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.     

Analysis of genetic relationships among perennial and annual Cicer species growing in Turkey using RAPD markers

Random amplified polymorphic DNA (RAPD) fragments were used to assess genetic relationships among Cicer spp. growing in Turkey. Seven 10-mer primers selected from a 50 random oligonucleotide primer set, depending on their ability to amplify genomic DNA in all species, were used to detect RAPD variation in 43 wild and cultivated accessions representing ten species. These primers yielded 95 reproducible amplification products, 92 of which were polymorphic. Pairwise genetic distances of accessions estimated according to Nei and Li (1979) were used to produce a dendrogram using UPGMA. The dendrogram contained two main clusters, one of which comprised accessions of the four perennial species (Cicer montbretii, Cicer isauricum, Cicer anatolicum and Cicer incisum) together with the accessions of the three annual species (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum), and the other cluster included the remaining three annual species (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Analysis of RAPD variation showed that C. incisum is the most similar perennial species to annuals, and C. reticulatum is the closest annual species to chickpea. These results generally agree with our allozyme study which was carried out using same Cicer collection and previous studies of relationships among annual species. The results also show that RAPD markers can be used to distinguish Cicer species and to survey genetic variation and relationships among taxonomic units in this genus.     

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.     

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 Karacada? 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.     

Systematic position of the genusCicer L. (Fabaceae) from data on DNA/DNA hybridization

This investigation was carried out in an attempt to determine the systematic position ofCicer via DNA/DNA hybridization. The data showed that members of the tribeVicieae were related toCicer than toTrifolium andOnonis. It is also showed thatPisum was the nearest species toCicer. Thus, the data presented in this work recommend the classification ofCicer underVicieae rather than a separate tribeCicerideae.     

Relative Embryo Growth Rates in the Annual Cicer L. Species

Early embryo growth rates were studied in the nine annual speciesof Cicer L., namely, C. arietinum L., C. bijugum Rech., C. chorassanicum(Bge.) M. Pop., C. cuneatum Rich., C. echinospermum Dav., C.judaicum Boiss, C. pinnatifidum J. and S., C. reticulatum Lad.and C. yamashitae Kit. The number of embryo cells increasedexponentially with time and was log linear in all the species.Species differed in their mean cell doubling time (MCDT). Cicerechinospermum and C. yamashitae had, respectively, the longestand the shortest MCDT which ranged from 9.67 to 16.15 h forthe nine species. Failure of successful interspecific hybridizationbetween C. arietinum and the wild annual species was only partlyexplained by differences in MCDT of the parental species. Relativegenetic closeness still plays the major role in determiningsuccess of interspecific hybridization in Cicer. Chickpea, Cicer, embryo, interspecific hybridization, suspensor     

Cladistic and phylogenetic analyses of the genus Cicer in Turkey

Fifteen taxa of the genus Cicer L. growing naturally in Turkey and out-groups were classified by phylogenetic and cladistic analysis. Taxa of the genus Cicer and the out-group taxa belonging to the closest genera Phaseolus L., Vicia L., Lathyrus L. and Ononis L., which are placed in Phaseoleae, Vicieae and Ononideae tribes, respectively, were used in molecular studies in order to derive their phylogenetic relationships. Morphological, palynological and seed characters were used on the basis of 143 traits. The micromorphological characters of seed and pollen grains were revealed by SEM. Lathyrus L. and Vicia L. species were used as out-groups for numerical analysis. Ten specimens were used for the measurements of metric characters related to the morphological structures of the taxa used for statistical analysis via PAUP and NTSYS-pc packages. Phylogenetic relationships between species and populations of the same species growing in different locations and their variations were determined using molecular methods performed on regions of the Inter Simple Sequence Repeat (ISSR). DNA was isolated from the collected samples, using modified CTAB protocols. ISSR was used for phylogenetic fingerprinting. The data were analyzed with NTSYS-pc package. Standardized data were used to generate a dendrogram that revealed the phylogenetic relationships of the taxa. Geographic distribution of the Cicer taxa appears to be closely related to the Anatolian Diagonal. As a result of this study, four new endemic taxa were added and evaluated for the first time.     

Random amplified polymorphic DNA (RAPD) analysis reveals genetic relationships among the annual Cicer species

 Random amplified polymorphic DNA markers were used to distinguish between nine different Cicer taxa representing the cultivated chickpea and eight other related annual wild species. Of the 75 random10-mer primers tested, only 8 amplified genomic DNA across all the species. A total of 115 reproducibly scorable RAPD markers were generated, all except 1 polymorphic, and these were utilized to deduce genetic relationships among the annual Cicer species. Four distinct clusters were observed and represented C. arietinum, C. reticulatum and C. echinospermum in first cluster followed by C. chorassanicum and C. yamashitae in the second cluster, while C. pinnatifidum, C. judaicum and C. bijugum formed the third cluster. Cicer cuneatum did not cluster with any of the species and was most distantly placed from the cultivated species. Except for the placement of C. chorassanicum and C. yamashitae, deduced species’ relationships agreed with previous studies. In addition, species-diagnostic amplification products specific to all the nine species were identified. The results clearly demonstrate a methodology based on random-primed DNA amplification that can be used for studying Cicer phylogeny and chickpea improvement. Received: 27 July 1998 / Accepted: 5 August 1998     

