Molecular Systematic of Some Brassicaceae Taxa in Egypt Based on Electrophoretic Isoenzymes Pattern and RAPD Markers

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1 Australian Journal of Basic and Applied Sciences, 3(3): , 2009 ISSN Molecular Systematic of Some Brassicaceae Taxa in Egypt Based on Electrophoretic Isoenzymes Pattern and RAPD Markers Amaal Hasan Mohamed Botany Department, Faculty of Science, Al-Azhar University (for Girls), Cairo, Egypt. Abstract: Molecular systematic for twenty two taxa belonging to 9 tribes of Brassicaceae in Egypt was studied by using electrophoretic pattern of three isoenzymes; Esterase (EC ), Peroxidase (EC ) and Acid phosphatase (EC ) as well as DNA (RAPD) markers techinque. The electrophoresis of three isoenzymes showed that esterase and acid phosphatase could be considered as positive markers while, peroxidase was detected as a negative marker. 87 bands were identified from specimens on electrophoretic gel of DNA (RAPD), 5 bands of them were unique and 82 bands were non unique. The percentage of polymorphic 100%, Data obtained from combination of isoenzymes and DNA were subjected to numerical analysis. The dendrogram resulting from analysis divided the studied taxa into two clusters. Cluster "I" is composed of two groups which include seven tribes with sixteen taxa while Cluster II" comprise the remainders. In essence, the use of fruits and seeds characters was unable to distinguish between even the closely related species. Molecular tools (isoenzymes and DNA (RAPD) markers were consider significant tools for varietal identification and studying evolutionary relationships even between closely related species. Key words: Brassicaceae, fruit and seed morphology, isoenzymes and DNA (RAPD) markers INTRODUCTION Brassicaceae (Cruciferae) consist of 330 genera and 3500 species; in all continents, mainly in temperate areas with highest diversity in Irano-Turanian, Mediterranean and west north America regions (Tai-yien et al., 1987). The family is represented in flora of Egypt by some 53 genera and 103 species (Boulos, 1999). Tai-yien et al., (1987) stated that the delimitation of genera in the Brassicaceae is often difficult because of the frequent independent evolution of what appear to be similar character states, the variability of a given character in one genus and its fixture in another and the inadequate sampling of material by most authors. They added that the most reliable determination of genera can be achieved when the material has both fruit and flowers and when keys for both are successfully used to reach the same genus. Several authors have tried to provide a natural system to classify family Brassicaceae into tribes [Bentham & Hooker (1862), Prantl (1891), Hayek (1911), Candle (1925), Post (1932), Janchen (1942), Rendle (1952) and Zohary (1966)]. These studies were based on a small number of morphological characters such as fruit shape, position of the embryo and cotyledons. However, most of these characters considered, e.g., fruit properties are subject to convergent evolution, at least on the tribal and subtribal level (Post, 1932 and Zohary, 1966). The problem becomes more difficult among the numerous genera with relatively similar linear-shaped fruits (Abdel Khalik et al., 2002). Reconstruction of phylogenies from molecular data has become an important and increasingly common approach in systematic. The product of such studies is a gene tree, hypothesizing relationships among genes or genomes. This gene tree may be fundamentally incongruent with the true species phylogeny, due to various biological phenomena such as introgression, lineage sorting, or mistaken orthology. In such circumstances all of the gene tree characters defining the relationships of molecular taxa (haplotypes) may be necessarily correlated, and the gene or genome may behave as a single species tree character (Doyle, 1992). Recently, the number of available markers in plant taxonomy and biosystematics has been fundamental for plants based on their evolutionary relationships. Molecular taxonomy has been accomplished traditionally through the use of isoenzymes, DNA (RAPD) and more in recent time through various types of molecular markers. Iso-enzymes electrophoresis analysis join with DNA (RAPD) markers are considered as new tools for varietal identification and studying evolutionary relationships even the closely related species. All molecular phylogenetic data show that species with similar fruits and seeds may be unrelated, whereas species with Corresponding Author: Amaal Hasan Mohamed, Botany Department, Faculty of Science, Al-Azhar University (for Girls), Cairo, Egypt. 1499

