# The Chromosome Number and rDNA Loci Evolution in _Onobrychis_ (Fabaceae)
## 4 Materials and Methods
### Chromosome Preparation and Fluorescence In Situ Hybridisation  
The chromosome preparations were made as described previously [97]. The 5S rDNA monomer that had been isolated from (clone pT2794 [98]) and labelled with digoxigenin-11-dUTP (Roche, Basel, Switzerland) to detect 5S rDNA loci was used. A 2.3-kb fragment of the 25S rDNA coding region of _Arabidopsis thaliana_[99] labelled with tetramethyl-rhodamine-5-dUTP (Roche) was used to detect the 35S rDNA loci. The probe labelling and FISH followed the Jenkins and Hasterok protocol [100]. The hybridisation mixture consisted \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Species** & **USDA \({}^{*}\) Collection Number** & **Voucher** & **GeneBank Accession** \\ \hline _O. biebersteinii_ Sirj. & PI 227377 & KTU154634 & OP288059 \\ \hline _O. vicifolia_ Scop. (1) & PI 1705831 \# & KTU154645 & OP288048 \\ \hline _O. vicifolia_ Scop. (2) & PI 200872 & KTU154636 & OP288055 \\ \hline _O. transcancasia_ Grossh. & PI 273771 \# & KTU154640 & OP288065 \\ \hline _O. arenaria_ (Kit) DC. & PI 273743 \# & KTU154639 & OP288054 \\ \hline _O. megataphros_ & PI 301107 & - & OP288064 \\ \hline _O. supina_ (Vill.) DC. & PI 383721 & KTU154646 & OP288047 \\ \hline _O. alba_ ([PERSON] \& Kit). Desv. subsp. & W6 19337 & KTU154647 & OP288049 \\ _laconica_ (Boiss.) [PERSON] & W617870 & KTU154654 & OP288053 \\ \hline _O. cyni_ [PERSON]. & W6 17800 & KTU154635 & OP288070 \\ \hline _O. altissima [PERSON]_ & PI 325448 & - & OP288067 \\ \hline _O. humilis_ (_[PERSON].) _[PERSON]_ & PI 319054 & - & OP288046 \\ \hline _O. stenorhiza_ D.C & PI 319056 & - & OP288066 \\ \hline _O. gracilis_ [PERSON] & W6 19496 & KTU154642 & OP288050 \\ \hline _O. caput-galli_ [PERSON][PERSON]. & PI 205304 & KTU154659 & OP288056 \\ \hline _O. persica_ [PERSON]. \& [PERSON]. & PI 380946 & KTU154638 & OP288071 \\ \hline _O. crista-galli_ [PERSON]. & PI 227040 & KTU154651 & OP288068 \\ \hline _O. iberica_ [PERSON]. & PI 219602 & - & OP288058 \\ \hline _O. sintenisii_ [PERSON]. & PI 314100 & KTU154632 & OP288057 \\ \hline _O. vassilczenkoi_ [PERSON]. & PI 678913 & KTU154641 & OP288063 \\ \hline _O. vaginalis_ [PERSON]. & PI 325444 & KTU154633 & OP288051 \\ \hline _O. chorossanica_ Bunge ex Boiss. & PI 314160 & KTU154658 & OP288061 \\ \hline _O. kachetica_ [PERSON]. \& [PERSON] & PI 314469 & KTU154649 & OP288062 \\ \hline _O. radiata_ (Desf.[PERSON] [PERSON]. & W6 24111 & KTU154650 & OP288074 \\ \hline _O. michauxii_ [PERSON]. & PI 380945 & - & OP288060 \\ \hline _O. ptolemica_ ([PERSON]) DC. & PI 215344 & KTU154655 & OP288073 \\ \hline _O. hyparygera_ [PERSON]. & PI 383719 & KTU154644 & OP288052 \\ \hline _O. grandis_ [PERSON] & PI 440568 & KTU154653 & OP288072 \\ \hline _O. subacanulis_ [PERSON]. & PI 219930 & KTU154643 & OP288075 \\ \hline _O. gaubae_ [PERSON]. & PI 380931 & - & OP288069 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: General characteristics of the analysed taxa and the GenBank accession numbers of the sequences obtained in this study. of 50% deionised formamide, 10% dextran sulphate, 2x SSC, 0.5% SDS (sodium dodecyl sulphate) and labelled probes (100 ng of each probe per slide). Hybridisation was conducted for 48 h at 37 \({}^{\circ}\)C in a humid chamber. Post-hybridisation washes (10% deionised formamide in 0.1x SSC at 42 \({}^{\circ}\)C; stringency 76%) were followed by the immunodetection of digoxigenated probes using FITC-conjugated anti-digoxigenin antibodies (Roche). The slides were mounted in Vectashield (Vector Laboratories, Newark, CA, USA) containing 2.5 ng/\(\mu\)L of DAPI (4\({}^{\prime}\),\(\delta\)-diamidino-2-phenylindole dihydrochloride). All images were acquired using a Zeiss AxioImager.Z.2 fluorescent microscope equipped with an AxioCam HMr camera (Zeiss, Oberkochen, Germany). The images were processed uniformly using ZEN 2.3 Pro (Zeiss). FISH experiments with 35S and 5S rDNA probes were conducted for 24 species, and the slides after FISH were used for chromosome counting. Chromosome counts were performed for five more species (_O. humilis_, _O. stenorhiza_, _O.