 700 bp amplicon was observed for all the 20 isolates obtained from C. bijugum conferring to MAT1.1 idiomorph. In contrast, for the isolates obtained from C. turcicum 14 isolates had a 700 bp amplicon for MAT1.1 and 6 isolates had a 500 bp amplicon for MAT1.2, thus representing both idiomorphs in a ~2:1 ratio. BLAST analysis of the ITS sequences showed 100% homology with the reference ITS sequences for A. rabiei except for 23 SVRC CT 09/22 and 23 SVRC CT 22/22 isolates showing 99.81 % similarity. All sequences were submitted to GenBank (OP967923, OP967924, OP967925, OP967926 and OP967927 for A. rabiei isolates from C. turcicum; OP981072, OP981073 and OP981074 for A. rabiei isolates from C. bijugum). A phylogenetic tree was constructed using MEGAX software and the Neighbor-Joining method, using the ITS sequences of A. rabiei, other Ascochyta spp. and Colletotrichum gloeosporioides. The A. rabiei isolates from C. turcicum and C. bijugum clustered together with A. rabiei sequences from the NCBI (Kumar et al. 2018). Twelve-day-old C. bijugum and C. turcicum seedlings were inoculated with 5 x 105 spore/mL concentration of spores from 5 C. turcicum and 3 C. bijugum isolates and put in plastic bags for 24 hours (Can et al. 2007). Pathogenicity tests were carried out in triplicate pots with four plants each for each isolate in a controlled climate chamber at 24±2 oC, 70% humidity under 12 hours light/dark conditions. The first symptoms were observed within 7 days after inoculation (dai) and severe Ascochyta blight symptoms developed on all plants by 21 dai. Cicer bijugum and C. turcicum are endemic Cicer species exhibiting narrow distribution in the Southeastern region of Republic of Türkiye. As a major biotic stress source, A. rabiei could be an important threat to Cicer spp (Abbo et al. 2003). To our knowledge, this is the first report of A. rabiei from C. bijugum and C. turcicum species.

Estimation of genetic diversity using seed storage protein (SSP) profiling in wild and cultivated species of Cicer L

The narrow genetic diversity of chickpea is a serious impediment to modern cultivar creation. Seed storage proteins (SSPs) are stable and have minimal or no degradation when subjected to isolation and SDS-PAGE. We have characterized SSPs of 436 chickpea genotypes, belonging to nine annual Cicer species, originated from 47 countries by SDS-PAGE and determined the extent of genetic diversity in chickpea through clustering. Based on scoring, a total of 44 bands (10 to 170kDa) were identified, which were all polymorphic. The least appeared protein bands were 11, 160 and 170kDa where band of 11 and 160kDa was present exclusively in wild type. Five bands were present in < 10% of genotypes. Bands appeared in 200-300 genotypes were suggested less polymorphic, on contrary bands present in 10-150 genotypes were suggested more polymorphic. Polymorphism of protein bands in context to their potential functions reported in literature were explored and suggested that the glubulins were most and glutelins were least abundant, whereas albumins with their known role in stress tolerance can be used as marker in chickpea breeding. Cluster analysis produced 14 clusters, interestingly three clusters contained only Pakistani genotypes and thus Pakistani genotypes appeared as a separate entity from the rest of the genotypes. Our results indicate that SDS-PAGE of SSPs is a powerful technique in determining the genetic diversity plus it is easily adaptable, due to its cost effectiveness in comparison to other genomics tools.