2 dramatically different fruits and seeds may be very closely related (Koch et al., 2001, 2003 and Beilstein et al., 2006). Among molecular markers, DNA (RAPD) has been extensively used in taxonomic researches as in Rodriguez et al., 1999, Gimenes et al., 2000, Fjellheim et al., 2001, Aliscioni et al., 2003, Singh et al., 2006 and Abbas et al., 2008). Most taxonomic affinity studies of Brassicaceae taxa growing in Egypt have focused mainly on morphology, anatomy, seed scan and limited work has done on seed proteins separation. Although the literature on the Random amplified polymorphic DNA (RAPD) on some species of Brassicaceae growing elsewhere other than Egypt is extensive (Al-Shehbaz (1984), Franzke et al.,1998, Koch et al., 2001 and 2003, Koch & Al- Shehbaz 2002, Koch and Bernardt (2004). O Kane & Al-Shehbaz 2003, Warwick et al., 2006 and Khosravi et al., 2008). It is intended in this work to compare between use of morphological characters of fruit and seed and use of molecular characterization in delimitation of some Brassicaceae taxa; since use of either of morphological characters or molecular structure for a proper delimitation of Brassicaceae taxa is matter of controversy. In the present study, both of morphology (fruit and seed) and molecular structure (electrophoresis of three isoenzymes: Esterase (EC ), Peroxidase (EC ) and Acid phosphatase (EC ) and RAPD markers) were evaluated as a tools of taxa delimitation and to assess relationships between 22 Brassicaceae taxa form flora of Egypt. Moreover, data obtained were subjected to numerical analysis MATERIALS AND METHODS The materials used in this study included 22 taxa of Brassicaceae collected fresh from different localities in Egypt (Table,1), and identified according to plant keys of Post (1932), Zohary (1966), Tãckholm (1974), Davis (1975) and Boulos (1999). Plant names were revised according to checklist of Boulos (1995) and Bisby et al., (2009). - Fruit and seed morphology: Fruit and seed details were examined with the aid of a binocular stereo microscope under incident light and photographed. - Isoenzyme electrophoresis: Seeds of the samples were collected, washed in distilled water, dried and ground to fine powder. Homogeneous polyacrylamide gel electrophoresis (PAGE) was conducted as the method outlined by Stegemann et al. (1988) for iso- enzyme analysis. The gels were stained after electrophoresis; the specific staining solution used for each studied isoenzyme was prepared according to Graham et al., 1964 and Jonathan and Wendel, Gel analysis: All gels resulted from isoenzyme electrophoresis were scanned using Gel Doc-2001 Bio-Rad system. - DNA Extraction: Genomic DNA of the samples under study were extracted according to the method outlined by Junghans and Metzlatt (1990), using extraction buffer containing 50mM Tris-HCL (ph=8.6), 100mM NaCl, 50mM EDTA, 0.5% SDS and 10μl â-mercaptoethanol for 10ml extraction buffer DNA was purified using phenol/chloroform/isoamyl alchohol (25/24/1 - v/v/v), precipitated using cold isopropanol and resolved in TE buffer. Polymerase Chain Reaction (PCR) Conditions: RAPD-PCR reactions were conducted using fifteen arbitrary 10-mer random primers (Operon Technologies, Inc., USA). However, only five primers gave scorable bands and are shown in (Table 2). The amplification was performed in a DNA thermal cycler ( Perkin Elmer Cetus), according to D'Ovidio (1993), under the following conditions: 1 cycle at 94ºC for 2 min followed by 35 cycles for 1 min. at 94ºC (denaturation), 1min. at37ºc (annealing), 2min. at 72ºC (elongation), 1 cycle at 72ºC for five min. and finally, 4ºC PCR products were resolved in 1% agarose gel electrophoresis with 1x TBE running buffer. The run was performed at 100V. for 90 min. The gel was stained with ethidium bromide and bands were detected on UV- transilluminator and photographed by Gel documentation system (Bio- RAD., GEL Doc Analyze 2000). 1500

