Molecular taxonomy and phylogeny of Silene species (Caryophyllaceae) using DNA based markers
DNA markers have provided valuable tools in various analyses ranging from phylogenetic analysis to the positional cloning of genes. Silene is a genus of flowering plant in the family Caryophyllaceae. This study compared the phylogenetic relationship within and between 16 Silene sections, including 24 species (Silenoideae: Caryophyllaceae) using some molecular markers such as Random Amplification of Polymorphic DNA (RAPD) Microsatellite Primed PCR (MP PCR) Anchored Microsatellite Primed PCR (AMP PCR) andSequence Related Amplified Polymorphism (SRAP) Phylogenetic analysis was carried out by parsimony, and neighbour joining model. Finally, our molecular analysis did not confirm morphological viewpoints completely. *Corresponding Author: Abdolhamid Angaji Angaji@khu.ac.ir Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220 6663 (Print) 2222 3045 (Online) Vol. 4, No. 4, p. 125 132, 2014 http:/www.innspub.net J. Bio. Env. Sci. 2014 126 Ghahremaninejad et al Introduction Silene L. is the largest genus in the family Caryophyllaceae with nearly 700 species world wide (Melzheimer, 1988) is mainly distributed in temperate regions of the northern hemisphere and has its principle centre of diversity in the Mediterranean region and the Middle East. The genus is mostly hermaphrodite although a few species are dioecious or gynodioecious (Bari, 1973; Greuter, 1995) The circumscription of the genus has long been controversial, with an expanding tendency during the past decades (Greuter, 1995; Chowdhuri, 1957; McNeill, 1978; Greuter et al.,1984; Sourkova, 1971) The genus Silene includes several important weedy species, some very beautiful horticultural plants and some medicinal plants (Swank, 1932; Vestal, 1952; Oxelman et al. 1995) The most important characters that separate sections are morphology and venation of the calyx, length and pubescence of the anthophore, shape and size of the corolla and coronal scales, capsule and seed. The genus Silene includes 98 species in Iran with 35 endemic to the Iranian plateau (26 species for Iran) The Iranian species are placed in 21 sections (Melzheimer, 1988) Major areas of species for the Iranian sections occur in west of the country (Edalatiyanet al. 2010) Genetic marker is any trait representing genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as signs or flags (Angaji, 2011) There are three major types of markers: morphological markers, which themselves are phenotypic traits or characters; biochemical markers, which include allelic variants of proteins and enzymes; and DNA (molecular) markers, which reveal sites of variation in DNA (Barh, 2013) Morphological variations, unfortunately, are not sufficient to differentiate between a large number of individuals, or between groups or subgroups within species, simply because the level of polymorphism is low. Besides, morphological characters or phenotypic expressions are affected by environmental changes, so stability of expression is low and sometimes uncertain. Certain disadvantages of morphological markers can be overcome by biochemical markers (Pratik, 2007) Biochemical markers are usually enzyme variants with the same functional role but structurally different. These are highly influenced by environment and relatively few biochemical assays are available (Angaji, 2011) DNA based markers offer several advantages over other markers. They are less affected by environmental factors. There are potentially an unlimited number of molecular markers available. Finally, they are much more polymorphic than other markers (Angaji, 2011; Singh et al. 2013) DNA markers may be broadly divided into four classes based on the method of their detection: 1) hybridization based e.g. Restriction Fragment Length Polymorphism (RFLP) 2) PCR based e.g. RAPD, MPPCR, AMP PCR, SRAP 3) molecular markers based on PCR followed by hybridization, and 4) DNA sequence based markers (Angaji, 2011; Fu et al. 2008) PCR based molecular markers offer the potential to reduce time, effort and expense required for molecular technology and these markers can be generally divided into single primer PCR methods and PCR methods using a pair of primers. This research was carried out by single primer PCR methods, such as MP PCR and AMP PCR, since no prior knowledge of DNA sequence is required for them as well as pair of primers method, for instance Sequence related amplified polymorphism (SRAP) due to more stringency compared to the former ones (Angaji, 2011; Li et al. 2001) Tandem arrays are widely distributed throughout plant and animal genomes that display high levels of J. Bio. Env. Sci. 2014 127 Ghahremaninejad et al genetic variation based on differences in the number of tandemly repeating units at a locus. In MP PCR and AMP PCR, detected polymorphisms are not due to microsatellite length variation in most cases, but are often caused by small insertion/deletion (indel) in intervening sequences (Angaji. 