Texas red 5 dctp

The protocol for fluorescence in situ hybridization (FISH) was adopted as previously described [43 (link)]. For FISH, repetitive DNA sequences pSc119.2 and a microsatellite (GAA)n oligonucleotide were labeled with Chroma Tide Alexa Fluor 488-5-dUTP (C11397, Invitrogen, Eugene, USA), and pAs1 was labeled with Texas Red-5-dCTP (NEL 426, Perkin-Elmer, Boston, USA). Anti-methylcytosine antibodies (1982501, Millipore, Temecula, USA) and anti-dimethyl-Histone H3 (Lys9) antibodies (07-441, Millipore, Temecula, USA) were used for immunofluorescence analysis using published protocols [5 (link), 42 (link)]. Slides were examined with an Olympus BX61 fluorescence microscope and digitally photographed. Immunofluorescence values of DNA methylation and H3K9me2 were estimated from homoeologous chromosomes of 30 cells using an Olympus cellSens Dimension and were averaged by the number of chromosomes in a given genotype.

Roots about 1.5∼2.0 cm long were taken from the seedlings and treated in N₂O gas for 2 h. These roots were then fixed in 90% (v/v) acetic acid and stored in 75% (v/v) alcohol at -20°C until use. Mitotic spread chromosome slides were prepared from the root-tips according to Zhang et al. (2013) (link). For each plant, chromosomes were counted for at least five well-spread metaphase cells.
Sequential florescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were performed according to a protocol reported previously (Zhang et al., 2013 (link)) with minor modifications. Briefly, Texas red-5-dCTP (PerkinElmer, NEL426001EA) was used for labeling the repeated sequence clone pAs1 (Rayburn and Gill, 1986 (link)) and genomic DNA isolated from A. tauschii, respectively. ChromaTide^TM Alexa Fluor^TM 488-5-dUTP (Thermo Fisher, C11397) was used for labeling the rye repeated sequence clone pSc119.2 and genomic DNA from T. urartu, respectively. In GISH, genomic DNA of A. bicornis was used as blocker. In both FISH and GISH, DAPI (Vector, H-1200) was also used to counterstain the chromosomes.
Both the FISH and GISH slides were examined under an Olympus BX63 fluorescence microscope and captured by Q-capture imaging software (QImaging, Version 2.90.1). Brightness, contrast and background were adjusted as an entirety in Adobe Photoshop CC.

Genomic DNA of the 12 mulberry accessions (M. notabilis, M. nigra, M. wittiorum ‘Ailaoshan No. 9’, M. cathayana ‘Huai302’, M. multicaulis ‘Heyebai’, M. alba ‘Baiyuwang’, M. atropurpurea ‘Lunjiao109’, M. alba var. pendula ‘Chuisang’, M. alba var. macrophylla ‘Dayezaoshengsang’, M. bombycis ‘Jianchi’, M. mongolica ‘Jimengsang’, and M. mongolica var. diabolica ‘Taiping No. 5’) used for probe labeling was extracted from young leaves using a DNAquick Plant System kit (TIANGEN BIOTECH, Beijing, China) according to the product manual. 25S rDNA sequences were amplified according to a previous study [29 ]. Genomic DNA and 25S rDNA probes used for GISH and FISH, respectively, were labeled with ChromaTide Alexa Fluor 488–5-dUTP (Thermo Fisher Scientific [Invitrogen], Massachusetts, USA) or Texas-red-5-dCTP (PerkinElmer, Massachusetts, USA) by a nick-translation method [50 (link)]. Briefly, the labelling system included 10 μL of DNA product (containing 2 μg genomic DNA or PCR product of the 25S rDNA sequences), 2 μL nick translation buffer, 2 μL dNTP (-dCTP or -dUTP) mix, 0.5 μL Texas-red-5-dCTP or ChromaTide Alexa Fluor 488–5-dUTP, 0.5 μL DNase I (100 mU/μL), and 5 μL DNA polymerase I (10 U/μL). After incubation at 15℃ for 2 h, the probes were purified in 2.5 volumes of 90% ethanol/10% sodium acetate mix (3 M, pH 5.2), and dissolved with 20 μL 2 × SSC and 1 × TE solution.

Seeds were germinated on moistened filter paper in Petri dishes. Actively growing roots were removed from seedlings and subjected to nitrous oxide treatment for 2 h, fixed in 90% acetic acid for 8 minutes and stored in 70% v/v ethanol. Chromosome preparation and FISH were performed following the protocols described by Kato et al.^{40 (link)} and Han et al.^{57 (link)}. Cytological observations were performed using a BX51 Olympus phase-contrast microscope (Olympus Corp., Tokyo, Japan). FISH with the DNA clones pAcTRT1 and pAcpCR2 was used to explore the clone distributions on P genome chromosomes in A. cristatum and wheat-A. cristatum derivative lines. The probes for 45S rDNA from wheat, rye tandem pSc200 repeat and Aegilops tauschii Coss. clone pAs1 were used to verify the order of the P genome chromosomes in diploid A. cristatum. The probes were labelled with Texas Red-5-dCTP or fluorescein-12-dUTP (PerkinElmer, Boston, MA, USA). The total volume of hybridization mixture per slide was 6 µl, containing 0.5 µl of 100 ng/µl probe, with the balance of the volume comprising 2 × SSC and 1 × TE. All the images were obtained using an Olympus AX80 fluorescence microscope (Olympus Corp., Tokyo, Japan) and processed with Adobe Photoshop CS 3.0 (Adobe, San Jose, CA, USA).