3 Numerical Analysis: All the examined specimens were used as operational taxonomic units OTU s and analyzed by means of Hierarchical Cluster analysis using Euclidean distance measuring similarity percent. The relationships between the studied taxa of Brassicaceae are demonstrated as dendrogram (Figs. 5) by using statistical program PRIMER (software, version 5). RESULTS AND DISCUSSION 1-Fruit and Seed Morphology: Table 3 and figures 1 & 2 summarize the morphological characters of mature fruits and seeds as clarified by aid of a binocular stereo microscope. 2-isoenzyme Electrophoresis: In the present investigation, three isoenzyme systems, Peroxidase (Prx.), Esterase (Est.) and Acid phosphatase (Acph.) were studied; the results are represented in Figures; 3A, B&C and Table 4. A-Esterase (EC ): Esterase electrophoretic patterns for the twenty two specimens are presented in Table 4 and illustrated in Figure 3-A. The total number of esterase bands obtained by scanning the gel was five distinguished patterns. These patterns indicate that, a total of five bands were identified in the investigated taxa, which were present in some taxa and absent in the others (polymorphic). Exceptional being the bands No. 1 (monomorphic), which were present in all taxa and may be considered positive markers. The highest number of esterase bands (5) was found in Lobularia maritime, Coronopus didymus, Capsella bursa-pastoris, Diplotaxis harra subsp. harra, Eruca sativa, Cakile maritima, Zilla spinosa subsp. spinosa and Enarthrocarpus lyratus which gave maximum gene / gene expression of esterase isoenzyme. On the other hand the lowest number (1 band) was recorded in Sisymbrium orientale, Sisymbrium irio, Eremobium aegyptiacum var. lineare, Matthiola incana, Matthiola longipetala subsp.livida, Farsetia aegyptia, Brassica tournefortii, Brassica nigra, Erucaria hispanica, Raphanus raphanistrum subsp.raphanistrum, Raphanus sativus and Moricandia nitens which, gave minimum gene / gene expression of esterase isoenzyme. B-Peroxidase (EC ): Four groups of peroxidase isoenzyme were electrophoretically detected in the studied specimens (Table 4 and Figure 3-B). The total number of peroxidase groups obtained by scanning the gel of the studied taxa was 1-3 bands. C- Acid phosphatase (EC ): Acid phosphatase electrophoretic patterns for the investigated samples are presented in Table 4 and Figure 3- C. The total number of acid phosphatase groups obtained by scanning the gel was 6 distinguished bands. The highest number of esterase bands (5) was found in Erucaria hispanica which gave maximum gene / gene expression of acid phosphatase isoenzyme. In contrast, the lowest number (1 band) was recorded in Sisymbrium orientale and Matthiola longipetala subsp.livida which gave minimum gene / gene expression of acid phosphatase isoenzyme. In essence, the results indicated that the esterase isoenzymes only have one monomorphic band and acid phosphatase isoenzyme produce one unique band. On the other hand, the percentage of polymorphism detected by three isoenzymes was more in peroxidase isoenzyme than in the other isoenzymes as shown in Table (5). 3. RAPD fingerprints: Randomly amplified polymorphic DNA (RAPD) technique has been used in many different applications involving the detection of DNA sequence polymorphisms. The study includes 5- tested primers to differentiate between the examined taxa (Tables 6 & 7 and Fig.4) The PCR products of the studied primers gave amplification products with the studied taxa. A total of 87 bands were generated using the tested primers, and all these were polymorphic with percentage 100 % (Table 8). The RAPD profiles for all samples generated amplification products size ranged from 2675 bp. to 100 bp. These products gave different bands and ranged from 5 to 8 for different samples. 1501