Interspecific relationships of the genus<Emphasis Type="Italic"> Cicer</Emphasis> L. (Fabaceae) based on<Emphasis Type="Italic"> trn</Emphasis>T-F sequences

The trnT-F region in chloroplasts was sequenced to elucidate interspecific phylogenetic relationships in the genus Cicer. Twenty-five species representing four sections and two outgroups were analyzed. A phylogenetic analysis revealed three major clades in the genus Cicer. Inferred phylogenetic relationships support multiple origins of annual species in the genus Cicer. Low variation within the most perennial species in the sequence regions suggests they may have originated during a period of rapid diversification after the genus arose. High levels of sequence divergence, biogeographical patterns and morphological traits between African and Eurasian groups of species suggest that Cicer may have independently diverged on each continent. Phylogenetic analysis of sequence data did not support the monophyly of the currently recognized sections and indicated the need for a revision of the infrageneric classification.Communicated by C. Möllers     

Isozyme polymorphism and phylogenetic interpretations in the genus Cicer L.

Summary Allozyme variation among 50 accessions representing the cultivated chickpea (Cicer arietinum L.) and eight wild annual Cicer species was scored and used to assess genetic diversity and phylogeny. Sixteen enzyme systems revealed 22 putative and scorable loci of which 21 showed polymorphism. Variation was prevalent between species (D_st = 0.510) but not within species (H_s = 0.050). No variation for isozyme loci was detected in the cultivated chickpea accessions. Cicer reticulatum had the highest proportion of polymorphic loci (0.59) while the loci Adh-2 and Lap were the most polymorphic over all the species accessions. The phylogeny of annual Cicer species, as determined by allozyme data, generally corroborated those based on other characters in previous studies. Cicer arietinum, C. reticulatum and C. echinospermum formed one cluster, while C. pinnatifidum, C. bijugum and C. judaicum formed another cluster. Cicer chorassanicum was grouped with C. yamashitae, whereas C. cuneatum formed an independent group and showed the largest genetic distance from C. arietinum.     

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 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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic relationships among perennial and annual<Emphasis Type="Italic"> Cicer</Emphasis> species growing in Turkey assessed by AFLP fingerprinting

AFLP markers were used to assess genetic relationships among Cicer species with distribution in Turkey. Genetic distances were computed among 47 Cicer accessions representing four perennial and six annual species including chickpea, using 306 positions on AFLP gels. AFLP-based grouping of species revealed two clusters, one of which includes three perennial species, Cicer montbretii, Cicer isauricum and Cicer anatolicum, while the other cluster consists of two subclusters, one including one perennial, Cicer incisum, along with three annuals from the second crossability group (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum) and the other one comprising three annuals from the first crossability group (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Consistent with previous relationship studies in the same accession set using allozyme and RAPD markers, in AFLP-based relationships, C. incisum was the closest perennial to nearly all annuals, and C. reticulatum was the closest wild species to C. arietinum. Cluster analysis revealed the grouping of all accessions into their distinct species-clusters except for C. reticulatum accessions, ILWC247, ILWC242 and TR54961; the former was found to be closer to the C. arietinum accessions while the latter two clustered with the C. echinospermum group. Small genetic distance values were detected among C. reticulatum accessions (0.282) and between C. reticulatum and C. arietinum (0.301) indicating a close genetic similarity between these two species. Overall, the AFLP-based genetic relationships among accessions and species were congruous with our previous study of genetic relationships using allozymes. The computed level of AFLP variation and its distribution into within and between Cicer species paralleled the previous report based on RAPD analyses. AFLP analysis also confirmed the presence of the closest wild relatives and previous projections of the origin of chickpea in southern Turkey. Results presented in this report indicate that AFLP analysis is an efficient and reliable marker technology in determination of genetic variation and relationships in the genus Cicer. Obviously, the use of AFLP fingerprinting in constructing a detailed genetic map of chickpea and cloning, and characterizing economically important traits would be promising as well.Communicated by P. Langridge     

Genetic relationships among annual species of Cicer(Fabaceae) using isozyme variation

In order to determine the pattern of genetic diversity within and among the species of Cicer and to estimate interspecific genetic relationships, allelic variation was assayed for 23 isozyme loci in 63 accessions of 11 species of Cicer using starch gel electrophoresis. The total allozymic variation observed in the genus (H _t )was equal to 0.60. When partitioned (G st), 96% of this allelic diversity was found among rather than within species. The allelic diversity among species (D st)and allelic diversity within species (H s)were equal to 0.58 and 0.02, respectively. Cicer reticulatum and C. pinnatifidum had the highest proportion of polymorphic loci (17.39%) and the highest mean number of alleles per locus (1.22 and 1.17, respectively). UPGMA cluster analysis of Nei's unbiased genetic distance revealed four genetic groups. One includes C. reticulatum, C. arietinum and C. echino spermum where the first 2 species represent a putative derivative-progenitor pair. A second cluster contains C. bijugum, C. pinnatifidum and C. judaicum. Cicer yamashitae, C. chorassanicum, C. anatolicum and C. songoricum form a third group. Finally, C. cuneatum, which has a very distinct isozyme profile and peculiar morphological features, is the only member of a fourth species group. This species grouping agrees partially with those obtained from crossability and cytogenetic studies. The results suggest that the annual habit arose from perennial progenitors at least twice in the genus Cicer.     

