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     

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.     

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.     

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.     

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

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

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.     

Transferability of sequence tagged microsatellite site (STMS) primers across four major pulses

The transferability of genome-specific sequence tagged microsatellite site (STMS) primers from field pea (P. sativum) and chickpea (C. arietinum) to other major pulses was examined. Overall, field pea STMS primers amplified products in most of the accessions in comparison to that of the chickpea STMS primers, which amplified products in relatively few accessions. The highest level of successful amplifications with a single primer was 89% for field pea and 33% for chickpea primers respectively. The potential transferability of the STMS primers among species, expressed as the total mean percentage of positive amplifications, was 53% for the field pea STMS primers and 9% for the chickpea STMS primers. The individual mean percentage of successful transferability of field pea STMS primers across lentil, vetch and chickpea/Cicer sp. accessions was 60%, 39% and 62%, respectively. Whereas, for the chickpea STMS primers successful transferability was 5%, 3% and 18% for lentil, vetch and field pea, respectively. The trnasferability of these STMS primers indicates a high level of sequence conservation in these regions across species. Together with their locus-specificity, co-dominant nature and potential to amplify multiple alleles, their transferability makes STMS markers a powerful tool for genetic mapping, diversity analysis and genotyping.     

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.     

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.     

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.     

Use of Random Amplified Polymorphic DNA Markers for Mapping the Chickpea Genome

Three interspecific crosses were developed using Cicer arietinum (ICC 4918) as the female parent and wild Cicer species [C. reticulatum - JM 2100, JM 2106 and C. echinospermum - ICCW 44] as the male parent. Cicer arietinum (ICC 4918) × C. reticulatum (JM 2100) cross produced the largest number of F₂ plants and was chosen for linkage mapping using Random Amplified Polymorphic DNA (RAPD) primers. A partial linkage map was constructed based upon the segregation of 36 RAPD markers obtained by amplification using 35 primers. The linkage map consists of two linkage groups with 17 linked markers covering a total of 464.9 cM. Analyses also revealed association of three morphological traits with linked RAPD markers. Out of seven morphological traits tested for association with linked markers in the segregating plants, four Quantitative trait loci (QTL) were detected for the trait leaf length and three QTLs each for the traits leaf width and erect plant habit.     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

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.     

Genetic relationships among the annual species of Cicer L.

Summary Genetic relationships between 7 annual species of the genus Cicer, including the cultivated chickpea, have been studied. These species were assigned to 3 crossability groups. In each group interspecific hybrids could be obtained but their fertility differed considerably in the various cross combinations. Crosses between members of different groups yielded no viable seeds. The possibility of gene transfer from the wild species to the cultivated chickpea C. arietinum was also assessed. Only two species could be considered for this purpose, C. reticulatum, which is the wild progenitor of the cultivated species, and C. echinospermum, which is in the secondary gene pool of C. arietinum. A unique postzygotic reproductive barrier mechanism was found between the members of Group II, C. judaicum, C. pinnatifidum and C. bijugum. It is based on a disharmony in the growth rate of the stigma and the anthers at the time of anthesis of the F₁ interspecific hybrid so that selfpollination is avoided. It is proposed that this kind of mechanism has been involved only when an effective spatial isolation between the three species had been obtained.     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Allelic variation at (TAA)n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm

A set of 12 randomly selected (TAA)_n microsatellite loci of the cultivated chickpea (Cicer arietinum L.) were screened in a worldwide sample comprising 72 landraces, four improved cultivars and two wild species of the primary gene pool (C. reticulatum and C. echinosperum) to determine the level and pattern of polymorphism in these populations. A single fragment was amplified from all the accessions with each of 12 sequence-tagged microsatellite site markers, except for one locus where no fragment was obtained from either of the two wild species. There was a high degree of intraspecific polymorphism at these microsatellite loci, although isozymes, conventional RFLPs and RAPDs show very little or no polymorphism. Overall, the repeat number at a locus (excluding null alleles) ranged from 7 to 42. The average number of alleles per locus was 14.1 and the average genetic diversity was 0.86. Based on the estimates obtained, 11 out of the 12 frequency distributions of alleles at the loci tested can be considered to be non-normal. A significant positive correlation between the average number of repeats (size of the locus) and the amount of variation was observed, indicating that replication slippage may be the molecular mechanism involved in generation of variability at the loci. A comparison between the infinite allele and stepwise mutation models revealed that for 11 out of the 12 loci the number of alleles observed fell in between the values predicted by the two models. Phylogenetic analysis of microsatellite polymorphism in C. arietinum showed no relationship between accession and geographic origin, which is compatible with the recent expansion of this crop throughout the world. Received: 18 September 1998 / Accepted: 2 December 1998     

ITS-PCR sequencing approach deciphers molecular phylogeny in chickpea

Twenty chickpea accessions belonging to ten different countries of the world have been subjected to phylogeny and length variations from nuclear ribosomal DNA (nr DNA). ITS1–5.8S–ITS2 regions of Cicer accessions were used for amplification in two sets, each comprising a reverse and forward ITS primers. Lengths of ITS-1 of C. arietinum and C. reticulatum ranged from 340 to 350 bp whereas that of ITS-2 from 400 to 410 bp. In all the 20 accessions investigated, GC content in ITS-1 ranged from 40 to 55% and in ITS-2 from 42 to 55%. Sequencing of polymerase chain reaction product from ITS-1 showed variability in 278 and 290 bp due to adenine and guanine nucleotide base pairs. BLAST search for ITS-1 region revealed highest homology (99%) with four strains of C. arietinum accessions. Whereas, ITS2 showed 100% homology with C. arietinum and 99% homology with that of C. echinospermum.     

Allozyme variation and phylogeny in annual species ofCicer (Leguminosae)

Allozymic variation at 30 isozyme loci was examined electrophoretically in nine annual and one perennial species ofCicer. While most of the accessions examined were monomorphic, species can be differentiated on the basis of their enzyme phenotypes. Several groups of species were identified based upon genetic distance values. For example,C. arietinum, C. reticulatum, andC. echinospermum shared the same alleles for most of the loci exmained. PerennialC. anatolicum is also closely related to this group. Similarly,C. judaicum, C. bijugum, andC. pinnatifidum formed another group. Two annual species,C. chorassanicum andC. yamashitae clustered together, whereasC. cuneatum was the most distantly related species. Correlations were found between genetic distances and geographic distribution. Results from enzyme electrophoresis tend to support the previously reported taxonomic treatments based upon crossability and morphological similarity. However,C. yamashitae, which has been classified in the second crossability group, is quite distinct genetically and morphologically from the remaining species of the group. An isozyme gene duplication observed in the genus suggested the monophyletic origin of the species examined in the present study.     

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.     

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