4 Table 1: List and Collection data of studied taxa of Brassicaceae (Post, 1932) Taxa Locality and date Lane No Groups Tribe Siliquosae Arabideae Matthiola longipetala (Vent.) DC subsp. livida (Delile) Maire. Cairo- Ismailia road, 3/ * Matthiola incana (L.)R.Br. Nasr City-Cairo, 3/ Brassiceae Moricandia nitens (Viv.) Durand & Barratte North coastal, Alex. 3/ Diplotaxis harra (Forssk.) Boiss. subsp. harra Cairo- Suez road, 3/ Brassica rapa L. Nasr City-Cairo, 3/ Al Sharkia, 3/2006 Nobaria, 3/ 2006 Brassica tournefortii Gouan. Nobaria, 3/ Marsa-matruh 3/2006 Brassica nigra (L.) Koch. Al Sharkia, 3/ Nobaria, 3/ 2006 Eruca sativa Mill. Cairo-Alex. Desert road, 3/ Al Sharkia, 3/2006 Nobaria, 3/ 2006 Nasr City-Cairo, 3/ Sisymbrieae Eremobium aegyptiacum (Spreng) Asch. & Schweinf. North coastal, Alex. 3/ ex Boiss. var. lineare (Delile) Zohary Sisymbrium irio L. Nasr City-Cairo, 3/ Al Sharkia, 3/2006 Nobaria, 3/ 2006 Sisymbrium orientale L. Cairo- Suez road, 3/ Lomentaeae Cakilineae Enarthrocarpus lyratus (Forssk.) DC. Al Sharkia, 3/ Nobaria, 3/ 2006 Erucaria hispanica (L.) Druce. Nobaria, 3/ Erucaria microcarpa Boiss. Marsa-matruh 3/ Cakile maritima Scop. Marsa-matruh 3/ Raphaneae Raphanus raphanistrum L. subsp. raphanistrum Al Sharkia, 3/ Nobaria, 3/ 2006 Cairo- Suez road, 3/2006 * Raphanus sativus L. Al Sharkia, 3/ Siliqulosae Alyssineae Lobularia maritima (L.) Desv. Nasr City-Cairo, 3/ Farsetia aegyptia Turra. Cairo- Suez road, 3/ Thlaspideae Coronopus didymus (L.) Sm. Nasr City-Cairo, 3/ Al-Zamalek, 4/ Isatideae Zilla spinosa (L.) Prantl subsp. spinosa. Cairo- Suez road, 3/ Lepidineae Capsella bursa-pastoris (L.) Medik. Al Sharkia, 3/

5 Table 2: List of Primers used in the present study and their nucleotide sequences Primer name Sequences 5 3 OPO -6 CCA CGG GAAG OPO-10 TCA GAG CGC C OPO-11 GAC AGG AGGT OPO-12 CAG TGC TGT G OPO-14 AGC ATG GCT C Table 3: Morphological characters of fruit and seed of the examined specimens of Brassicaceae Taxa Esterase Taxa Sisymbrium irio Sisymbrium Eremobium Matthiola longipetala Matthiola incana Diplotaxis harra Characters orientale aegyptiacum var. lineare subsp. livida subsp. harra Fruit Type 1. siliqua 2. silicle lomenta Shape 1. Linear 2. oblong or linear 3. elliptical or ovate orbicular 5. obcordate Terete 2. Slightly compressed 3. Flattened ± 4-angled 5. Globose Texture 1. smooth 2. Hairy 3. rough- ribbed rough-tubercled Beak length 1. Long (more than 6mm) 2. short (2-6 mm) Very short or Absent (0 2 mm) horn 1. with Horn like 2. without horn seed 2. seedless Seed Seed/cell1. winged 2. wingless many seed seed in 1-row 2. in 2-row one in each cell oblong ellipsoid 2. oblong-ovate 3. globose orbiculate 5. kidney shaped Color 1. brown 2. pale brown 3. dark brown yellowish brown 5. orange-brown Taxa Peroxidase Taxa Brassica rapa Brassica Brassica Eruca sativa Moricandia Farsetia aegyptia Characters tournefortii tournefortii nitens Fruit Type 1. siliqua 2. silicle lomenta Shape 1. Linear 2. oblong or linear 3. elliptical or ovate orbicular 5. obcordate Terete 2. Slightly compressed 3. Flattened ± 4-angled 5. Globose Texture 1. smooth 2. Hairy 3. rough- ribbed rough-tubercled Beak length 1. Long (more than 6mm) 2. short (2-6 mm) Very short or Absent (0 2 mm) horn 1. with Horn like 2. without horn seed 2. seedless

