Introduction
The genus Cicer comprises 43 annual and perennial species. The nine annual species have been subdivided into 4 groups by means of crossability, karyotype (Ocampo et al., 1992; Abbo et al., 1994), isozyme (Tuwafe et al., 1988; Gaur and Slinkard, 1990 a; b; Ahmad et al., 1992; Labdi et al., 1996), seed storage protein (Ahmad and Slinkard, 1992) and DNA markers (Choumane et al., 2000). The first group contains the cultigen Cicer arietinum (chickpea) and its presumable progenitor C. reticulatum and C. echinospermum. The perennial C. anatolicum has also been grouped with these species based on isozyme similarities estimates (Kazan and Muehlbauer, 1991) and sequence tagged microsatellite sites (STMS) conservation (Choumane et al., 2000). The second group comprises C. bijugum, C. pinnatifidum, C. judaicum and C. yamashitae. The remaining two annuals, C. cuneatum and C. chorassanicum, cannot be crossed with each other or with any other species, therefore make the third and fourth group (Kazan and Muehlbauer, 1991).
Chickpea is the third most important pulse crop of the world, providing high quality protein in a vegetarian diet. A part of the human consumption, it's also used as feed for livestock and contributes substantially to soil nitrogen (Patankar et al., 1999). The productivity of chickpea is, however, restricted, due to various biotic and abiotic stresses. Ongoing crop cultivar improvement is dependent on a continued supply of genetic variability. Resistance genes from alien resources do become necessary. This is most easily accessed from the primary gene pools of a particular crop. Several wild species of Cicer possess disease resistance and other characters of value in chickpea improvement (Singh and Ocampo, 1997; Reddy and Nene, 1978). Therefore, a clear understanding of the genetic relationships among various species is essential for successful and efficient utilization of the genetic variability present in related wild species. The advent of molecular techniques has opened up new avenues for studying genetic variability between losely related species and accessions. They allow the direct detection of variation at DNA level.
In 1990, a technique for detecting polymophism at DNA level using the polymerase chain reaction (PCR) with single short oligonucleotides primer of arbitrary sequences was described (Williams et al., 1990). This method allows the amplification of discrete fragments of DNA to produce random amplified polymorphic DNAs (RAPD). It enables the detection of informative genetic markers at a large number of loci in both coding and non-coding regions of the genome (Williams et al., 1990; 1993; Quiros et al.,1993). The RAPD procedure has been used in many different crops and for many purposes: to identify DNA markers for resistance to downy mildew of lettuce (Paran et al., 1991), rust and common bacterial blight resistance genes of common bean ( Bai et al., 1997), Fusarium vascular wilt and frost injury in Lentil (Eujayl et al., 1998; 1999), to identify varieties in grapeveine (Vidal et al., 1999) to genotype pathotype diversity in ascochyta blight pathogen (Udupa et al., 1998), to determine genetic diversity, molecular phylogenics and systematics (Kazan et al. 1993; Howell et al., 1994; Orozco-Castillo et al., 1994; Marillia and Scoles, 1996; Tartineni et al., 1996; Baum et al., 1997; Choumane et al., 1998; Ferguson et al., 1998; Mignouna et al., 1999; Forapani et al., 1999; Jain et al., 1999; Rodriguez et al., 1999; Moller and Schaal, 1999)
The objectives of this study were to assess the feasibility of the RAPD approach for genotyping the annual species, estimating the genetic diversity existing between and within them and establishing a phylogenetic relationships among the annual Cicer species. The over-all aim of our work is the characterization of marker-assisted of the primary and secondary gene pool of chickpea for the improvement of chickpea.
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Materials And Methods:
Plant Material
Seeds from eight wild Cicer species and chickpea accessions were obtained from the Genetic Resource Unit (GRU) of the International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria. Seedlings were grown in the plastic house. Five accessions per species and three plants per accession were used for analysis. Species and accession identification numbers are listed together with their geographical origin in Table 1.
Dna Extraction, Rapd Amplification And Agarose Gel Electrophoresis:
Total DNA was extracted from young leaves from each plant using the CTAB method (Rogers and Bendich, 1985).
DNAs from all genotypes were amplified using twenty primers , except C. chorrassanicum which was analyzed with only 15 primers from Operon Technologies Inc. (Alamenda, California., USA) (Table 2). All amplification reactions were performed using a DNA thermo-cycler Perkin Elmer Cetus 9600, according to Williams et al., (1990), in a final volume of 15?l and contained 10 pmol of the 10-mer Operon primers, 40 ng of template DNA, 200 ?m of each d NTPs, 1.5 mM MgCl2 and 1unit of Taq DNA polymerase. After initial denaturation for 4 min. at 94oC the reaction was subjected to 40 cycles of 30sec. at 94oC, 1min. at 36oC and 2 min. at 72oC , followed by 10min. at 72oC. Amplification products were separated by electrophoresis on 1.2% agarose gels made in 1X TAE buffer (0.04 M Tris-Acetate, 0,001M EDTA, pH=8) under constant voltage. Gels were visualized by ethidium bromide staining.
