Evaluation of Genetic Diversity of Iris Genotypes (Iris spp) Using ISSR

Some of Iris species are growing in different parts of the Iran as wild species. Iris species have important medicinal and horticultural properties. Understanding of the genetic variation within and between populations is essential for the establishment of effective and efficient methods for conservation of the plants. Genetic variation studies are fundamental for the management and conservation of this species. The use of molecular markers is a powerful tool in the genetic study of populations. The use of DNA marker, such as AFLP, SSR, RAPD and ISSR represents an alternative method in detection of polymorphism. ISSRs are highly variable, require less investment in time, money and labor than other methods. ISSR can generate higher percentages of polymorphic loci than other PCR methods. These can serve as an efficient tool for phylogenetic studies. ISSRs had reported that used in studies of cultivated species to produce genetic linkage maps and to determine the relatedness of lines of agriculturally important species. ISSR analysis involves the PCR amplification of regions between adjacent, inversely oriented microsatellites, using a single simple sequence repeat (SSR) motifs (dinucleotide, trinucleotide, tetranucleotide or penta nucleotides). Therefore, little is known about the genetic variability of the Iranian Iris ssp .The objectives of this study were to evaluate genetic diversity among genotypes using ISSR markers and the degree of polymorphism generated from ISSR technique as a pre-requisite for their applicability to population genetics studies in Iris ssp.
To evaluate genetic variations in some wild Iris genotypes, Iris kopetdaghensis ¡Iris songarica and Iris fosteriana were collected from some parts of Khorasan province. Genomic DNA was extracted from young leaves following the cetyltrimethylammonium bromide (CTAB) procedure. Extracted DNA concentration was quantified by using the spectrophotometer and qualified using agarose gel electrophoresis. A total of 16 primers were initially screened against two plants selected from different regions and finally six primers for final analysis was selected based on consistent (CA)8G ¡(CT)8RG ¡(TC)8C ¡(TG)8G ¡ (AC)8YG and (AG)8YT, strong amplification products, production of polymorph, reproducible fragments between replicate Polymerase Chain Reaction (PCR). The ISSR amplification reactions contained 30-50 ηg of genomic DNA, 2.5 μL 1 × buffer, 2 mM MgCl2, 200 μM of each dNTP (Fermentas), 10 μM primers and 0.2 U Taq DNA polymerase (Fermentas), with the final volume adjusted to 25μL with H2O bidest. ISSR reaction products were separated on 1.5% horizontal agarose gels, in TBE buffer and visualized under ultraviolet light after staining in 0.5μg/mL ethidium bromide. Digital photo was taken with gel documentation system. The 100 bp DNA ladder plus molecular weight marker was used to compare the molecular weight of amplified products. Amplified products were scored for the presence (1) or absence (0) of bands and binary matrices were assembled for the ISSR markers. The binary matrices were subjected to statistical analyses using NTSYS-pc software version 2.02.
Six ISSR primers produced 126 bands across the 16 genotypes, of which 119 were polymorphic. The number of amplified fragments varied from 16 [primer (CA)8G)] to 24 [primer (TC)8C and (AC)8YG)] across the genotypes. The average polymorphic bands per primer were 19.4. The percentage of polymorphism for primers ranged from 76 to 100, with an average of 94.4.The amplified bands genotypes related to a species the same banding pattern was observed but there was lower similarity between the species. Our data indicated that ISSR technology can detect considerable polymorphisms (76.4 %) in our genotypes, suggesting that it will be useful in characterization and fingerprinting of Iris germplasm. The results of this study also provide fundamental evidence demonstrate that ISSR marker is a simple, informative, reproducible and suitable approach to evaluation of molecular diversity and phylogenetic relationships in Iris spp. The highest genetic similarity was between species Iris kopetdaghensis and Iris fosteriana. This study revealed a significant variation especially between Iris kopetdaghensis and Iris songarica.
The results of cluster analysis showed that molecular markers able to identify the species and genotypes within a species from each other. Results of this study showed that the use of molecular markers in breeding programs, especially fingerprinting is useful for lily. ISSR molecular markers have proved to be an efficient tool for studying genetic diversity and management of lily germplasm. . Also the result showed these genotypes have high genetic diversity, and the success in Iris breeding programs use to recommend Iranian local Iris.