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(Rubiaceae) and Related Species Using ISSR and RAPD Markers

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(Rubiaceae) and Related Species Using ISSR and RAPD Markers Biologia 69/3: 300—310, 2014 Section Botany DOI: 10.2478/s11756-013-0314-z Genetic diversity and relationships among Egyptian Galium (Rubiaceae) and related species using ISSR and RAPD markers Kadry Abdel Khalik1,2*, Magdy Abd El-Twab3 &RashaGalal3 1Botany Department, Faculty of Science, Sohag University, Sohag 82524, Egypt; e-mail: [email protected] 2Biology Department, Faculty of Science, Umm-Al-Qura University, Saudi Arabia 3Botany and Microbiology Department, Faculty of Science, Minia University, Egypt Abstract: Genetic diversity and phylogenetic analyses of 24 species, representing nine sections of the genus Galium (Rubi- aceae), have been made using the Inter Simple Sequence Repeats (ISSR), Randomly Amplified Polymorphic DNA (RAPD), and combined ISSR and RAPD markers. Four ISSR primers and three RAPD primers generated 250 polymorphic ampli- fied fragments. The results of this study showed that the level of genetic variation in Galium is relatively high. RAPD markers revealed a higher level of polymorphism (158 bands) than ISSR (92 bands). Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrograms were compared. Six clades can be recognized within Galium, which mostly corroborate, but also partly contradict, traditional groupings. UPGMA-based dendrogram showed a close relationship between members of section Leiogalium with G. verum and G. humifusum (sect. Galium), and G. angusti- folium (sect. Lophogalium). Principal coordinated analysis, however, showed some minor differences with UPGMA-based dendrograms. The more apomorphic groups of Galium form the section Leiogalium clade including the perennial sections Galium, Lophogalium, Jubogalium, Hylaea and Leptogalium as well as the annual section Kolgyda. The remaining taxa of Galium are monophyletic. Key words: Galium; genetic diversity; ISSR; RAPD; Rubiaceae Introduction distributed in temperate and tropical regions of the world (Willis 1985; Mabberley 1987). Galium itself is Rubiaceae is the fourth-largest angiosperm family, com- problematic taxonomically, because taxa from different prising approximately 660 genera and 11,500 species sections exhibit similar habit, many species are widely and classified into 42 tribes (Robbrecht & Manen 2006; distributed and polymorphic, and species groups often Soza & Olmstead 2010a). Most of the family is trop- are poorly differentiated both morphologically and geo- ical and woody. Rubieae is the only tribe centered in graphically (Schischkin 2000). This genus was described temperate regions, but has cosmopolitan distribution. by Linnaeus (1753) who established the occurrence of Most of its members are herbaceous and adapted to 26 species. He divided them into two groups according xeric habitats (Robbrecht 1988; Jansen et al. 2000). to fruit type (glabrous and hispid). Boissier (1881) con- Rubieae is a monophyletic group, sharing both mor- sidered 90 species and divided them into three sections phological and molecular synapomorphies (Manen et al. (Eugalium, Aparine and Cruciata) and 11 subsections. 