Intraspecific versus interspecific crosses for superior progeny in Cicer species

AbstractBoth intraspecific and interspecific crosses are a well‐known breeding strategies to generate variations or assemble desirable traits in economically important plants. Interspecific crosses have been mined to improve resistance to (a)biotic stresses in chickpea (Cicer arietinum L.) in a limited number of research studies but have never been crosschecked for yield and yield criteria in intraspecific versus interspecific crosses. Comparison of superior progeny or superior recombinant inbred lines (RILs) and better progeny than their best parent for yield, yield criteria, and resistance to (a)biotic stresses in intraspecific versus interspecific crosses was the objectives of this study. Also, associations between superior progeny and genetic distance based on simple sequence repeat (SSR) markers were assessed among annual Cicer species including cultivated chickpea, C. reticulatum Ladiz. and C. echinospermum P.H. Davis. Each two genotypes of C. reticulatum and C. echinospermum were crossed with three genotypes of cultivated chickpea. Not only superior progeny for yield and yield criteria, but also superior RILs that were cold tolerant were detected higher in interspecific crosses than in intraspecific crosses. On the other hand, plant height, first pod height, and seed size were higher in intraspecific crosses than in interspecific crosses, indicating that seed size and traits suitable for combine harvest can be increased via intraspecific crosses. A significant association was found between superior progeny and genetic distance among Cicer species. It was concluded that interspecific crosses in Cicer species may increase yield and resistance to (a)biotic stresses, whereas intraspecific crosses may increase plant height and seed size.

Identification of promising chickpea interspecific derivatives for agro-morphological and major biotic traits

The wild Cicer species is well-known for having climate-resilient and productivity-enhancing traits of interest. Therefore, wide hybridization could be used as a realistic strategy for introgressing prospective traits from wild species into the cultivated gene pool. The present study was, thus, undertaken to evaluate F7 chickpea interspecific derivatives derived from Cicer reticulatum Ladiz. and C. echinospermum P. H. Davis wild annual Cicer species. As a result, a set of six interspecific crosses were advanced using the single seed descent (SSD) method of breeding. The F7 generation of these crosses was assessed in two diverse agro-ecological regions of India. The data revealed a wide range of variation with respect to seed yield and its important component traits, which resulted in the identification of the most promising derivatives carrying desirable characters as indicated by range, mean, and coefficient of variation. Further, fruitful heterosis was also estimated as promising selection criteria for identifying superior lines for earliness and high seed yield, including resistance against prevailing stresses (ascochyta blight, botrytis gray mold, dry root rot, and fusarium wilt). The superior derivatives carrying putative characters could be recommended for further breeding and selection of genetic materials for developing suitable genotypes.

Cicer turcicum: A New Cicer Species and Its Potential to Improve Chickpea.

Genetic resources of the genus Cicer L. are not only limited when compared to other important food legumes and major cereal crops but also, they include several endemic species with endangered status based on the criteria of the International Union for Conservation of Nature. The chief threats to endemic and endangered Cicer species are over-grazing and habitat change in their natural environments driven by climate changes. During a collection mission in east and south-east Anatolia (Turkey), a new Cicer species was discovered, proposed here as C. turcicum Toker, Berger & Gokturk. Here, we describe the morphological characteristics, images, and ecology of the species, and present preliminary evidence of its potential utility for chickpea improvement. C. turcicum is an annual species, endemic to southeast Anatolia and to date has only been located in a single population distant from any other known annual Cicer species. It belongs to section Cicer M. Pop. of the subgenus Pseudononis M. Pop. of the genus Cicer L. (Fabaceae) and on the basis of internal transcribed spacer (ITS) sequence similarity appears to be a sister species of C. reticulatum Ladiz. and C. echinospermum P.H. Davis, both of which are inter-fertile with domestic chickpea (C. arietinum L.). With the addition of C. turcicum, the genus Cicer now comprises 10 annual and 36 perennial species. As a preliminary evaluation of its potential for chickpea improvement two accessions of C. turcicum were field screened for reproductive heat tolerance and seeds were tested for bruchid resistance alongside a representative group of wild and domestic annual Cicer species. C. turcicum expressed the highest heat tolerance and similar bruchid resistance as C. judaicum Boiss. and C. pinnatifidum Juab. & Spach, neither of which are in the primary genepool of domestic chickpea. Given that C. arietinum and C. reticulatum returned the lowest and the second lowest tolerance and resistance scores, C. turcicum may hold much potential for chickpea improvement if its close relatedness supports interspecific hybridization with the cultigen. Crossing experiments are currently underway to explore this question.

