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VELICEVICI GIANCARLA, MADOŞĂ EMILIAN, ŞUMĂLAN RADU, CIULCA SORIN,POPESCU SORINA, PETOLESCU CERASELA high degree of genetic relationship among the different varieties, and the high degree of polymorphism within barley varieties. Molecular markers had been proved to be valuable tools in the characterization and evaluation of genetic diversity within and between species and populations. The advent of the polymerase chain reaction (PCR) favored the development of different molecular techniques such as random amplified of polymorphic DNA (RAPD) [1,31], simple sequence repeats (SSR or microsatellite) [17], the sequence-tagged sites (STS), random amplified microsatellite polymorphism (RAMP), and inter-simple sequence repeat polymorphic DNA (ISSR) [24 ]. Those molecular markers had been used in barley for detecting genetic diversity (Ciulca, 2010), genotype identification, and genetic mapping [10, 17, 27]. Genetic analysis with RAPD markers is relatively easy, fast and efficient, therefore it has been extensively used to determine genetic diversity among barley varieties and to identify the best quality ones for malting [9, 10, 27]. Nevertheless, there are few studies about genetic characterization of hull-less barley [32]. Determination of genetic variability among genotypes is useful for both practical applications in a breeding program and cultivar protection. A variety of approaches, from morphological data to molecular markers including RAPD [4, 13, 33], AFLP [1], SSR [14, 15 ] and ISSR [13, 22 ] markers have been applied for assessment of genetic variability among genotypes. ISSR markers, which involve PCR amplifications of DNA using a primer, composed of a microsatellite sequence anchored at 3′ or 5′ end by 2–4 arbitrary, could be used to assess genetic diversity [28]. ISSRs had been used for cultivar identification for potatoes [23], wheat [21], bean [18], and barley [10, 26]. The understanding and knowledge of genetic variation and genetic similarities present within individuals or populations are useful for the efficient use of genetic resources in a breeding program. The breeder can use the genetic similarity information to complement phenotypic information in the development of breeding populations [11]. In this study, we evaluated genetic diversity using RAPD and ISSR markers, in order to establish a base line to assist future conservation and breeding programs of this species. Also we aimed to report the usefulness of RAPD and ISSR for the assessment of genetic diversity. Materials and methods Plant material. Ten leaves from each of the 19 cultivars were collected separately and stored immediately at-80 o for DNA extraction. The cultivars numbers and country of origin are listed in (Tab.1). Table 1. Studied barley cultivars and their geographical origins code. Genotypes Locatian code. Genotypes Location 1 Orizont Romania 11 Plaisant Hungary 2 Dana Romania 12 Tas Hungary 3 Precoce Romania 13 Secura Austria 4 Adi Romania 14 Dina Germany 5 Mădălin Romania 15 DH19/1 Romania 6 Andrei Romania 16 DH 254/10 Romania 7 Regal Romania 17 DH 260/18 Romania 8 Compact Romania 18 DH 260/12 Romania 9 Gerbel Germany 19 DH 261/22 Romania 10 Lyric France 7494 Romanian Biotechnological Letters, Vol. 17, No. 4, 2012
The use of RAPD and ISSR markers for genetic diversity among some barley cultivars DNA extraction Total genomic DNA from fresh leaves was extracted using cetyltrimethyl ammonium bromide (CTAB protocol). DNA concentration was determined by both spectrophotometry at 260 nm and by 2% agarose gel electrophoresis. Primers A set of 13 RAPD primers was screened using ten DNA samples from each cultivar and on the basis of that preliminary data, 9 polymorphic RAPD were chosen, and 7 primers were tested for ISSR, and 5 were chosen. In table 2 were presented the sequences of the RAPD and ISSR primers used for detection of the genetic polymorphism. Table 2. Primers sequences used for PCR analysis Primers RAPD code Nucleotide sequences Primers ISSR code Nucleotide sequences (5’-3’) (5’-3’) P-1 ACACAGAGGG HB-12 (CAC) 3 GC P-16 TCGGCGGTTC HB-14 (CTC) 3 GC P-17 CTGCATCGTG HB-15 (GTG) 3 GC P-27 CCGTGCAGTA 810 (GA) 8 T OPA-05 AGG GGT CTT G 811 (GA) 8 C OPA-11 CAA TCG CCG T OPG-02 GGCACTGAGG E-6 AAGACCCCTC A-16 AGCCAGCGAA RAPD- PCR reaction The amplifications were carried out in a 25 μl PCR buffer containing: GoTaq® Green Master Mix ready-to-use solutions (GoTaq® DNA Polymerase, dNTP, MgCl2, and reactions buffers at optimal concentrations for efficient amplification of DNA templates), RAPD primers, DNA template and Nuclease-Free Water. GoTaq® Green Master Mix contained two dyes (blue and yellow) that allow monitoring of progress during eletrophoresis. DNA amplification was carried out using a Thermalcycler by Corbett and reactions were submitted to the following PCR program: preliminary DNA denaturation step at 94°C for 4 min, followed by 35 cycles at 94°Cfor 1 min., 35°C for 1 min. and 72°C for 2 min. A final extension for 4 min at 72°C was included. The RAPD products were separated by 2% agarose gels electrophoresis (3V cm-1), which run with 1xTAE buffer. The PCR marker (1000-50bp) was also run on each gel as a molecular weight standard. Photo documentation was performed under UV light using a photo imaging system. ISSR-PCR reaction The amplifications were carried out in a 25 μl PCR buffer containing: Go Taq® Green Master Mix ready-to-use solutions (GoTaq® DNA Polymerase, dNTP, MgCl2, and reactions buffers at optimal concentrations for efficient amplification of ADN templates), ISSR primers, DNA template and Nuclease-Free Water. GoTaq® Green Master Mix contains two dyes (blue and yellow) that allow monitoring of progress during electrophoresis. DNA amplification was carried out using a Thermalcycler by Corbett and reactions were submitted to the following PCR program: preliminary DNA denaturation step at 94°C for 5 min, followed by 40 cycles at 94°C for 30s, a primer annealing step at 52°C for 45 s. and an extension at 72°C for 2 min, then a final extension was carried out at 72°C for 6 min. The ISSR products were separated by 2% agarose gels electrophoresis (3V cm-1), which run with 1xTAE buffer. The standard marker used as marker of ADN quantity was PCR marker. Photo documentation was performed under UV light using a photo imaging system. Romanian Biotechnological Letters, Vol. 17, No. 4, 2012 7495
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