6 Table 3: Continue Seed Seed/cell 1. winged 2. wingless many seed seed in 1-row 2. in 2-row one in each cell oblong ellipsoid 2. oblong-ovate 3. globose orbiculate 5. kidney shaped Color 1. brown 2. pale brown 3. dark brown yellowish brown 5. orange-brown Taxa Acid phosphatase Taxa Lobularia Z. spinosa Coronopus Capsella Cakile Enarthrocarpus R. raphanistrum Raphanus Erucaria Erucaria Characters maritima subsp. didymus bursa-pastoris maritima lyratus subsp. sativus hispanica microcarpa spinosa raphanistrum Fruit Type 1. siliqua 2. silicle lomenta Shape 1. Linear 2. oblong or linear 3. elliptical or ovate orbicular 5. obcordate Terete 2. Slightly compressed 3. Flattened ± 4-angled 5. Globose Texture 1. smooth 2. Hairy 3. rough- ribbed rough-tubercled Beak length 1. Long (more than 6mm) 2. short (2-6 mm) Very short or Absent (0 2 mm) horn 1. with Horn like 2. without horn seed 2. seedless Seed Seed/cell 1. winged 2. wingless many seed seed in 1-row 2. in 2-row one in each cell oblong ellipsoid 2. oblong-ovate 3. globose orbiculate 5. kidney shaped Color 1. brown 2. pale brown 3. dark brown yellowish brown 5. orange-brown The results revealed that five unique (specific) bands are recorded: 2 bands of primer opo6 with mol. size 645bp and 105bp (Cakile maritima and Matthiola longipetala subsp.livida); 2 bands of primer opo 11 with mol. size 2075bp and 1090 bp (Coronopus didymus and Sisymbrium orientale) and one band of primer opo14 with mol. size 1895 bp (Sisymbrium orientale).twenty nine polymorphic bands which can be used as positive marker are present in Sisymbrium orientale, Sisymbrium irio, Eremobium aegyptiacum var. lineare, Matthiola incana, Matthiola longipetala subsp.livida, Farsetia aegyptia and absent in all the reminders. While six polymorphic bands are absent in Sisymbrium orientale, Sisymbrium irio, Eremobium aegyptiacum var. lineare, Matthiola incana, Matthiola longipetala subsp.livida, Farsetia aegyptia and present in all the remainder. 1504

7 Table 4: Electrophoretic patterns of the three isoenzymes of studied taxa of Brassicaceae EST.1 EST.2 EST.3 EST.4 EST.5 Prx.1 Prx.2 Prx.3 Prx.4 Acph.1 Acph.2 Acph.3 Acph.4 Acph.5 Acph.6 Sisymbrium orientale Sisymbrium irio Eremobium aegyptiacum var. lineare Matthiola incana Matthiola longipetala subsp.livida Farsetia aegyptia Lobularia maritima Coronopus didymus Capsella bursa-pastoris Diplotaxis harra subsp. harra Eruca sativa Cakile maritima Zilla spinosa subsp. spinosa Enarthrocarpus lyratus Brassica rapa Brassica tournefortii Brassica nigra Erucaria hispanica Erucaria microcarpa Raphanus raphanistrum subsp.raphanistrum Raphanus sativus Moricandia nitens Table 5: Number, types and percentage of the total polymorphism generated by the three isoenzymes of the studied taxa. Isoenzyme Monomorphic bands Polymorphic bands Total bands % of polymorphism Unique bands Non-unique bands Peroxidase % Esterase 1 _ % Acid phosphatase _ % Table 6: Molecular sizes of amplified DNA bands using Opo- 6 and Opo- 10 primers with the 22 studied Brassicaceae taxa Band No. Opo bp * Band No. Opo bp *(Refer to table (1) for plant names) 1505

