Abstract: RAPD, ISSR and RFLP were used to study phylogenetic relationships among ten species belong to six genera (Asteraceae) in Egypt. Hundred and thirty one bands were resulted after 15 RAPD primers. The average of similarity coefficient was 0.0-0.286. The DNA of Matricaria recutita L., Achillea fragrantissima (Forssk.) Sch. Bip and Artemisia arborescens L., showed 60, 30 and 23 bands, respectively. Glebionis coronaria (L.) Cass. ex Spach, Art. judaica L., Cotula cinerea Del, Anacyclus monanthos (L.) Thell and Achillea santolina L., showed 7, 5, 3, 2 and 1 bands, respectively, while Cotula barbata DC. and Mat. aurea (Loefl.) Sch. Bip showed no bands. Eighteen bands were resulted after four ISSR primers. The average of similarity coefficient was 0.0-0.25. The DNA of Ach. fragrantissima, Mat. recutita, Art. arborances, Gle. coronaria and Ana. monanthos showed 6, 4, 4, 3 and 2 bands, respectively, while Cot. barbata, Cot. cinerea, Mat. aurea, Ach. santolina and Art. judaica showed no bands. Twenty six RFLP bands were detected after EcoR I and BamH I. The average of similarity coefficient was 0.0-0.5. The band products of the restricted DNA of Ach. fragrantissima, Art. arborescens, Art. judaica, Mat. aurea, Gle. coronaria, Cot. barbata and Cot. cinerea were 6, 4, 5, 4, 3, 2 and 2 bands, respectively, while Ach. santolina, Ana. monanthos and Mat. recutita showed no bands. Based on the present study the species were classified into Mat. recutita as out group; Ach. fragrantissima and Art. arborescens as related group and a group of seven species were closely genetical distance.
INTRODUCTION
Tribe Anthemideae is one of the largest tribes of the family Asteraceae with 1741 species predominantly distributed in Eurasia, North and South Africa, with fewer species in North America and Australia (Bremer and Humphries, 1993) contains many major ornamental and medicinal plants (Tang et al., 2000). Many species are weeds of cultivated lands, disease resistant and have other desirable agronomical traits including Artemisia, Chrysanthemum and Matricaria (Silva, 2004). The principal taxonomic problems within the tribe are entirely of relationships between genera and also circumscription of the genera (Bremer and Humphries, 1993).
Chrysanthemum sensu lato consisted of 27 genera has been taxonomically placed in the subtribe Chrysantheminae O. Hoffm., the tribe Anthemideae Cass (Shih and Fu, 1983) while it has been placed in different subtribes in the tribe Anthemideae (Bremer and Humphries, 1993). The main centers of distribution of Chrysanthemum sensu lato are two regions; one in the Mediterranean area and the other in China and Japan, which are considered as ancestors of the Mediterranean species (Dowrick, 1952). Chrysanthemum sensu lato performs a polyploid series which plays an important role in the evolution of the species (Kondo and Abd El-Twab, 2002; Kondo et al., 2003; Abd El-Twab and Kondo, 2003). In plant evolutionary studies and breeding there is often a need to discriminate between the genomes of closely related genera or species and to identify the ancestors of polyploid species (Orgaard and Heslop-Harrison, 1994; Abd El-Twab and Kondo, 2003). In Egypt, the species of Chrysanthemum sensu lato are represented by 11 genera and 31 species, several species are very rare and became very difficult to find in the natural habitats nowadays (Tackholm, 1974; Zareh, 2009).
