Achillea is one of the most recent genera of the Asteraceae family which exists throughout the world (Rechinger 1963). More than 100 species have been identified in this genus. Many of those who used these plants reported properties such as anti-inflammatory, anti-rheumatic, antiseptic, antispasmodic, analgesic, astringent, carminative, diaphoretic, digestive, expectorant, hypotensive, stomachic and etc. (Balbir et al. 2012). These plants are native to Europe and Western Asia but are also found in Australia, New Zealand, and North America (Rechinger 1963).

Achillea millefolium has a high genetic and phenotypic variation in Iran (Farajpour et al. 2012, Ebrahimi et al. 2012). The basic chromosome number is often reported in different species of Achillea is x= 9; however, the diversity in chromosome numbers and ploidy levels are frequently occurring in the genus (Ebrahim et al. 2012). Polyploid taxa have originated in many clades including 4x, 6x and 8x species, and as a result, several Achillea species show high morphological variability (Sheidai et al. 2009). Biste (1987) explained worthy diversity in leaf width, height, shoot number, and stomata length in different populations of the same species.

In most of the chemotypes in Achillea sp, camphor, borneol (Rohloff et al. 2000 and Mockute and Judzentiene 2003) and 1.8-Cineole (Saeidnia et al. 2004; Barghamadi et al. 2009 and Azizi et al. 2010) have been detected. Among a number of data that can be obtained by chromosome studies: karyotype structure, karyotype asymmetry, chromosome banding, FISH, GISH and chromosome painting (Stace 2000, Levin 2002, Graphodatsky et al. 2011, Guerra 2012), the most popular, cheap and widely used approaches is that concerning karyotype asymmetry (Peruzzi and Eroğlu 2013).

Achillea millefolium has been cytologically analyzed extensively in different regions of the world (Felfoldy 1947, Mizianty and Frey 1973, Pireh and Tyrl 1980, Lavrenko et al. 1991, Guo et al. 2012, Bala and Gupta 2013). Three cytological studies have been reported in Iran and showed the following ploidy levels: tetraploid 2n= 4x= 36, hexaploid 2n= 6x= 54 and octoploid 2n= 8x= 72 (Farsi et al. 2000, Ebrahim et al. 2012, Sheidai et al. 2009). The aims of this study were (1) to determine the chromosome numbers of four Achillea millefolium accessions and (2) to find any relationship between the karyotype characteristics and asymmetrical index with ploidy levels.

The aerial parts of the four Achillea millefolium accessions were collected from three provinces in north, west and south of Iran (Table 1). Voucher samples were deposited at the herbarium of Research Institute of Forests and Rangeland (RIFR) of Tehran, Iran. Seeds were germinated on moist filter paper in Petri dishes. Actively growing root tips, 1 to 2 cm length were cut from the germinating seeds and pretreated with 8-hydroxyquinoline (0.002M) for 2 to 4 hours and fixed in Farmer (1:3, glacial acetic acid : ethanol 95%) for 24 hours at 4° C. Thereafter, the root tips were hydrolyzed in 1 N NaOH at 60° C for 5-10 minutes, stained for 45 minutes in esterase stain at 30° C, and squashed in 45% glacial acetic acid. Finally, the chromosome images were obtained with photomicroscope.

Karyotypec characteristics such as differences of range relative length (DRL), mean chromosome length (MCL), and mean arm ratio (MAR) were calculated using MICROMEASURE (Version 3.3) Software (Reeves 2001). Stebbin’s classification was calculated (Stebbins 1971). Cluster analysis was performed to differentiate the accessions according to the Ward’s method SPSS software for Windows 20.0 (SPSS Inc., Chicago, IL, USA).

The results of the present study illustrated a new ploidy level (2n= 2x= 18) in Iranian Achillea millefolium accessions. Cluster analysis indicated that accessions can be classified based on ploidy levels.