8 Table 7: Molecular sizes of amplified DNA bands using Opo-11, Opo- 12 and Opo-14 primers with the 22 studied Brassicaceae taxa. Band No. Opo bpl * Band No. Opo bp * Band No. Opo bp * *(Refer to table (1) for plant names) 1506

9 Table 8: Number, types of amplified DNA bands and percentage of total polymorphism generated by five primers in all studied taxa. Primer code Monomorphic bands Polymorphic bands Total bands Polymorphic Unique bands Non unique bands Opo-6 _ % Opo % Opo-11 _ % Opo % Opo-14 _ % Fig. 1: The morphological features of fruits of some taxa of Brassicaceae. a- Eremobium aegyptiacum var. lineare, b- Matthiola incana, c-enarthrocarpus lyratus, d- Diplotaxis harra subsp. harra, e- Raphanus sativus, f- Farsetia aegyptia, g- Capsella bursa-pastoris, h- Zilla spinosa subsp. spinosa and i-coronopus didymus (scale=1cm). Fig. 2: The morphological features of seeds of some taxa of Brassicaceae. a- Eremobium aegyptiacum var. lineare b- Diplotaxis harra subsp. harra c- Enarthrocarpus lyratus d- Capsella bursa-pastoris e- Matthiola incana f- Zilla spinosa subsp. spinosa g- Farsetia aegyptia h- Lobularia maritima, i- Raphanus sativus and j- Coronopus didymus 1507

10 Fig. 3: Electrophoretic patterns of the three isoenzymes of the 22 studied taxa of Brassicaceae 1-22 (Refer to table (1) for plant names) Opo6 Opo

11 Opo11 Opo12 Opo14 Fig. 4: DNA polymorphism generated by five primers from the genomic DNA of the 22 Brassicaceae taxa. (Refer to table (1) for plant names, M: Marker) Fig. 5: Dendrogram illustrating the relationships of 22 studied taxa of Brassicaceae based on the combination of isoenzymes and DNA data. 4. Cluster Analysis: In the present work, all isoenzyme and DNA data are discussed by the use of Hierarchical cluster analysis. The resulted dendrogram opposed the previous classification in most limits Post, 1932 and Zohary, The dendrograms prove the close relationship between Sisymbrium irio & Eremobium aegyptiacum var. lineare, Matthiola longipetala subsp. livida & Farsetia aegyptia, Enarthrocarpus lyratus & Eruca sativa, Cakile maritima & Lobularia maritima and Erucaria hispanica & Erucaria microcarpa. The dendrogram resulting from analysis divided the studied taxa into two clusters. Cluster "I" is composed of two groups which comprising seven tribes with sixteen taxa at the taxonomic distance level The group "A" includes Erucaria hispanica, Erucaria microcarpa, Brassica rapa, B. tournefortii, B.nigra, Raphanus raphanistrum subsp.raphanistrum and R. sativus, which split off other species at the taxonomic 1509

