Fluorescein 12 dutp

Probes for FISH were generated using a modified conventional PCR: the reaction mix with a final volume of 25 μl contained 0.1 mM each of unlabelled dATP, dCTP and dGTP and 0.03 mM of dTTP; 0.5 μl one fluorophore conjugated dUTP (cyanine 3-dUTP (Enzo), cyanine 5-dUTP (Enzo) or fluorescein-12-dUTP (Thermo Fisher Scientific)); 1× Taq-buffer; and 0.625 U GoTaq DNA Polymerase (Promega). Each PCR amplification was performed using 0.5 μg of genomic DNA template from testes, 34 PCR cycles and a 30-s extension step to obtain appropriately sized probes for FISH. After cycling the reaction, 25 μl PCR mix was combined with 5 μl sheared salmon sperm DNA (1 mg ml⁻¹; Thermo Fisher Scientific), 3 μl 3 M sodium acetate, pH 5.2 and 80 μl 100% cold ethanol, and kept overnight at −20 °C for probe precipitation. After spinning and supernatant removal, the pellet was dissolved in 25–30 μl of 50% formamide and stored at −20 °C before use.

L. mollis genomic DNA was labeled with fluorescein-12-dUTP (Thermo Scientific) using Random Primers DNA Labeling System (Invitrogen). With the labeled L. mollis genomic DNA as probe, GISH was performed for the 10 addition lines following a protocol described for Triticeae species³³ , with slight modifications: steps 3–9 of slide pre-hybridization were skipped and the probe was denatured at 100 °C for 5 min instead of 75 °C for 10 min. After hybridization, the slides were viewed and photographed with an Olympus BX61 automated fluorescence microscope (Olympus).

One microliter of 1 mM fluorescein-12-dUTP (Thermo Fisher Scientific), 2 µl dNTP mix (1 mM dATP, dCTP, and dGTP and 0.5 mM dTTP [Thermo Fisher Scientific]), 0.5 µl DreamTaq DNA polymerase (Thermo Fisher Scientific), 5 µl DreamTaq buffer, 1 µM prMB013, 1 µM prMB014, and 2 ng pDG1664 (43 (link)) in a total reaction mixture volume of 50 µl were used for fluorescein-labeling PCR. Thirty-five cycles of a standard DreamTaq PCR protocol were used. A longer extension time of 3 min was used for a 2,300-bp product. After PCR, samples were incubated for 2 h with 0.5 µl DpnI per 50-µl PCR sample (FastDigest; Thermo Fisher Scientific). PCR samples were purified using a Macherey-Nagel PCR kit. Samples were stored at −20°C. Samples were protected from light at all times. The label incorporation of fluorescein-dUTP lies between 1 and 3 pmol/µl as measured using the NanoDrop (Thermo Fisher Scientific).

Slides of mitotic chromosomes were prepared from imaginal discs of fourth instar larvae following published protocols^{3 (link),70 ,71 (link)}. BAC clones were obtained from the University of Liverpool¹⁹ or from a previously described BAC library^{72 (link)}. BACs were plated on agar plates (Thermo Fisher) and a single bacterial colony was used to grow an overnight bacterial culture in LB broth plates (Thermo Fisher) at 37 °C. DNA from the BACs was extracted using Sigma PhasePrep TM BAC DNA Kit (Sigma-Aldrich, NA-0100). BAC DNA for hybridization was labelled by nick translation with Cy3-, Cy5-dUTP (Enzo Life Sciences) or Fluorescein 12-dUTP (Thermo Fisher). Chromosomes were counterstained with DAPI in Prolong Gold Antifade (Thermo Fisher). Slides were analysed using a Zeiss LSM 880 Laser Scanning Microscope at 1,000× magnification. We note that localization of the M-locus to 1p11 is supported by both FISH and genomic analyses, but is contrary to a previously published placement at 1q21^{17 (link)}.

Fluorescent in situ hybridization of BACs to metaphase chromosome spreads were performed as previously described^{74 ,75 (link)}. A Qiagen Large Construct kit (Qiagen Science, 12462) was used to extract bacterial artificial chromosome (BAC) DNA for E24C3 and E12A6, previously associated with sex^{29 (link)}. Probes for in situ hybridization were labeled by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) as described previously⁷⁴ and hybridization of BAC probes was performed as previously described for axolotl chromosomes^{40 (link)}.
Phenol-chloroform extraction in 1.2X SSC was used to isolate repetitive DNA fractions from female salamander tissue⁷⁶ . DNA was denatured for 5 minutes at 120 °C, re-associated at 60 °C for 1 hour to obtain C_ot DNA. Microtubes containing the DNA were placed on ice for 2 minutes, then transferred to a bead bath at 42 °C for 1 hour with 5X S1 nuclease buffer and S1 nuclease for a concentration of 100 units per 1 mg DNA. DNA was precipitated with 0.1 volume of 3M sodium acetate and 1 volume isopropanol at room temperature, tubes were inverted several times and centrifuged at 14,000 rpm for 20 minutes at 4 °C. DNA was washed with 70% ethanol, centrifuged at 14,000 rpm for 10 minutes at 4 °C, air dried and solubilized in TE buffer.