Ideal molecular markers are stable and detectable of all plant tissues regardless of growth, differentiation and defense status. These markers should be insensitive to the environment, should lack pleiotropic and epistatic effects and be abundant. Most molecular markers that target nucleic acids fit these requirements and have become important tools for detection and measurement of genetic variation and biodiversity in plants. While a wide range of markers is available scan nucleic acids by amplification, such as Random Amplified Polymorphic DNA (RAPD), which had been developed by using a single arbitrary primer (10 mer) and amplifying DNA by Polymerase Chain Reaction (PCR), the resulting DNA markers can be easily separated on an agarose gel by electrophoresis (Williams et al., 1990; Wolff and Rijn, 1993; Huang et al., 2000) those selectively amplify highly informative genomic regions, such as Simple Sequence Repeat (SSR) marker analysis (Litt and Luty, 1989; Weber and May, 1989) and such as those Restriction Fragment Length Polymorphism (RFLP) analysis (Botstein et al., 1980) which could be used to guide the introgression of target genes from related species (Wolff et al., 1994). In Chrysanthemum traditional techniques markers such as morphological and cytological markers are not useful for breeding analysis (Wolff et al., 1994) therefore another technique for identifying molecular marks as RAPD had been developed and applied (Williams et al., 1990). RAPD method has advantages of being sensitive, quick to perform and applicable to many samples (Babaoglu, et al., 2004). ISSR is a method for specific details (Zietchiewicz et al., 1994) characterized as hyper variable expressed as different variants within population and among different species dispersed throughout the various genomes and circumvents the challenge of characterizing individual loci that other molecular approaches requires (Srivastava et al., 2007). These procedures have the advantages of being sensitive, quick to perform and applicable to many sample. These primers are used as genetic markers in detection of polymorphism without the specific knowledge of nucleotide sequence (Waugh and Powell, 1992).
Molecular DNA markers such as RAPD, ISSR and RFLP methods have never been conducted on the Egyptian species in Chrysanthemum sensu lato. Therefore, this study was conducted in order to; 1) find suitable and reproducible molecular DNA markers, 2) use the DNA markers to assess genetic diversity within and divergence among the congeneric species, 3) to compare between the efficiency of the techniques on our materials and 4) contribute towards a better understanding of the genome relationships and evolution of the species in Chrysanthemum sensu lato distributed in Egypt.
MATERIALS AND METHODS
Plant materials: The plants that were collected by the authors from their natural habitats and used in this study were Achilliea fragrantisma (Forssk.) Sch. Bip, Ach. santolina L., Anacyclus monanthos (L.) Thell., Artemisia arborescens L., Art. judaica L., Glebionis coronaria (L.) Cass. ex Spach., Cotula barbata DC. Cot. cinerea Del and Matricaria aurea (Loefl.) Sch. Bip. and Mat. recutita L. The dried plant materials were preserved in the Herbarium of the Department of Botany and Microbiology, Faculty of Science, Minia University, El-Minia City, Egypt. This study was conducted during 2007-2009, in the Department of Botany and Microbiology, Faculty of Science, Minia University, Egypt.
Plant genomic DNA extraction: Following Sharma et al. (2003) and Abd El-Twab and Zahran (2008) total genomic DNA was extracted from young leaves of the tested taxa. The leaves were kept in fixing solution for 60-90 min (95% alcohol). Treated leaves with the fixative were removed from the solution, allowed to evaporate until dry and homogenized with a mortar and pestle on ice. The homogenized tissue was transferred to preheated 2% CTAB DNA extraction buffer; was incubated in a water bath at 60°C for 1.0 h, occasionally mixing by gentle inversion of the tubes; was removed from water bath and added same volume of chloroform-isoamylalcohol (24:1). Mix by inversion for 10 min Spin at 10,000 rpm for 10 min. The upper aqueous phase was transferred to another tube and this process was repeated until the chloroform-isoamylalcohol layer became clear. Twice the volume of absolute alcohol was added and centrifuged briefly to precipitate the DNA. The DNA pellet was washed with 70% alcohol and the tubes were left to dry at room temperature. The dried DNA was dissolved in TE buffer. 1/10 v of 10X RNAs was added to 0.5 mL of crude DNA, mixed thoroughly but gently and incubated at 37°C for 1 h. 0.3-0.4 mL of chloroform-isoamylalcohol (24:1) was added and mixed thoroughly for 15 min and centrifuged at 10,000 rpm for 10 min. The supernatant was removed to a new tube to precipitate the DNA by using double the quantity of 95% alcohol and centrifuged at 10,000 rpm for 15 min. The pellet was washed with 70% alcohol and left to dry at room temperature. The DNA was redissolved in TE buffer, diluted 1000 times in TE buffer and quantified by taking the Optical Density (OD) at L260 nm with a spectrophotometer. Readings at L260 and L280 was taken to obtain the L260/L280 ratio as an indicator of DNA purity (Sambrook et al., 1989). The purified DNA was observed on 1.5% Agarose gel after staining with Ethidium Bromide to ascertain its integrity.
