# The Chromosome Number and rDNA Loci Evolution in _Onobrychis_ (Fabaceae)
## 1 Introduction  
The genus _Onobrychis_ Mill. (Fabaceae) comprises approximately 162 perennial and annual species distributed in temperate zones of North America, the Middle East and Europe [1, 2]. The majority of _Onobrychis_ species are restricted to Iran, Anatolia and Caucasus, suggesting that these areas were the centre of diversity of the genus [3]. [PERSON] [4], based on floral characteristics, divided the genus into two subgenera: _Onobrychis_ Mill (including sections _Onobrychis_, _Dendrobychis_ DC., _Lophobrychis_ Hand.-Mazt., _Hemicydolrychis_ (Sirj.) [PERSON]) and _Sisrosema_ Bunge (including sections _Anthyllium_ Nab., _Afghanicae_ Sirj., _Heliborychis_ Bunge ex Boiss., _Hymenobrychis_ DC.). Recent molecular phylogenetic analyses indicate that _Onobrychis_ is a monophyletic taxon and can be further divided into two main evolutionary lineages, each corresponding to the redefined subgenus _Onobrychis_ and _Sisrosema_[5]. Among _Onobrychis_, _O. vicitiglia_ (sainfoin) is relatively widespread and well known as a cultivated species, mainly used as a forage crop or as a valuable pollen and nectar source for honey production. This crop has a few agronomically useful characteristics, such asdrought and cold tolerance and nitrogen fixation [2]. Although the agronomical value of _O. vicifolia_ has been emphasised in the literature, the genomic structure of sainfoin and its cultivated and wild relatives remains largely unexplored [2; 5], and exact phylogenetic relationships in the genus are unclear. Therefore, the potential utilisation of wild genetic resources in _O. vicifolia_ breeding programmes is limited. Nuclear genome size, a fundamental biological character, was estimated until now only for one _Onobrychis_ representative, _O. vicifolia_, which possessed 1C DNA = 1.25 pg (the amount of DNA in the unreplicated haploid nucleus) [1]. To date, cytogenetic analyses in _Onobrychis_ have been mainly restricted to chromosome number reports. Few studies on karyotype structure and evolution have shown that _Onobrychis_ species have, in general, short and mostly metacentric or submetacentric chromosomes [6]. Two basic chromosome numbers, \(x=7\) and \(x=8\), and species on two ploidy levels, diploids (\(2n=2x=14\) or \(16\)) and tetraploids (\(2n=4x=28\) or \(32\)) were reported in the genus [6; 7; 8; 9; 10]. Two mechanisms, dysploidy and polyploidy, are responsible for the polymorphisms in chromosome number in most analysed plant genera [11; 12; 13]. No common trends in the evolution of chromosome numbers among analysed taxa were observed; however, polyploidy and descending dysploidy seem to be most often reported [14; 15; 16; 17]. A serious weakness of the analyses of _Onobrychis_ karyotypes is the paucity of chromosome markers, which has limited the identification of individual chromosomes and makes a comparative analysis very difficult. Fluorescence in situ hybridisation (FISH) often provides informative chromosome markers with a single copy and repetitive sequences as DNA probes [18; 19; 20]. Ribosomal DNA (rDNA)-based sequences are the markers of the first choice in comparative analyses of chromosomes, especially in wild and neglected plant species [21; 22; 23]. The genes encoding 35S ribosomal RNA (18S-5.8S-25S rRNA) and 5S rRNA usually are present in separate, unlinked loci as tandem arrays in the genomes of angiosperms [24]. In most plant genomes, low numbers of rDNA loci were observed. The median plant karyotype has two loci of 5S rDNA in the interstitial chromosome regions and two loci of 3 SS rDNA in the subterminal position [25]. In many cases, rDNA sequences were shown to be useful chromosome markers that enable the unambiguous identification of some chromosomes in karyotypes of species from various angiosperm families [26; 27; 28]. In addition, among diploid _Hedysarum_ species (\(2n=14\)), the genus closely related to _Onobrychis_, the application of 5S and 25S rDNA probes enables the identification of two to four out of seven chromosomes [29].