A cluster analysis was performed using the unweighted pair group method with arithmetic average (UPGMA) based on simple matching coefficient using XLSTAT software version 2012.3.01 (2). Data were analyzed with the NTSYS-pc software package version 2.10 (29). An additional step of 7 min at Only clear and repeatable amplification products were scored as 1 for present bands and 0 for absent ones. The thermal cycler conditions for PCR reactions were an initial denaturation of 1 min at 94☌ followed by 40 cycles comprising 1 min at 94☌, 1 min at 35-37 ☌ (annealing temperature was optimized for each primer) (Table 2), and 1 min and 30 s at 72☌. PCR amplification was carried out in a PTC- 10096V Thermocycler (MJ Research, Inc, USA). DNA amplification was carried out in 25 μL reactions containing 20 ng of template DNA, 0.2 mM dNTPs, 10μM primer, 2.5 μL of 10× PCR Buffer (CinnaGen, Iran), 3 mM of magnesium chloride, 17.2 μL ddH 2 O and 1.5 unit of Taq polymerase (CinnaGen, Iran). Thirty RAPD primers were initially screened and finally 19 primers that produced scorable polymorphic bands were selected for further analysis (Table 2). The two-way Mantel test (21) for goodness of fit for the UPGMA cluster was also performed using the NTSYS ver. establishing relationships among genotypes using the Genalex ver 6.5 software (28). The extracted DNA was diluted to 20ng/μl and stored at -20☌ for PCR amplification. The DNA concentration was determined spectrophotometrically (Nano Drop 1000) at 260 nm and its quality was checked by electrophoresis on 0.8 % agarose gel. Principal coordinate analysis (PCo) was also carried out for studying correlations among the variables and From each genotyepe, five young leaves were taken and total genomic DNA was isolated from leaves using the CTAB (hexadecyltrimethylammonium- bromide) method (22). A pair-wise similarity matrix was generated based on simple matching coefficient method using software NTSYS ver. All of the 61 morphological characters were converted to bi- and multi-state code. According to the criteria provided by protocols of International Plant Genetic Resources Institute (IPGRI) 61 morphological characters (qualitative or quantitative) of flowers, leaves, fruits and seeds were determined (13). This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Referred to inĬAB Abstracts, Czech Agricultural and Food BibliographyĪll content is made freely available for non-commercial purposes, users are allowed to copy and redistribute the material, transform, and build upon the material as long as they cite the source. Similarity CheckĪll the submitted manuscripts are checked by the CrossRef Similarity Check. Keywords: clover isozymes morphology PAGE polymorphism References:Īsk for email notification. The rich isozyme variability present among the Algerian clover species indicates that they can provide good gene resources for breeding. The clustering pattern of isozyme markers was incongruent with the groupings based on quantitative traits. A considerable number of species-specific zymograms were detected which can be used for the species identification. The pairwise Jaccard’s similarity coefficient ranged between 0.10 and 0.60, indicating that the collection represents genetically diverse species. Phenotype diversity of isozyme markers ranged from 0.07 to 0.61 with an average of 0.31 based on the polymorphic information content. The two isozyme systems analysed, esterase (EST) and glutamate oxaloacetate transaminase (GOT), proved polymorphic. No significant relationship between the environment of the collection site and morphological features was detected. Most of morphometric characters contributed to the discrimination of the species. Genetic variation within and among fifteen Trifolium species represented by 157 accessions was assessed using morphological and isozyme markers.
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