Random Amplified Polymorphic DNA (RAPD) analysis: Some of the RAPD primers and procedure followed Huang et al., 2000 and Abd El-Twab and Zahran (2008). PCR amplification was done with several primers using 100 ng of genomic DNA (Table 1).
| Table 1: | RAPD and ISSR primers sequence, GC content (%) used in the present study |
The 25 μL PCR mixture contained 2.5 μL of buffer (Taq DNA polymerase complete High Specificity Reaction buffer; as above); 2.5 μL dNTPs (from 10-mM stock, Bioron International, Germany); 12 ng primers (Operon Nippon EGT Co. Ltd.) 1 U DFS-Taq DNA polymerase (Bioron International, Germany) and 100 ng of DNA. The thermal cycler (Thermo Hybaid) was operated as follows: 1 cycle at 95°C for 5 min; 40 cycles at 95°C, 36°C and 72°C for 40 sec, 1 min and 2 min respectively; and a final amplification at 72°C for 10 min.
Inter Simple Sequence Repeats (ISSR) analysis: ISSR procedure followed Dogan et al. (2007) and Abd El-Twab and Zahran (2008). PCR amplification was carried out with several primers using 100 ng of genomic DNA (Table 1). The 25 μL PCR mixture contained 2.5 μL of buffer (Taq DNA polymerase complete high specificity reaction buffer (10X) containing 500 mM KCl, 100 mM Tris HCl pH 8, 0.1% 20 and 15 mM MgC12, Bioron International, Germany); 2.5 μL dNTPs (from 10 mM stock, Bioron International, Germany); 12 ng primers (Operon Nippon EGT CO. LTD.) 1 U DFS-Taq DNA polymerase (Bioron International, Germany); and 100 ng of DNA. The thermal cycler (Thermo Hybaid) was operated as follows: 1 cycle at 94°C ~ for 1.5 min; 35 cycles at 9, 40 and 72°C for 40, 45 sec and 1.5 min respectively; 1 cycle at 94°C for 45 sec; 1 cycle at 44°C for 45 sec and a final amplification at 72°C for 5 min.
Restriction Fragment Length Polymorphism (RFLP: EcoR I and BamH I) analysis
EcoR I analysis: The digestion of the genomic DNA was carried out according
to the manufacturer's protocol (SibEnzyme LTD E057). EcoRI from an Escherichia
coli strain carries the cloned EcoRI gene from Escheruchia coli, was
used to digest total genomic DNA of the plant materials and use the products
as molecular marker. Reaction mixture contain 15 μL of genomic DNA, 15
μL buffer, 5 μL of EcoRI enzyme (SibEnzyme LTD E057) and 15 μL
dist. H2O. One unit is defined as amount of enzyme required to digest
1 μg of λ DNA over night at 37°C in total reaction volume of 50
μL. Enzyme is inactivated by incubation at 65°C for 20 min.
BamH I analysis: The digestion of the genomic DNA was carried out according to the manufacturer's protocol (SibEnzyme LTD E021). BamH I source from An E. coli strain carries the cloned BamH I gene from Bacillus amyloliquefaciens H, was used to digest total genomic DNA of the plant materials and use the products as molecular marker. Reaction mixture contain 15 μL of genomic DNA, 15 μL buffer, 5 μL of BamH I enzyme (SibEnzyme LTD E021) and 15 μL dist. H2O. One unit is defined as amount of enzyme required to digest 1 μg of λDNA over night at 37°C in total reaction volume of 50 μL. Enzyme is inactivated by incubation at 65°C for 20 min.