12 distance of about The group "B" includes Diplotaxis harra subsp. harra, Eruca sativa Enarthrocarpus lyratus, Lobularia maritima, Cakile maritima (3.58 distance level), Zilla spinosa subsp. spinosa (3.87 distance level), Capsella bursa-pastoris (3.92 distance level) and Coronopus didymus (4.06 distance level). Brassica sp. (tribe Brassiceae) with linear siliquosae fruit is more related to Erucaria sp. (tribe Cakilineae) and Raphanus sp. (tribe Raphaneae) with lomentaeae fruit. Farsetia aegyptia (tribe Alyssineae) with siliqulosae, oblong or linear fruit is more related to Matthiola longipetala subsp.livida (tribe Arabideae) with siliquosae and elliptical or ovate fruit Present data give a result supports the view of Koch et al.( 2001& 2003) and Beilstein et al., 2006 and confirm the difficulty of using fruit and seed characters as indicators of relationship. The tribal classification proposed by Post, 1932 and Zohary, 1966 still in widespread use is shown to be a poor reflection of relationship. REFERENCES Abbas, S.T., I.A. Farhatullah, K.B. Khan, Marwat and I. Munir, Molecular and biochemical assessment of Brassica napus and Indigenous campestris species. Pak. J. Bot., 40(6): Abdel Khalik, K., L.J.G. Measen, W.J.M. Koopman and R.G. Van Den Berg, Numerical taxonomic study of some tribes of Brassicaceae from Egypt. Plant Syst. Evol., 233: Aliscioni, S.S., L.M. Giussani, F.O. Zuloaga and E.A. Kellogg, A molecular phylogeny of Panicum (Poaceae: Paniceae): Tests of monophyly and phylogenetic placement within the Panicoideae. Amer. J. Bot., 90: Al-Shehbaz, I.A., The tribes of Cruciferae (Brassicaceae) in the South eastern United States. J. Arnold Arb., 65: Beilstein, M.A., I.A. Al-Shehbaz and E.A. Kellogg, Brassicaceae Phylogeny and trichome evolution Amer. J. Bot. 93(4): Bentham, G. and J.D. Hooker, (1862.Genera plantarum 1: London. Bisby, F.A., Y.R. Roskov, T.M. Orrell, D. Nicolson, L.E. Paglinawan, N. Bailly, P.M. Kirk, T. Bourgoin, J.van Hertum, Eds (009. Species 2000 and ITIS Catalogue of life: 2009 Annual Checklist. CD-ROM; Species 2000: Reading, U.K. Boulos, L., Flora of Egypt.Checklist. Al Hadara publishing Cairo, Egypt., pp: 383. Boulos, L., Flora of Egypt. vol. 1 (Azollaceae Oxalidaceae ). Al Hadara publishing Cairo, Egypt Candle, A.B., The Classification of flowering plants. Cambridge, England. II: Davis, P.H., Flora of Turkey., 1: Edinburgh. Doyle, J.J., Gene trees and species trees: molecular systematics as one-character taxonomy. Syst. Bot., 17(1): D Ovidio, R., Single-seed PCR of LMW glutenin genes to distinguish between durum wheat cultivars with good and poor technological properties. Plant Molecular Biology, 22(6): Fjellheim, S., R.E. Elven and C. Brochmann, Molecules and morphology in concert, II. The Festuca brachyphylla complex (Poaceae) in Svalbard. Amer. J. Bot., 88(5): Franzke. A., K. Pollman, W. Bleeker, R. Kohrt and H. Hurka, 1998.Molecular systematics of Cardamine and allied genera (Brassicaceae): ITS and non-coding chloroplast DNA. Folia Geobot, 33: Gimenes, M.A, C.R. Lopes, M.L. Galgaro, J.F.M. Valls and G. Kochert, Genetic variation and phylogenetic relationships based on RAPD analysis in section Caulorrhizae, genus Arachis (Leguminosae). Euphytica, 116: Graham, R.C., U. Lundholm and M.J. Kamovsky, Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J. Histochem Cytochem, 13: Hayek, A.V., Entwurf eines Cruciferen-systems auf phylogenetischer grundlage. Beih. Bot. Centralbl., 27: Janchen, E., Das System der Cruciferen. Osterr. Bot. Zeit., 91: Jonathan, F.W. and N.F. Wendel, Visualization and interpretation of plants isozymes.1n:isozymes in Plant Biology: D.E. Soltis and P.S. Solits (eds) London, Champan and Hall, Junghans, S. and M. Metzlatt, A simple and rapid method for the preparation of total plant DNA. Biotechniques, 8: 176. Khosravi, A.R., S. Mohsenzadeh, K. Mummenhoff, Phylogenetic position of Brossardia papyracea (Brassicaceae) based on sequences of nuclear ribosomal DNA. Feddes Repertorium, 19:

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