2011; Davilaet al. 1999; Sharma et al. 1995) The rapidly growing amount of gene in databases opened the door to the development of DNA profiling strategies that are directed at specific coding DNA regions of interest. SRAP is a new marker system based on PCR reaction and more reproducible, stable and less complex compared with other molecular marker techniques. Two primers are used each of which consists of the following three elements: 1) an arbitrary filler sequence of 10 to 11 bases at 5' end, 2) the sequence motifs CCGG and AATT in the forward and reverse primer, respectively, and 3) three selective bases at the 3' end. The rationale behind the primer architectures is that exon sequences are known to be more GC rich than other regions of the genome. In contrast, the core sequencesof the second primer (AATT) is designed to bind to AT rich sequences, which are preferentially found in non coding regions(Angaji, 2011; Fu et al. 2008; Ferriol et al. 2003; Budak et al. 2004; Budak et al. 2004; Esposito et al. 2007; Fu et al. 2008) Material and method Plant material and DNA extraction: Herbarium specimens were provided from Herbarium of Kharazmi University (FAR) and Central Herbarium of Tehran University (TUH) The plant samples were used from the below specimens (Table1) The problem of DNA extraction is still an important issue in the field of plant molecular biology. Numerous protocols for DNA extraction from plants have been published (Doyle et al. 1990; Scott et al. 1996; Csaikl et al. 1998; Sharma et al. 2000; Li et al. 2001; Pirttila et al. 2001; Drabkova et al. 2002; Shepherd et al. 2000; Mogg et al. 2003; Haymes et al. 2004) Total DNAs were isolated from leaf tissue by the CTAB method (Saghai Maroof et al. 1984) which is modified by Doyle et al. in1987. Fifty primers were used for amplification, including MP PCR, AMP PCR, SRAP and RAPD. Among them, 36 markers were polymorphic (Table 2, 3) Single primer PCR methods In this study 28 RAPD, MP PCR and AMP PCR primers were polymorphic. Pair of Primers method In this research, SRAP markers were applied as functional markers. Eight polymorphic SRAP were found. PCR amplification and separation for scorning Each PCR amplification reaction micro tube (50mL) contained 5mL of DNA template, 4 mL primer, 28 mL water, and 13 mL Taq DNA polymerase Master MIX Red which cosists of dNTPs, Tris HCL pH 8.5, (NH4)2SO4, MgCl2, 0.2% Tween 20 and Taq polymerase. The PCR amplification was carried out in a Peltier thermal cycler in 35 cycles, denaturation at 95degC for1min, primer annealing at 42degC for 1 min and extension at 72degC for 2 min. Amplification products were analyzed by electrophoresis through 8% (w/v) agarose gel and the gel was cast and run using TAE at 76 V for 30 min and stained by ethidium bromide. Gels were visualized under UV light and photographed and then PCR amplification products were scored as present (1) or absent (0) The data were analyzed by PAUP version 0.4 and shown as Neighbour joining tree. Result Cladistics Following cladogram is according to result, which obtained from the molecular researches on 24 Silene species. This cladogram has shown an interesting shape that caused species placed at different locations. J. Bio. Env. Sci. 2014 128 Ghahremaninejad et al Table 1. List of Specimens were used. Species Author Collector Herbariums numbers Herbarium S.apetala Willd. Atayi 3650 [FAR] S.astro iranica Rech.f. Aell. Esfand Veiskarami 23960 [TUH] S.aucheriana Boiss. Bidarlord 2246 [FAR] S.cappadocica Boiss. Heldr Bidar 2249 [FAR] S.chlorifolia SM. Bidar 2248 [FAR] S.commelinifolia Boiss. without collector 23250 [FAR] S.compacta Fisch. Ghahreman et al. 35576 [TUH] S.conoidea L. without collector 8995 [FAR] S.dichotoma Ehrh Ghahreman&Mozaffarian 17431 [TUH] S.italic (L. Pers without collector 4182 [FAR] S.latifolia Poir Moradi Siadati 40178 [TUH] S.linearis Decne Ghahreman Mozaffarian 5612 [TUH] S.macrowizicii Schischk. Eslami 29572 [TUH] S.marschallii C.A.Mey. Ghahreman Sheikholeslami 6271 [TUH] S.multifida (Adams) Rohrb. Ghahreman et al. 17527 [TUH] S.nana Kar. Kir. Ghahreman et al. 28488 [TUH] S.noctiflora L. Rezvanian 23098 [TUH] S.odontopetala Fenzl Bidar Lord 2266 [FAR] S.persica Boiss. without collector 023254 [FAR] S.schafta J.G.Gmel. ex Hohen Attar Dadjou 14689 [TUH] S.spergulifolia (Willd. M.B. Bidar 2259 [FAR] S.swertiifolia Boiss. Gilani 8221 [FAR] S.tenella C.A.Mey. Bidar 2261 [FAR] S.vulgaris (Moench) Garcke Eslami 29566 [TUH] Discussion In this study, the results of molecular analysis of 24 species of genus Silene were compared with morphological classification, and similarities and differences were reported. The molecular phylogenetic analysis showed that Silene is divided into two major clades with strong support (Fig. 1) and not correspond to previous informal grouping (Melzheimer, 1988) One group contains S.italica and S.linearis. Poly