Gel-electrophoretic analysis: Gel electrophoretic followed Abd El-Twab and Zahran (2008) was used to determine the presence/absence of the total genomic DNA and size of the DNA fragments after RAPD, ISSR and RFLP loaded using loading buffer in 1.5% Agarose Gel, which carry DNA from negative to positive side. DNA was stained in gel by ethidium bromide (0.5 ~μg mL-1), which combined with DNA fragments and give violet light under UV light, at that time; photographs were taken using a digital system.
Analytic programs and calculations
Calculation of DNA concentration and purity: The total genomic DNA concentration
μl mL-1 and purity were calculated by the equations: 50x260
ODx100/1000 and 260/280 L, respectively (Sambrook et al.,
1989).
Data analysis: RAPD and ISSR markers produce DNA amplification signals that can be converted into measurements of similarity or dissimilarity (DNA electrophoretic patterns contain visible bands assigned to specific positions in an individual lane). Pairwise similarity of the genotypes or genetic phenotypes represented in the different lanes can be quantified using indexes or coefficients of similarity. These estimators define genetic distances that portray DNA divergence between organisms in phenetic and cladistic analysis (Huang et al., 2000). For each primer, the consistent amplified products were recorded. The polymorphic fragments (RAPD and ISSR) were named by the primer code followed by the size of the amplified fragment in base pairs. The presence of a specific product (RAPD, ISSR and RFLP) was noted whatever the intensity of the band. Each marker was assumed to correspond to a locus with two alleles (presence or absence of the band). A similarity index S, expressing the probability that RAPD, ISSR and/or RFLP in one taxon is also found in another was calculated according to Nei and Li (1979) for all possible pairwise comparisons between taxa.
Calculation of the similarity coefficient for each primer: 2Nab/Na+ Nb. Nab (means the shared bands between the a,b), Na (bands in species a), Nb (bands in species b) (Nei and Li, 1979; Huang et al., 2000).
Calculation of the similarity matrix (Jaccard): Using SPSS program version 8.0 for windows.
Cluster analysis: PAST computer program was used for a hierarchical clustering analysis based on the unweighted pair group method with arithmetic mean to generate a dendrogram and to describe relationships among genotypes.
RESULTS
Twenty six primers were used for the RAPD analysis and 18 primers for the ISSR, while EcoRI and Bam HI were used for the RFLP analysis to investigate the pattern of genetic variation among ten species related to six genera, which were Ach. fragrantissima, Ach. santolina, Ana. monanthos, Art. arborescens, Art. judaica, Gle. coronaria, Cot. barbata, Cot. cinerea, Mat. aurea and Mat. recutita in Chryanthemum sensu lato growing wild in Egypt. 15 RAPD primers and four ISSR revealed a polymorphism (Table 1). Each of these primers was tested on all samples studied and were selected for genotype analysis because their patterns were reproducible and stable, monomorphic loci were not recorded. Polymorphic bands were selected for identifying the genetic similarity for the group of species. Genetic distances were calculated for all the species studied at RAPD, ISSR and RFLP methods, dendrograms were obtained with the PAST computer program.
One hundred and thirty one reproducible polymorphic bands were resulted after 15 RAPD-PCR primers; those bands were used for studying the genetics similarity among the species. The average similarity coefficient was ranged from 0 to 0.286 (Table 2). The DNA of Mat. recutita, Ach. fragrantissima and Art. arborescens showed 60 (45.8%), 30 (22.9%) and 23 (17.5%) bands on gel electrophoresis, respectively.
| Table 2: | RAPD similarity matrix based on similarity coefficient of the amplified bands for the 10 species |
| Table 3: | ISSR similarity matrix based on similarity coefficient of the amplified bands for 10 species |
| Table 4: | RFLP similarity matrix based on similarity coefficient of the cutting bands for 10 species |
The DNA of Gle. coronaria, Art. judaica, Cot. cinerea, Ana. monanthos and Ach. santolina showed low number of bands 7 (5.34%), 5 (3.81%), 3 (2.29%), 2 (1.52%) and 1 (0.79%) bands, respectively, while the DNA of Cot. barbata and Mat. aurea showed no bands.