Genetic relationships in the genus Cicer L. as revealed by polyacrylamide gel electrophoresis of seed storage proteins

Summary Total seed storage protein of the cultivated chickpea, C. arietinum L., and eight other wild annual Cicer species (all 2n = 16) was separated and compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The seed-protein profile was a conservative and species-specific trait. Relative interspecific similarities of protein patterns were estimated using Jaccard's similarity index, and a cluster analysis was performed. The resultant dendrogram generally agreed with the limited data already available on interspecific relationships in Cicer based on morphological characteristics, crossability, genome pairing in hybrids, karyotypes and isozyme analysis. The difference between the profiles of C. judaicum and C. pinnatifidum supported the idea that they are indeed two separate species. The closest relative of C. arietinum was C. reticulatum, followed by C. echinospermum and other species, while C. cuneatum was the farthest relative. In general, C. cuneatum was also genetically the farthest removed from any other species. The suggestion that C. reticulatum is the wild progenitor of the cultivated chickpea was therefore further supported.     

Morphological study of pollens and seeds on annual taxa of the genus Cicer L. (Leguminosae)

Abstract

Morphobiometric analyses of pollens and seeds of 29 accessions and 2 cultivars belonging to 8 annual taxa of the genus Cicer L. revealed 6 pollen and 7 seed types. Keys of identification were established on the basis of the characters which distinguished the morphotypes. Taxa were classified into 4 groups which confirmed their cytogenetic relationships.     

Phylogenetic relationships among annual and perennial species of the genus <Emphasis Type="Italic">Cicer</Emphasis> as inferred from ITS sequences of nuclear ribosomal DNA

The cladistic analysis of the DNA sequences of the internal transcribed spacers of ribosomal cistrons (ITS1 and ITS2) for 20 species of Cicer L. (among which all the annuals), shows that various sections of the genus are not monophyletic. Annual species do not form a clade: C. arietinum, in fact, is closely related to both C. echinospermum and C. reticulatum, whereas C. bijugum, C. judaicum, and C. pinnatifidum form a separate clade. The annual C. cuneatum is sister group to the perennial C. canariense and both are archaic species within the genus. C. yamashitae is, on the contrary, the only annual species belonging to a group of perennials, within which close relationships are evident between C. graecum and C. montbretii as well as among a group of mainly Asian species.     

Evaluation of seven housekeeping genes for multilocus sequence analysis of the genus Mesorhizobium: Resolving the taxonomic affiliation of the Cicer canariense rhizobia

Cicer canariense is a wild chickpea that can be nodulated by Mesorhizobium strains belonging to nine different genomic groups or genospecies. In this study, multilocus sequence analysis (MLSA) of seven protein-coding genes, recA, glnII, dnaK, rpoB, gyrB, truA and thrA, was used to resolve the phylogenetic relationships and taxonomic affiliation of 27 representative strains from all the genotypes. Individual phylogenies were mostly congruent, although there were a few discrepancies. Some genes were more discriminative than others, but concatenation of the seven genes produced a robust phylogeny of the genus Mesorhizobium. MLSA gave good support for the taxonomic affiliations of most of the genomic groups to previously recognized species and delineated several potential new species. Five genospecies found in C. canariense nodules showed average nucleotide identity values for seven genes (ANIg7) of >96% and they could be assigned to previously described Mesorhizobium species. Two large closely related genomic groups had M. caraganae as the closest species and they shared ANIg7 values in the 94–95% range, suggesting that they could be different subspecies or sister species. The predominant genospecies represented a novel monophyletic lineage that was well resolved from all currently recognized species of Mesorhizobium, with the highest ANIg7 below 92%. Other single strains represented potential new species.     

Phytoalexin production by Ononis species

Following exposure to short wavelength (254 nm) ultra-violet light, the detached leaflets of Passaea (Ononis) ornithopodioides and 31 species and subspecies of Ononis have been found to accumulate substantial quantities of the isoflavonoid (pterocarpan) phytoalexin, medicarpin. Apart from P. (Ononis) ornithopodioides, O. cristata, O. fruticosa, O. pubescens and O. rotundifolia, leaf tissues of all the species investigated similarly contained small amounts of the related fungitoxic pterocarpan, maackiain. Isoflavan phytoalexins common in genera such as Medicago and Trifolium (tribe Trifolieae) were absent from both Ononis and Passaea. The phytoalexin data suggest that Passaea should probably be combined with Ononis and, in conjunction with information on constitutive isoflavonoids, that Ononis itself should be assigned to the Trifolieae rather than to the distinct tribe Ononideae. Chemical evidence for and against an especially close taxonomic association between Ononis and Cicer (tribe Cicereae) is also briefly discussed.     

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.