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Geranium subg. Robertium (Geraniaceae) comprises eight sections, of which sect. Batrachioidea contains four species centred in Eurasia, Mediterranean region and the Himalaya Mountains. Three species of Geranium pusillum, G. molle and G. pyrenaicum occur in Iran show some degree of morphological overlaps that make the species delimitation difficult. Moreover, hybrids are known to be formed between these species elsewhere. Till present time, there has been no detailed information available on molecular phylogeny and genetic structure of these species in the country. Therefore, the present study was conducted with the aim to investigate species delimitation by both morphological and molecular data and to reveal genetic diversity and population structure in these three Geranium species. For this study, 216 randomly collected plants from 30 geographical populations in three Geranium species were used. We encountered extensive within species genetic and morphological diversity. ISSR molecular markers could not delimit the studied species. STRUCTURE analysis revealed the occurrence gene flow between these species. The Mantel test showed no correlation between genetic distance and geographical distance of the populations studied. Statistical analysis showed no significant difference between ITS and rbcL sequences and phylogenetic tree was constructed based on combined data set which separated outgroups from the studied species. Genetic affinity of the studied species has been discussed.

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Species delimitation is essential since species is regarded as the basic unit of analysis in nearly all biological disciplines, such as ecology, biogeography, conservation biology, and macroevolution. The genus Geranium (Geraniaceae) comprises about 350 species distributed throughout most parts of the world. The subg. Robertium comprises 30 species which are arranged in 8 sections. This subgenus is represented in Iran by 10 species. These species are grouped into 5 sections. In spite vast distribution of many Geranium species that grow in Iran, there are not any available report on their genetic diversity, mode of divergence and patterns of dispersal. Therefore molecular (ISSR markers) and morphological studies of 147 accessions from 10 species of Geranium (subg. Robertium), that were collected from different habitats in Iran were performed. The aims of the present study are: 1) to find the diagnostic value of ISSR markers in delimitation of Geranium species, 2) to find the genetic structure of these taxa in Iran, and 3) to investigate the species inter-relationship. The present study revealed that combination of morphological and ISSR data can delimit the species. AMOVA and STRUCTURE analysis revealed that the species of subg. Robertium are genetically differentiated but have some degree of shared common alleles.

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Acta Botanica Hungarica
S. Esfandani-Bozchaloyi
M. Sheidai
, and
M. Hassanzadeh Kalalegh

The genus Geranium (Geraniaceae); with about 320 species throughout the temperate regions, is chemically characterised by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. It is necessary to optimise the extraction protocols to reduce the effects of the presence of these compounds to the lowest level.

The present study compares the plant genomic DNA extraction Kit (DNP™ Kit), CTAB DNA extraction method by Murray and Thompson and Sahu et al., from the extracting DNA point of view Geranium species. The results showed significant differences in DNA contents between the three methods. Quantity and quality of extracted genomic DNAs were compared by employing the spectrophotometer, Nano-Drop, agarose gel electrophoresis, and polymerase chain reaction (PCR) methods and molecular marker such as (ITS and trnL-F) and ISSR. The method of Sahu et al., provided the best results (200 ng/µL) in terms of quantity and quality of DNA, therefore, this method was taken and optimised for DNA extraction. Our results proposed that this method could be effective for plants with same polysaccharides, proteins and polyphenols components. The advantage of this method is that it omits the use of liquid nitrogen and toxic phenols which are expensive. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Geranium species group and the reliability of this method has been discussed.

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