Characterization of resistance to ascochyta blight of selected wild Cicer germplasm

The resistance of 24 perennial Cicer accessions to Ascochyta rabiei (Pass.) Labr. was assessed under field and greenhouse conditions. Two perennial and two annual Cicer accessions with superior resistance were examined and compared with susceptible (‘CDC Xena’) and resistant (‘CDC Frontier’) domesticated Cicer arietinum L. (chickpea) cultivars in terms of pathogen colonization, symptom development, host autofluorescence, and hydrogen peroxide generation. Colonizing hyphae on the four wild Cicer genotypes were pigmented, but hyaline on cultivated chickpea. The morphology of colonizing hyphae differed on the six genotypes, with wider hyphae observed on the most resistant genotype, Cicer anatolicum Alef. PI 383626. Inoculation of leaves of the top five nodes indicated that infection rates were highest on the youngest unfurled leaf for C. anatolicum PI 383626, Cicer oxyodon Boiss. &amp; Hoh. PI 561103, Cicer bijugum Rech.f. ILWC 260, and ‘CDC Frontier’. Observations of hyphal pigmentation, modified hyphal growth, and low infection rates on fully developed leaves of wild Cicer plants suggest that components of the plant surface play an important role in disease resistance. Localized autofluorescence in response to infection was observed in the two perennials, but not in the other genotypes examined. The distinct resistance strategy observed in these perennial accessions make them interesting but challenging candidates for introgression of resistance into cultivated chickpea.

Exploitation of wild annual Cicer species for widening the gene pool of chickpea cultivars

AbstractIntrogression of unadapted genes from the wild Cicer species could contribute to the widening of genetic base of important traits such as yield, yield attributes and resistance to major biotic and abiotic stresses. An attempt was made successfully to intercross two wild annual Cicer species with three cultivated chickpea cultivars. Four interspecific cross‐combinations were made, and their true hybridity was ascertained through morphological and molecular markers. These cross‐combinations were also studied for some important quantitative traits under real field conditions. The range, mean and coefficient of variation of agro‐morphological traits were assessed in the parental lines, their F1 and F2 generations to determine the extent of variability generated in cultivated chickpea varieties. A high level of heterosis was recorded for number of pods/plant and seed yield/plant in F1 generation. Three cross‐combinations of ‘Pusa 1103’ × ILWC 46, ‘Pusa 256’ × ILWC 46 and ‘Pusa 256’ × ILWC 239 exhibited substantially higher variability for important yield‐related traits. The present research findings indicate that these wild annual Cicer species can be easily exploited to broaden the genetic base of cultivated gene pool for improving seed yield as well as adaptation.

Allelic diversity between and within three wild annual Cicer species

Most wild Cicer species have narrow eco-geographic amplitude. Likewise, domesticated chickpea suffers from severe adaptive limitations due to its unique evolutionary history. The wild progenitor may offer only limited adaptive allelic variation for improving the chickpea crop. Therefore, there is a need to explore allelic diversity between and within annual Cicer sp. that span diverse natural habitats. Here we characterized the allelic diversity between and within wild populations of C. pinnatifidum, C. judaicum and C. cuneatum spanning most of their documented native range in Turkey, Israel and Ethiopia. Eco-geographical analysis resulted in clear separation between the collection sites of C. cuneatum in east Africa and the other two east Mediterranean species. Analysis of molecular variance shows that only 18 % of the allelic variation accounts for differences between the three species, while 34 % was contributed from difference between populations. Interestingly, most (48 %) of the allelic variation was detected among accessions within populations. PCoA analysis confirmed the independent taxonomic and indeed the genetic integrity of the two east Mediterranean sister species C. pinnatifidum and C. judaicum. Conservation of large rich populations seems a more effective strategy than the preservation of small thin populations of annual Cicer sp. Given the relatively narrow geographic range of most annual Cicer sp., accessing germplasm lines from ecologically distinct habitats emerges as the most promising strategy for the identification of useful adaptive allelic variation.

