Assessment of Genetic Diversity in Iranian Fennels Using ISSR Markers

Fennel (Foeniculum vulgare Mill.) is an important medicinal plant with used for various purposes in different industries. In this study 25 different ecotypes of fennel from all over Iran were collected and their genetic diversity studied by seven ISSR primers. Seven ISSR primers generated 52 amplified fragments, of which 49 were polymorphic. The highest similarity coefficient among the ecotypes was between Chahestan and Haji abad whereas the minimum similarity coefficient observed between Fozveh and Moqan. In most cases, classifications were consistent with their geographical distribution for some ecotypes (like Givi and Khalkhal in close distance) and although with similarity in climate (like Damavand and Alamot with same climate). This study revealed that ISSR marker could properly separate these ecotypes based on geographical distribution and similarity in climates and showed the wide genetic diversity among Iranian fennels. In term of conservation program, it is highly recommended at least one conservation program for several accessions with near genetical distance should be conducted.


Introduction
Bitter Fennel (Foeniculum vulgare Mill.) known as Raziyane in Persian is one of the oldest herbs and possesses beneficial medicinal effects, belongs to the Apiaceae family and native to Mediterranean regions (Hornok, 1992).
Fennel is used for various purposes in the food, cosmetic, and medical industries.Fennel essential oil has a valuable antioxidant, and has antibacterial, anticancer and antifungal activity (Lucinewton et al., 2005;El-Awadi & Esmat, 2010).The increasing commercial value of fennel necessitates the need to identification, recognizing and conservation the existing diversity.
Genotype, environment and genotype by environment (G * E) interactions are major factors that influence phenotypic diversity in plants (Olle & Bender, 2010;Hadian et al., 2010).Fennel has been cultivated as a medicinal and spice plant for a long time in different areas of Iran and, due to adaptation to the local environments; different genotypes show genetic diversity (Heywood, 2002).Knowledge of present diversity can guide appropriate selection schemes for taxonomic and breeding programs and also in conservation programs in fennel (Judzentiene & Mockute, 2010).
In recent years, DNA-based molecular markers have been used for assessment of the genetic diversity between germplasm in many plant species.Inter simple sequence repeat (ISSR) markers (Zietkiewicz et al., 1994) have been used with success to identify and determine relationships at the species, population and cultivar levels in many plant species, including several aromatic and medicinal plants (e.g.Sangwan et al., 1999;Nan et al., 2003).This method is widely applicable because there are rapid, inexpensive, require small amounts of template DNA and, unlike SSR markers, do not require prior knowledge of DNA sequences (Godwin et al., 1997(Godwin et al., ). 2012)).Genetical diversity of some fennel ecotypes of Pakistan have been investigated by Zahid et al (2009) using RAPD molecular markers and showed a considerable genetical diversity in Pakistani fennels.
The present work aimed to determine the efficiency of ISSR marker for the identification of genetical diversity in Iranian fennels.

Plant Materials and DNA Isolation
A total of 25 ecotypes of bitter fennel (Foeniculum vulgare Mill) from 25 Iranian locations consisting of different climates (with different features in term of humidity, temperature, altitude and longitude) were collected (Table 1).The seeds were sawed in pots and at the length of 30 cm of height, sampling for DNA extracting was done.All plant materials were frozen in liquid nitrogen immediately after harvesting then was transferred to genomic lab.Total genomic DNA was extracted from frozen leaves (30 mg per ecotype) following the CTAB procedure according to Murray and Thompson (1980) with minor modifications.The A260/280 ration ranged between 1.8 and 1.9 DNA integrity was confirmed by gel electrophoresis on 0.8% agarose on TBE buffer (50 mM Tris, 50 Mm boric acid, 2.5 mM EDTA, pH 8.3).DNA content was quantified using spectrophotometer and diluted to a 2 ng/µL working solution.A relationship between genetic diversity and geographic distribution and climatic similarity has been observed in several species of aromatic plants of Anthemideae sub-family including Artemisia annua (Sangwan et al., 1999), Tanacetum vulgare (Keskitalo et al., 2001), three Achillea species (Farajpour et al., 2011) and Achillea millefolium (Farajpour et al., 2012) and this is reasonable because climate that always in near place is same, in term of genetical changes is one of the most important and effective agent on genetical information in different way (like mutation and natural selection).
The 7 ISSR primers with sufficient polymorphism these ecotypes based on their geographical and climatical properties have properly separated and it is recommendable for medicinal plants.The present study revealed the wide genetic diversity among 25 ecotypes of Iranian fennels.These results may help to understand the genetic diversity and structure of fennel with possible applications in breeding programs.F. vulgar Mill is facing great pressure because of economic activities and if some conservation measures are not adopted, the species could become endangered.Genetic diversity data are important for conservation and management of rare and endangered species.With considering of wide distribution of fennel in Iran it is highly recommended at least one conservation program for several accessions with near genetical distance (like Damavand and Alamot) should be conducted.

Table 1 .
Geography profile of Fennel collecting origins Foeniculum vulgare