Eighteen reproducible polymorphic bands were resulted after four ISSR-PCR primers; those bands were used for studying genetics similarity. The average similarity coefficient was ranged from 0 to 0.25 (Table 3). The DNA of Ach. fragrantissima, Mat. recutita, Art. arborances, Gle. coronaria and Ana. monanthos showed 6 (33.3%), 4 (22.2%), 4 (22.2%), 3 (16.6%) and 1 (0.76%) bands, respectively for each species, while the DNA of Cot. barbata, Cot. cinerea, Mat. aurea, Ach. santolina and Art. judaica did not produce any bands after PCR .
Twenty six reproducible bands and 7 smears of RFLP were resulted after using EcoR I and BamH I to digest the total DNA to detect the fragments length polymorphism among the species on Gel Electrophoresis: EcoR I and BamH I cut the genomic DNA of most of the taxa and produced 13 bands each, 3 and 4 smears respectively. The cutting products of DNA of Ach. fragrantissima was six (23.08%) bands, Art. arborescens four (15.39%) bands and four smears, Art. judaica five (19.23%) bands, Mat. aurea four (15.39%) bands, Gle. coronaria three (11.59%) bands and three smear, Cot. barbata two (7.69%) bands and Cot. cinerea two (7.69%) bands, while the cutting product of Ach. santolina, Ana. monanthos and Mat. recutita were no bands. The similarity coefficient was ranged from 0.0 to 0.5 (Table 4).
Based on the genetic similarity of RAPD, ISSR and RFLP the species were classified into separated groups: RAPD divided the species into three groups, the group 1 has Mat. recutita as out group (clade 1); group 2 has Art. arborescens and Ach. fragrantissima (clade 3) and group 3 has the other seven species (clade 4); Cot. barabata and Mat. Aurea (clade16) showed identical genetical similarity (Fig. 1). The ISSR classified the species into three groups; group 1 has Art arborescens and Ach. fragrantissima (clade2) as out group; group 2 Mat. recutita (clade 4); group 3 has the other seven species of which Cot cinerea, Ach. santolina, Art. judica, Cot. barbata and Mat. aurea (clade 9) showed identical genetical similarity (Fig. 2).
| Fig. 1: | Dendogram resulting from cluster analysis of RAPD based genetics distance matrix for 10 species |
| Fig. 2: | Dendogram resulting from cluster analysis of ISSR based genetics distance matrix for 10 species |
| Fig. 3: | Dendogram resulting from cluster analysis of RFLP ( EcoR I and BamH I) based genetics distance matrix for 10 species |
| Fig. 4: | Dendogram resulting from cluster analysis of RAPD, ISSR and RFLP based genetics distance matrix for 10 species |
The RFLP classified the species into three groups; group 1 has Art. judaica and Ach. fragrantissima (clade 2); group 2 has Art. arborescens (clade 4) and group-3 has the other 7 species of which Mat. recutita, Ach. santolina and Ana. monanthos (clade 12) showed identical genetical relationship (Fig. 3).
The dendogram resulting from the genetic similarity of RAPD, ISSR and RFLP combined together; the species were classified into the following groups: group 1 Mat. recutita as out group (clade1), group 2 Ach. fragrantissima separated from Art. arborescens (group 3) and group 4 has the other seven species that showed closely genetically distance.
| Fig. 5: | Dendogram resulting from cluster analysis of morphological characters, chromosomal number, RAPD, ISSR, RFLP genetics distance matrix for 10 species |
DISCUSSION
RAPD banding patterns have been used for elucidating relationships between various congeneric species (Adams and Demeke, 1993; Campos et al., 1994; Burmmer et al., 1995; Spooner et al., 1996). In this study, for the first time, molecular data have been employed to the present materials that are distributed in Egypt for detecting the taxa relationships, in addition, we combined these molecular markers to the previously studied morphology and chromosome number (Abd El-Twab and Zahran (2008); Zareh, 2009) as markers for elucidating relationships and for generating phylogenetic hypotheses for the plant congeneric species. Elucidating phylogenetic relationships among congeneric species in Egypt is necessary for understanding adaptive radiation and the evolution of characters in the closely related yet often morphologically and ecologically divergent species.