1994; Natali et al. 1995, 1996; Bremer 1996; Andersson Ehrendorfer et al. (1976) recognized 145 species for Eu- & Rova 1999; Bremer & Manen 2000; Nie et al. 2005; ropean flora, classifying into 10 sections. Ehrendorfer & Backlund et al. 2007; Bremer & Eriksson 2009). How- Sch¨onbeck-Temesy (1982) listed for the flora of Turkey ever, classification and identification within Rubieae 101 species divided into 10 sections. have been problematic, especially for the larger genera In Egypt, Tackholm (1974) named 12 species of Asperula and Galium. A number of taxa within Aspe- Galium, Boulos (1995, 2000) recognized only 10 species. rula appear morphologically similar to Galium, differing Abdel Khalik et al. (2007; 2008a, b, c) studied 13 Egyp- only in corolla tube length, and these have been trans- tian taxa of Galium by different means such as mor- ferred from Asperula to Galium (Ehrendorfer 1958; Na- phological characters, including vegetative parts, flow- tali et al. 1995; Ehrendorfer et al. 2005; Abdel Khalik ers, fruits, seeds, pollen grains, anatomical structure. & Bakker 2007). Numerical analysis was conducted, and they classified Galium L. is one of the largest genera of Ru- these species into groups. bieae with more than 400 species included into 16 sec- Molecular markers are useful in identifying the tions containing annual and perennial herb that are maximally diverse parental genotypes through an eval- * Corresponding author c 2013 Institute of Botany, Slovak Academy of Sciences Genetic diversity and relationships among Galium 301 Table 1. List of the studied species of Galium sited according to traditional Boissier (1881), more recent traditional (Ehrendorfer & Sch¨onbeck-Temesy 1982; Ehrendorfer et al. 2005) and a recent phylogenetic classification based on molecular data (Soza & Olmstead 2010b). No. Taxon Voucher Boissier (1881) Ehrendorfer & Soza & Olm- Present study Sch¨onbeck-Temesy stead (2010b) RAPDs + (1982); Ehrendor- ISSRs fer et al. (2005) 1 Galium aparine L. Egypt, Gebel Elba, Gebel Sect. Aparine Sect. Kolgyda Clade III, sub- Group 6 Ekwal, Abdel Khalik et Subsect. Leucaprinea clade A al., s.n. (SHG) 2 Galium album Mill. XX-0-ULM-2004-F-13: –Sect.Leiogalium Clade III, sub- Group 1 subsp. pycnotrychum No. 2012/451 (ULM) clade D (Braun) Krendl. 3 Galium album Mill. DE-0-B-2040707: No. 2160 –Sect.Leiogalium Clade III, sub- Group 4 A subsp. album (B) clade D 4 Galium angustifolium Kew Garden, Millennium –Sect.Lophogalium Clade VII Group 4 B subsp. angustifolium seed bank, serial number: 0377463(K) 5 Galium asparagifolium GR-0-B-2713189: No. 2162 –Sect.Leiogalium –Group4B Boiss. and Heldr. (B) 6 Galium canum Req. Palestine, Wadi Sawaanit, Sect. Eugalium Sect. Jubogalium –Group4B in rocks, P.H. Davis 5038 Subsect. Chromogalia (K) 7 Galium circae Krendl GR-0-B-2647080: No. 2163 –Sect.Leiogalium -Group4B (B) 8 Galium glaucum L. DE-0-B-1961900:No. 2167 –Sect.Leiogalium –Group4B (B) 9 Galium grande Mc RSABG, EX: 21746, No. –Sect.Baccogalium –Group3 Clatchie 21747 (RSABG) 10 Galium humifusum M. Kew Garden, Millennium –Sect.Galium –Group4B Bieb seed bank, serial number: 0243746(K) 11 Galium lucidum All. IT -0-B-2612099: No. 2169 –Sect.Leiogalium Clade III, sub- Group 1 (B) clade D 12 Galium mollugo (L) All. Netherlands, Gelderland, Sect. Eugalium Sect. Leiogalium Clade III, sub- Group 1 NW of Wolfheze.Open low Subsect. Leiogalia clade D vegetation on sandy soil, C.C.H. Jongkind 5226 (WAG) 13 Galium murale L. Egypt, in cultivated land, Sect. Aparine Sect. Kolgyda Clade III, sub- Group 4 B near Maruit,Letuneux 197 Subsect. Apera clade B (K) 14 Galium obliquum Vill. Kew Garden, Millennium –Sect.Leptogalium –Group4A seed bank, serial number: 0058584 (K) 15 Galium odoratum (L.) DE-0-B-1870409: No. 2172 –Sect.Hylaea Clade III, sub- Group 4 A Scop. (B) clade A 16 Galium parisiense L. Morocco,40kmS.of Sect. Aparine Sect. Kolgyda Clade III, sub- Group 4 A Tiznit on the road to Subsect. Xanthapari- clade B Bou-Izakarm, alt. 1000 nea m, W.J. de Wild and J. Dorgelo 1948 (WAG). 