Karyological Properties of Some Onobrychis Taxa Belonging to A Hymenobrychis Section Growing Naturally in Turkey

In this study, chromosome numbers and morphologies of six Onobrychis taxa [Onobrychis tournefortii (Willd.) Desv., Onobrychis albiflora Hub-Mor., Onobrychis hypargyrea Boiss., Onobrychis radiata (Desf.) Bieb., Onobrychis meschetica Grossh, and Onobrychis galegifolia Boiss.] were investigated with the squash preparation method. These taxa belong to the Hymenobrychis section, including two endemic species naturally grown in Turkey. First, karyotype analysis of the Onobrychis meschetica was performed. Then mitotic metaphase and basic chromosome numbers of the taxa were determined in 2n=14 and x= 7, respectively. Chromosomes showed differences as median to submedian according to centromer position. Except O. meschetica, all of the taxa generally included a satelite on chromosome I or IV.


Introduction
The Onobryhis genus is a member of Fabaceae family and it has 170 annual and perennial taxa which dispersed in the interior of minor Asia, which includes Central Anatolia (Turkey), the entire Transcaucasus, the higher parts of Iran, and Turkmenistan.Turkey, one of the most important diversity center of the genus, has 55 Onobrychis taxa and 28 of them are endemic (Hedge, 1970;Aktoklu, 2001;Avcı and Kaya, 2013).This taxa is well-adapted in arid and semi-arid lands of Turkey because of its deep roots.They can be safely grazed since their hay does not cause bloat in ruminant animals contrary to alfalfa (Pupilli et al., 1989;Li et al., 1993;Çeliktaş et al., 2006).Phylogenetic relationships have not been clearly disclosed by using morphological parameters in taxonomic studies due to closely related and excessive number of taxa and cross pollination (Avcı et al., 2016).The genus is obviously subdivided in two subgenus namely Onobrychis with three sections and Sisyrosema with two sections based on morphological and molecular DNA techniques in Turkey (Davis et al., 1988;Aktoklu, 2001;Emre et al., 2007;Arslan and Ertugrul, 2010;Avcı et al., 2014;Avcı ABUŞ andAVCI / JAFAG (2018) 35 (2), 103-110 et.al., 2016).Therefore, there are still some problems about grouping of sections under subgenus and the distribution of taxa.It is important to confirm previous studies with detailed cytotaxonomic studies.
There are limited karyological studies on studied taxa of Hymenobrychis section.Akçelik et al. (2012); Arslan et al. (2012); Sepet et al. (2011);Hejazi et al. (2010); Tekin et al. (2016) performed karyological properties of O. tournefortii, O. hypargyrea; O. galegifolia; O. radiata and on O. albiflora.However, there is no studies on O. meschetica.The aim of this study is to reveal of cytotaxonomic properties of these taxa belonging to Hymenobrychis section by using different populations unlike previous studies.Thus, the taxonomic status and phlogenetic relationship of taxa were defined more clearly.

Material and Method
The root tips were used from seedling obtained by germinating ripe seeds which were collected from natural terrains of Turkey and they are listed in Table 1.The ripe seeds with hard characteristics were abraded by mechanical scarification with sandpaper and were germinated at 20 ± 1 °C on wet filter paper, as described by Avcı and Kaya (2013).Root tips of 1-2 cm in length were pretreated with 0.5% α-Bromonaphthalene at room temperature for 4 h.A fixation, 10% formaldehyde:1% chromic acid (1:1) solution was used at room temperature for 16 hours.Hydrolysis was performed with 1 N NaOH solution at 60°C and hydrolysis time changed between 7 to 13 minutes depending on taxa.The root tips were dyed with hematoxylin-iron for 3-4 h and then treated with 1% cellulase enzyme (Celulase Onozuka RS) to soften hardened root tips for two minutes and squashed in a droplet of 45% acetic acid and lactic acid (10:1) (Hejazi et al., 2010).
The best 5 images obtained from different metaphase plates, were captured with a Zeiss Aixo Vision microscope integrated camera and carpological measurements were performed with Zeiss Axio Vision software.The following parameters were measured to determine the karyotype characteristics of the taxa: long arm (LA), short arm (SA), satellite (SAT), total chromosome length (TCL) [LA+SA], relative length percentage (RL%) [100xTotal length of each chromosome/total length of all chromosomes], arm ratio (AR) [LA/SA], centromeric index (CI%) [100xSA/ (LA+SA)].Karyotype formula was determined as described by Levan et al. (1964).Karyograms were arranged based on the size of chromosomes.

Results and Discussion
According to obtained results, the somatic chromosome number was 2n=14 and the basic chromosome number was x=7 (Table 2) and all chromosomes were varied from metacentric to submetacentric in terms of position of centromeric constriction (Table 3).The basic chromosome number of Onobrychis genus was observed as either x=7 or x=8 in many studies (Abou-El-enain, 2002;Akçelik et al., 2012;Ghanavati et al., 2010;Hejazi et al., 2010;Massoud et al., 2010;Sepet et al., 2011).All chromosomes of O. tournefortii were median type according to position of centromeric constriction, and chromosome 1 had a satellite (Fig 1).The somatic chromosome number (2n=14) of this taxon was confirmed by Akçelik et al. (2012) and Arslan et al. (2012).However, chromosome types varied between median to submedian and satellite was not exist in their studies.3, Table 2).This species had 4 median (4 m) and 3 submedian (3 sm) chromosomes and chromosome 1 included a satellite (Fig 3).Akçelik et al. (2012) and Sepet et al. (2011) were confirmed in terms of somatic chromosome number, centromeric position and satellite in this study.
O. radiata and O. meschetica species are morphologically very similar to each other.O. meschetica was recorded as a new species in Turkey first time by Aktoklu (2001).In this study, there were some similarity in terms of somatic chromosome number (2n=14) and total chromosome length.galegifolia varied from 1.80-3.80µm and total chromosome length was 20.33 µm (Table 3, Table 2).Also, chromosomes of this species varied from median (6 m) to submedian (1 sm) according to centromeric position, and chromosome 1 had a satellite (Fig 6).

Table 1 .
The taxa used in the study and their locations and coordinates Çizelge 1. Araştırmada kullanılan taksonlar, konumları ve koordinatları

Table 2 .
Karyotype characteristics of 6 taxa of Onobrychis.2n, somatic chromosome number; x, basic chromosome number; TLC, total chromosome lenght; KF, Karyotype formula; m, metacentric; sm, submetacentric While, the lowest mean values of long arm, short arm and total chromosome length were 1.24, 0.88 and 2.25 µm in O. tournefortii, the highest averages of them were 2.32, 1.40 and 3.91 µm in O. hypergyrea.However, the arm ratio mean value was varied from 1.32 µm in O. radiata to 1.66 µm in O. hypergyrea.When, the lowest centromeric index average was 35.35 in O. hypergyrea unlike other averages, the highest value was 59.74 in O. meschetica.Although all of the taxa generally included a satelite on chromosome I (O.tournefortii, O. hypergyrea and O. radiata) or IV (O.albiflora), a satelite was not found in O. meschetica (Table 3).