Table 1: Species and number of accessions used in this study and their geographical distribution
Table 2: Name and sequences of Operon primers used in the analysis of Cicer species
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Results And Discussion
Inter And Intraspecific Variability:
Twenty Operon primers were used to detect specific bands and to estimate the genetic distance between the different wild species. The reproducibility of the results were assured by observing the same results three times for some randomly selected samples. Abundant polymorphism was detected by all primers used in this study, but only distinct bands were used for analysis. The number of fragments amplified by a primer ranged from 8 with OPA-05 to 16 with OPO-05 with an average of 11.3 polymorphic fragments per primer.
Most of the primers generated specific patterns, allowing the distinction between C. arietinum and the others species. These patterns varied in number and size of amplification products detected. For example, 11 polymorphic fragments were detected with OPC-04, resulting in 7 different patterns (Table 3). Therefore, use of one primer such as OPC-04 is sufficient to distinguish between different species and produce specific pattern for each species. C. arietinum, C. reticulatum and C. echinospemum, which represent the first crossability group, were grouped at 0.3 genetic similarity with this primer (OPC-04). More primers are required to distinguish a species from another. These kinds of primers are very informative as they can produce specific markers which could be easily used in the distinction between parents in an inter-specific cross and in the analysis of the derived segregating populations.
Table 3: Number and size of fragments amplified with OPC-04 in the different annual species from Cicer.
Filled cells in the table reflect an amplified product at the molecular size mentioned. Empty cell refect the absence of amplification product.
The analysis of the different genotypes with twenty primers provided an inetersting number of specific markers. These results are presented in Table 4, where the useful primers that distinguish between C. arietinum and other species of interest are displayed.
To confirm the specificity of these markers, 15 plants from five accessions of each species were analysed. Some special fragments were considered as specific markers because they were present in all genotypes of a certain species and absent in other species. This analysis demonstrated different level of variability existing within a species. The highest value of intra-specific genetic variability was detected in C. pinnatifidum while C. arietinum accessions and plants showed a very low level of variability.
Table 4: Analysis of DNA from different wild Cicer species with 20 Operon primers.
+ indicate that the primer concerned is able to distinguish between C.arietinum and the given species.
Genetic Relationships Between The Annual Species Of Cicer:
The DNA from the nine annual species was amplified with fifteen Operon primers. After the separation of amplification products on agarose gels, bands were scored as present (1) or absent (0) according to their molecular weight. 169 polymorphic fragments were scored. Fragments with the same molecular weight , although, they could be different in their sequences, were considered similar. These results were used as a matrices to estimate the genetic distance and the index of similarity between C. arietinum and the wild annual species. Statistical analysis was performed using Numerical Taxonomy and Multivariate Analysis System (NTSYS-pc, Rohlf,1993) and Nei's coefficient was used for genetic dissimilarity ( Nei and Li, 1979). Cluster analysis was carried out using the unweighted pair group mean average method (UPGMA) (Sneath and Sokal, 1973). Based on the calculated genetic distance presented in Table 5, an estimation of the relationships with different species could be concluded. It showed that the smallest genetic distance was observed between C. arietinum and C.reticulatum (0.204) while the highest one was found between C. arietinum and C.cuneatum (0.894).
Table 5: Genetic distance between the annual species of Cicer based on RAPD data.
Based on genetic dissimilarity given in table 5 a dendrogram of annual species was generated (Figure 1). Although all wild species were clearly separated from the cultivated C. arietinum , the nine species were regrouped into four distinct clusters. The first group assembled C. arietinum, C. reticulatum and C. echinospermum with stronger relationship of C. arietinum with C. reticulatum, which is considered as the presumable ancestor of C. arietinum, than that with C. echinospermum. The second group includes C. judaicum, C. pinnatifidum and C. bijugum where C. pinnatifidum is more close to C. judaicum than C. bijigum. The third group includes C.chorassanicum and C.yamashitae, and the species C.cuneatum form alone the fourth group, which is distinct from all other species.
Figure 1: Dendrogram of genetic relationships between the annual species of Cicer based on RAPD data:
At a first glance these data seem to reflect phylogenic relationships between the species as determined by other criteria (Kazan and Muehlbauer, 1991; Labdi et al., 1996; Choumane et al., 2000) . The tree established with RAPD results was identical to that based on isozymes (Labdi et al., 1996), but vary slightly where compared to the results based on STMS marker, where no great distance was detected between the species from the second and the third groups( Choumane et al., 2000).
In all previous phylogenic trees, the species forming the first crossability group were regrouped together. This was also confirmed in this study, which supports the idea that C. reticulatum is the progenitor of C. arietinum and that C. echinospermum split off in an earlier stage of evolution. On the other hand, C. cuneatum is placed as the most distant species to the cultigen C.arietinum. These results add an evidence to support the doubts that this species (C. cuneatum) belongs to Monocicer section, where all annual species (except C. chorassanicum) are placed.
Despite some criticisms about the RAPD technique (Rieseberg, 1996; Karp et al..,1997), our experience showed that RAPD pattern can be reproduced and results obtained with this technique agreed with those obtained by biochemical and other molecular techniques. RAPD provides an efficient and inexpensive way to generate molecular data and produces important inter-specific markers which could be a very useful tool in an inter specific breeding program in the genus Cicer.
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