17 Galium scabrifolium GR-0-B-2312983: No. 2176 -Sect.Leiogalium –Group1 (Boiss.) Hausskn (B) 18 Galium schultesii Vest. RO-0-B-0751486: No. 2178 –Sect.Leiogalium –Group4B (B) 19 Galium setaceum Lam. Egypt, Gebel Elba, Wadi Sect. Aparine Sect. Jubogalium –Group5 subsp. setaceum. Yahameeb, N: 22◦ 12 Subsect. Xanthapari- 28,E:36◦ 20 12,Alt. nea 600m, Abdelkhalik 2033 (SHG). 20 Galium sinaicum Egypt, Wadi El Arbaeein, Sect. Eugalium Sect. Jubogalium –Group2 (Delileex Decne) Boiss. Wadi Gragena, St. Ka- Subsect. Chromogalia trein 19.6.2005 Abdel Khalik et al. sn. (SHG) 21 Galium spurium L. Egypt, Sohag city, in gar- Sect. Aparine Sect. Kolgyda –Group4B subsp. spurium. den near Akhmim bridge, Subsect. Leucaprinea Elkordy 1 (SHG). 302 K. Abdel Khalik et al. Table 1. (continued) No. Taxon Voucher Boissier (1881) Ehrendorfer & Soza & Olm- Present study Sch¨onbeck-Temesy stead (2010b) RAPDs + (1982); Ehrendor- ISSRs fer et al. (2005) 22 Galium tricornutum XX-0-BONN-23771 Sect. Aparine Sect. Kolgyda Clade III, sub- Group 3 Dandy (BONN) Subsect. Camptopoda clade A 23 Galium uniflorum Kew Garden, Millennium –Sect.Bataparine –Group4B Michx. seed bank, serial number: 0532334(K). 24 Galium verum L. DE-0-BONN-14328 Sect. Eugalium Sect. Galium Clade III, sub- Group 1 (BONN) Subsect. Chromogalia clade D Table 2. Characteristics of RAPD and ISSR primers sequence and amplification products generated by the studied taxa. Methods Primer name Sequence 5-3 Number of polymorphic Size of DNA fragments (bp) fragments of DNA Primer A GGTGCGGGAA 40 300–1000 RAPD Primer B GTTTCGCTCC 57 200–1200 Primer C GTAGACCCGT 61 200–1500 ISSR 13 GAGGAGGAGGC 15 500–1000 ISSR 15 GTGGTGGTGGC 22 300–1000 ISSR ISSR 16 AGAGAGAGAGAGAGT 50 100–1500 ISSR 17 ATATATATATATATATAG 5 200 Total number of polymorphic bands 250 – uation of genetic diversity which is useful in culti- found by using RAPD and ISSR as the combined results var identification, seed purity analysis and breeding. would be more credible to analyze the genetic structure Among the various molecular markers, Random Am- of Galium species of Egypt and related species. plified Polymorphic DNA (RAPD) and Inter-Simple Se- quence Repeat (ISSR) are simple and quick techniques Material and methods and have become popular as their application does not need any prior information about the target sequences Plant materials in the genome, high-efficiency and sharp sensibility, and The samples of Gallium seeds were taken from wild popu- these techniques have now been widely used for line lations and some herbarium specimens. Voucher specimens identification and genetic diversity. These markers have of the populations studied are deposited in the herbarium of the Department of Botany of Sohag University (SHG) been used for DNA fingerprinting, conservation biol- (Table 1). ogy (Martin & Sanchez-Yelamo 2000; Li et al. 2005), to identify and determine relationships at the species, Plant genomic DNA extraction population and cultivar levels in many plants (Pezh- Total genomic DNA was extracted from germinated seeds. manmehr et al. 2009; Manica-Cattani et al. 2009; Nan These were first ground into a fine powder in liquid nitro- et al.
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