Characterization and Evaluation of Wild Annual Cicer Species for Agro‐morphological Traits and Major Biotic Stresses under Northwestern Indian Conditions

ABSTRACTSystematic characterization and evaluation of the wild gene pool for breeding purposes is a common practice in an increasing number of cultivated crop species. Substantial yield improvement of cultivated chickpea (Cicer arietinum L.) has been limited by the loss of useful genes for higher yield and lack of resistance to major biotic stresses. In the present study, a total of 88 wild accessions of six annual Cicer species viz., C. reticulatum Ladizinsky, C. echinospermum P.H. Davis, C. judaicum Boiss., C. pinnatifidum Jarb. &amp; Spach, C. bijugum Rech. f., and C. yamashitae Kitam., along with three check varieties (controls) of cultivated chickpea namely, Pusa 256, Pusa 1103, and JG 11, were characterized and evaluated for phenological and agro‐morphological traits, including their reaction to the major fungal diseases [Ascochyta blight caused by Ascochyta rabiei (Pass.) Labr. and Botrytis gray mold caused by Botrytis cinerea Pers. ex. Fr.] and root knot nematode (Meliodogyne incognita). Wild Cicer accessions exhibited variation for some morphological traits, including plant pigmentation in C. reticulatum, C. judaicum, and C. pinnatifidum; number of leaflets leaf−1 in C. reticulatum, C. echinospermum, C. judaicum, C. pinnatifidum, and C. bijugum; and seed color in C. reticulatum, C. judaicum, and C. pinnatifidum. Likewise, variation for agro‐morphological traits was also studied, that is, earliness, number of branches plant−1, and number of pods plant−1, etc. Some of these were comparable with the cultivated genotypes. Pattern of within‐species diversity was also estimated using Ward's Minimum Variance Dendrogram and principal components analysis for important agro‐morphological traits. Among the wild Cicer species, C. judaicum and C. pinnatifidum had the greatest number of resistant accessions including multiple resistance for Ascochyta blight, Botrytis gray mold, and root knot nematode. Variability among wild annual Cicer accessions is invaluable and can be exploited following introgression into cultivated chickpea. It will also help in the flow of useful genes from the wild to the cultivated gene pool and generating additional variation for use in chickpea improvement.

Combining Ascochyta blight and Botrytis grey mould resistance in chickpea through interspecific hybridization

Ascochyta blight (AB) caused by Ascochyta rabiei (Pass.) Labr. and Botrytis grey mould (BGM) caused by Botrytis cinerea (Pers. ex Fr.) are important diseases of the aerial plant parts of chickpea in most chickpea growing areas of the world. Although conventional approaches have contributed to reducing disease, the use of new technologies is expected to further reduce losses through these biotic stresses. Reliable screening techniques were developed: ‘field screening technique’ for adult plant screening, ‘cloth chamber technique’ and ‘growth chamber technique’ for the study of races of the pathogen and for segregating generations. Furthermore, the ‘cut twig technique’ for interspecific population for AB and BGM resistance was developed. For introgression of high levels of AB and BGM resistance in cultivated chickpea from wild relatives, accessions of seven annual wild Cicer spp. were evaluated and identified: C. judaicum accessions 185, ILWC 95 and ILWC 61, C. pinnatifidum accessions 188, 199 and ILWC 212 as potential donors. C. pinnatifidum accession188 was crossed with ICCV 96030 and 62 F9 lines resistant to AB and BGM were derived. Of the derived lines, several are being evaluated for agronomic traits and yield parameters while four lines, GL 29029, GL29206, GL29212, GL29081 possessing high degree of resistance were crossed with susceptible high yielding cultivars BG 256 to improve resistance and to undertake molecular studies. Genotyping of F2 populations with SSR markers from the chickpea genome was done to identify markers potentially linked with AB and BGM resistance genes. In preliminary studies, of 120 SSR markers used, six (Ta 2, Ta 110, Ta 139, CaSTMS 7, CaSTMS 24 and Tr 29) were identified with polymorphic bands between resistant derivative lines and the susceptible parent. The study shows that wild species of Cicer are the valuable gene pools of resistance to AB and BGM. The resistant derivative lines generated here can serve as good pre-breeding material and markers identified can assist in marker assisted selection for resistance breeding.