Art. arborescens is a morphologically highly variable species (or mixture of species) with gray-green to silver leaves. It is native to the various habitats of the Mediterranean region, where it occurs as a shrub to one meter in height. The wild species of Art. arborescens may have originated in North Africa or the Middle East (Tucker et al., 1993) and was reported by Delile in Egypt (Pickering, 1854). Abd El-Twab and Zahran (2008) found that the chromosome complement of the Art. arborescens was the longest among the studied taxa and evolutionary was not clear based on the structure of the karyotype, while in the present study the Art. arborescens is always clustering with Ach. fragrantissima indicating closely relationship with it more than other species among the group of species, therefore more studies should be applied on the expected closely related species and comparing the results to justify the species relationship.
The genus Matricaria was established by Linnaeus (1753) and placed by Lessing (1932) and Hoffmann (1894) within their extended sub-tribe Chrysanthemineae. Reitbrecht (1974) placed it in Matricaria-group with Anthemis and Tripleurospermum. Matricaria is represented in Egypt by three species (Tackholm, 1974) this genus already segregated into several genera of which Chlamydophora Ehrenb. Ex Lessing is represented in Egypt by Chlamydophora tridentata. In the present study, Mat. recutita showed genetically distantly relationship as out group of the other 9 species. Therefore, the related genera to Matricaria such as Anthemis, Chlamydophora and Tripleurospermum should be subjected to more studies using the present molecular markers to investigate the genetic similarity and relationships with this genome complex of the species. Previous study (Abd El-Twab and Zahran (2008)) found that the chromosome complement of Mat. recutita had the most variable and advanced karyotype among the diploid species. Based on the present study, Mat. recutita was found to have the highest genetics variable markers and is considered as the most advanced among the present plant group followed by Ach. fragrantissima and Art. arborescens, those three congeneric species considered as the most advanced among the ten species of the present study.
Molecular markers (RAPD) was employed in plant systematic, population biology and constitution of plant genome maps successfully (Williams et al., 1990; Welsh and McClelland, 1990; Spooner et al., 1997; Wolfe and Liston, 1998). Based on the karyotype evolutions as important aspects of the whole evolutionary processes (Imai et al., 1986; Abd El-Twab and Zahran (2008)) and as an isolating mechanism in speciation (Imai et al., 2001). The tetraploid Ach. santolina was considered as the most advanced species, while, among the diploid species, Gle. coronaria was considered the most primitive and Mat. recutita the most advanced (Abd El-Twab and Zahran (2008)). The present study could show the following: (1) Not only supports the speculation of Abd El-Twab and Zahran (2008) for considering the diploid Mat. recutita is the most advanced species among the diploids, but also the most advanced among the whole ten species under investigation; (2) Ach. santolina showed the lowest number of molecular markers variation, this might lend us support to speculate that the ancestor parents of this species is not among the present plant group. Since Ach. fragrantissima the expected related diploid ancestor to Ach. santolina, had quite complex genome and considered genetically distant; (3) Ach. fragrantissima is genetically more closely related to Art. arborescens than to Art. judica, which is genetically distant from Ach. santolina. (4) Mat. recutita and Mat. aurea showed genetically distant to each other, since there was no closely clustering between the two species after using the molecular markers methods supporting genetically and taxonomical separation of the two species into two different genera; (5) Mat. aurea showed genetically closely related with both cot. barbata and cot. cinerea supporting genetically and taxonomical conjugation with the genus cotula and (6) Ach. santolina, Ana. monanthos, Cot. barbata, Mat. aurea, Cot. cinerea, Gle. coronaria and Art. judaica have genetically closely relationships, which might indicate gene flow amongst them.