IPBS-Retrotransposons-based genetic diversity and relationship among wild annual Cicer species

Lack of requisite genetic variation in cultivated species has necessitated systematic collection, documentation and evaluation of wild Cicer species for use in chickpea variety improvement programs. Cicer arietinum has very narrow genetic variation, and the use of a wild relative in chickpea breeding could provide a good opportunity for increasing the available genetic variation of cultivated chickpea. Genetic diversity and the relationship of 71 accessions, from the core area of chickpea origin and domestication (Southeastern Turkey), belonging to five wild annual species and one cultivated species (Cicer arietinum) were analysed using iPBS-retrotransposon and ISSR markers. A total of 136 scorable bands were detected using 10 ISSR primers among 71 accessions belonging to 6 species, out of which 135 were polymorphic (99.3 %), with an average of 13.5 polymorphic fragments per primer, whereas iPBS detected 130 bands with 100 % polymorphism with an average of 13.0 bands per primer. C. echinospermum and C. pinnatifidum were the most diverse among species, whereas C. arietinum exhibited lower polymorphism. The average polymorphism information contents (PIC) value for both marker systems was 0.91. The clustering of the accessions and species within groups was almost similar, when iPBS and ISSR NeighborNet (NNet) planar graphs were compared. Further detailed studies are indispensable in order to collect Cicer germplasm, especially C. reticulatum, from southeastern Turkey particularly, from Karacadag Mountain for preservation, management of this species, and to study their genetic diversity at molecular level. This study also demonstrates the utility and role of iPBS-retrotransposons, a dominant and ubiquitous part of eukaryotic genomes, for diversity studies in wild chickpea and in cultivated chickpea.

Comparison of genetic variation and differentiation among annual Cicer species using start codon targeted (SCoT) polymorphism, DAMD-PCR, and ISSR markers

Three molecular markers, including start codon targeted (SCoT) polymorphism, directed amplification of minisatellite-region DNA polymerase chain reaction (DAMD-PCR), and inter simple sequence repeat (ISSR) markers, were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 38 accessions of eight annual Cicer species. The results were as follows: (1) the highest level of detected polymorphism was observed for all three marker types; (2) the rate of diversity for the three marker techniques was approximately equal, and the correlation coefficients of similarity were statistically significant for all three marker systems; (3) the three molecular markers showed relatively similar phylogenetic grouping for examined species. Diversity analysis showed that Cicer reticulatum is the closest wild species to the cultivated chickpea, and this finding supports the hypothesis that C. reticulatum is the most probable progenitor of the cultivated species. C. bijugum, C. judaicum, and C. pinnatifidum were clustered together, and in other clusters C. yamashitae and C. cuneatum were grouped close together. To our knowledge, this is the first detailed comparison of performance among two targeted DNA region molecular markers (SCoT and DAMD-PCR) and the ISSR technique on a set of samples of Cicer. The results provide guidance for future efficient use of these molecular methods in genetic analysis of Cicer.

Hybridisation of Australian chickpea cultivars with wild Cicer spp. increases resistance to root-lesion nematodes (Pratylenchus thornei and P. neglectus)

Australian and international chickpea (Cicer arietinum) cultivars and germplasm accessions, and wild annual Cicer spp. in the primary and secondary gene pools, were assessed in glasshouse experiments for levels of resistance to the root-lesion nematodes Pratylenchus thornei and P. neglectus. Lines were grown in replicated experiments in pasteurised soil inoculated with a pure culture of either P. thornei or P. neglectus and the population density of the nematodes in the soil plus roots after 16 weeks growth was used as a measure of resistance. Combined statistical analyses of experiments (nine for P. thornei and four for P. neglectus) were conducted and genotypes were assessed using best linear unbiased predictions. Australian and international chickpea cultivars possessed a similar range of susceptibilities through to partial resistance. Wild relatives from both the primary (C. reticulatum and C. echinospermum) and secondary (C. bijugum) gene pools of chickpea were generally more resistant than commercial chickpea cultivars to either P. thornei or P. neglectus or both. Wild relatives of chickpea have probably evolved to have resistance to endemic root-lesion nematodes whereas modern chickpea cultivars constitute a narrower gene pool with respect to nematode resistance. Resistant accessions of C. reticulatum and C. echinospermum were crossed and topcrossed with desi chickpea cultivars and resistant F4 lines were obtained. Development of commercial cultivars with the high levels of resistance to P. thornei and P. neglectus in these hybrids will be most valuable for areas of the Australian grain region and other parts of the world where alternating chickpea and wheat crops are the preferred rotation.

Development of chickpea EST-SSR markers and analysis of allelic variation across related species

Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.

Sympatric ascochyta complex of wild Cicer judaicum and domesticated chickpea

The aim of this study was to isolate, identify and characterize ascochyta blight pathogens from Cicer judaicum, a wild annual Cicer species which grows in Israel and other Mediterranean countries in sympatric distribution with legume crops, and determine their virulence and aggressiveness to other wild and domesticated legumes. Native C. judaicum plants exhibited symptoms resembling ascochyta diseases of grain legume crops. Two distinct pathogens were isolated and identified as Phoma pinodella and Didymella rabiei using morphological and molecular tools; their infectivity was verified using Koch's postulates. The virulence of these pathogens was examined on 13 legume species, of which P. pinodella was virulent to Pisum sativum, P. fulvum, C. judaicum, C. arietinum, C. reticulatum, C. pinnatifidum and C. bijugum. Didymella rabiei infected all these Cicer species, but not the other legume species tested. Aggressiveness of the pathogens was tested on wild and domesticated chickpea and pea. Didymella rabiei isolated from C. judaicum had significantly higher (P &lt; 0·001) aggressiveness than P. pinodella from C. judaicum on both wild and domesticated chickpea. Disease severity on the former species ranged from 62·5% to 70% and on the latter from 41% to 56%. Phoma pinodella isolates from C. judaicum were more aggressive on C. arietinum and P. sativum than on C. judaicum and P. fulvum. Results of the current study suggest that C. judaicum may serve as an alternative host to ascochyta pathogens that endanger chickpea and possibly other crops and wild species growing in close proximity.

Identification of duplicates and fingerprinting of primary and secondary wild annual Cicer gene pools using AFLP markers

Wild annual Cicer gene pools contain valuable germplasm for chickpea improvement programs. Previous research showed that duplication might exist in accessions collected from these gene pools, which would hinder chickpea breeding and related research. AFLP (amplified fragment length polymorphism) markers were used to fingerprint the world collections of the primary and secondary gene pools including C. reticulatum Lad., C. bijugum K.H. Rech., C. judaicum Boiss. and C. pinnatifidum Jaub. et Sp. Duplicates were detected in a total of 24 accessions in both the gene pools, highlighting the necessity to fingerprint the germplasm. Genotypic difference was detected as gene pool specific, species specific and accession specific AFLP markers. These were developed into fingerprinting keys for accession identification between and within species and gene pools. Use of AFLP markers to detect duplicates and to identify accessions is a reliable method which will assist in the characterisation and use of wild annual Cicer germplasm in chickpea improvement programs. We recommend the procedure presented in this paper as a standard approach for the precise genetic identification and characterisation of future world collections of wild Cicer, to keep germplasm integrity and to benefit chickpea breeding and related research programs.

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.

Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus

BackgroundChickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits.ResultsAn EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested.ConclusionGene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses.

Geographical patterns of genetic variation in the world collections of wild annual Cicer characterized by amplified fragment length polymorphisms

Cicer reticulatum, C. echinospermum, C. bijugum, C. judaicum, C. pinnatifidum, C. cuneatum and C. yamashitae are wild annual Cicer species and potential donors of valuable traits to improve chickpea (C. arietinum). As part of a large project to characterize and evaluate wild annual Cicer collections held in the world gene banks, AFLP markers were used to study genetic variation in these species. The main aim of this study was to characterize geographical patterns of genetic variation in wild annual Cicer germplasm. Phylogenetic analysis of 146 wild annual Cicer accessions (including two accessions in the perennial C. anatolicum and six cultivars of chickpea) revealed four distinct groups corresponding well to primary, secondary and tertiary gene pools of chickpea. Some possible misidentified or mislabelled accessions were identified, and ILWC 242 is proposed as a hybrid between C. reticulatum and C. echinospermum. The extent of genetic diversity varied considerably and was unbalanced between species with greatest genetic diversity found in C. judaicum. For the first time geographic patterns of genetic variation in C. reticulatum, C. echinospermum, C. bijugum, C. judaicum and C. pinnatifidum were established using AFLP markers. Based on the current collections the maximum genetic diversity of C. reticulatum, C. echinospermum, C. bijugum and C. pinnatifidum was found in southeastern Turkey, while Palestine was the centre of maximum genetic variation for C. judaicum. This information provides a solid basis for the design of future collections and in situ conservation programs for wild annual Cicer.