Applied RAPD (Huang et al., 2000; Chikkaswamy et al., 2007) and ISSR (Kar et al., 2008; Srivastava et al., 2007; Yao et al., 2008; Su et al., 2006) in many plants in different families insure the efficiency of RAPD and ISSR in genomic identification. Compare between RFLP and RAPD in the genetics variation of mitochondrial and nuclear genomes was done (Nakajima et al., 1997). There are several advantages of RAPD and ISSR methods relative to RFLP. The RAPD and ISSR require far smaller quantities of genomic DNA than needed for RFLP analysis. RAPDs reveal high levels of polymorphism even within and among species that show little RFLP (Witkus et al., 1994). In the present study, comparing between the three molecular marker methods of RAPD, ISSR and RFLP found that RAPD was the most effective method to fingerprint the genetic similarity and variability among the taxa, ISSR primers was not so much effective to fingerprint the genomes of all species, so more ISSR primer screening for this group is recommended. RFLP alone was not effective to find the variable genetical separation among the group, since some species did not produce any DNA bands. While segregating all the resulted makers together could justify the genetic similarity and genome variability among the taxa.
According to the genetic similarity of morphological characters of the taxa (Zareh, 2009) and chromosome number (Abd El-Twab and Zahran (2008); unpublished data) the species were classified into two groups: the group 1 has Gle. coronaria, Mat. reutita, Mat. aurea and Cot. cinerea and group 2 has Art. arborescens, Art. judaica, Cot. barata, Ana. monanthos, Ach. santolina and Ach. fragrantissima. Adding the markers of the morphological characters and chromosome numbers to the molecular DNA markers of this study did not change the species clustering except changing the sister cluster of Mat. aurea between Cot. barbata and Cot. cinerea.
Addressing evolution by combination of different experimental approaches might reveal new insights, such as the re-interpretation of chromosome number, size and morphology among the polyploidy species in Chrysanhemum senu lato (Kondo and Abd El-Twab, 2002; Abd El-Twab and Kondo, 2003). Hence, this study aims to contribute towards a better understanding of the phylogenetical evolution of closely related species and genera in Chrysanthemum sensu lato distributed in Egypt. The information that has been presented here and in the previous study (Abd El-Twab and Zahran (2008)) is a forward step towards understanding the variation and phylogenetical relationships among the species. We still in need for more chromosome and molecular studies employing classical cytogenetics, molecular and molecular cytogenetics methods, which might be an important source of information for analyzing taxonomical relationships and evolution of the taxa. Polymorphisms between and within the congeneric species were determined by using RAPD, ISSR and RFLP markers, which are useful for genotype identification, genotype relationship and genetic variability. Obviously, the polymorphism generated by the selected primers reflected genetic isolation between some species and variability among other species genotypes. In Chrysanthemum sensu lato, DNA methodologies have become a clear and powerful impact on understanding of the origin, evolution and genome relationships among the plant species. However, the routine use of molecular DNA markers for identification of plant collections might be very important to get more and better understanding about the genome relationships of the related plant species. Therefore, more studies are planned on the chromosome complements and genomes to clarify and justify the species taxonomical relationships, polyploidization, phylogenetical relationships and evolution of the species in Chrysanthemum sensu lato. Also, we are planning to extend our studies by using the RAPD, ISSR and RFLP markers on the other congeneric species in Chrysanthemum sensu lato distributed in Egypt; also to use those molecular markers for studies of genetic differentiation among the wild species, to identify areas of maximum diversity and to estimate genetic variability in natural populations.
ACKNOWLEDGMENT
We thank Prof. Dr. Hassan S.K.M., head of Department of Botany and Microbiology for his valuable suggestions and support during the course of this study. This study was funded in part by Minia University, for which we are grateful.