Genescan 500 liz

Pri-mir-30c-1: SRSF3 complexes were assembled as described for SHAPE analysis. After protein incubation samples were subsequently subject to primer extension using fluorescent primer (NED) 5′-CTAGATGCATGCTCGAGCG-3′.pri-miR-30c (ref. 34 (link)). Primer extension products were extracted with phenol, and ethanol precipitated pellets were resuspended in 5 μl of HI-Di formamide (ThermoFisher), which included 0.5 μl of GeneScan 500 Liz dye size standards. The products were separated by capillary electrophoresis and analysed by GeneMarker software.

Total genomic DNA was isolated from young leaves using the DNeasy Plant Kit (Qiagen) following the protocol provided by the manufacturer. Nine pear SSR primer combinations (Yamamoto et al., 2002a (link),b (link)) were used (Table S2). PCRs were carried out with the Type-it Microsatellite PCR Kit (Qiagen) containing 1X Type-it master mix with 0.2 μM of each forward and reverse primer and 20 ng of DNA and H₂O to a final volume of 20 μl. Amplification was performed as follow: an initial step at 95°C for 5 min followed by 30 cycles at 95°C for 30 s, 54–62°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min.
PCR products were separated and analyzed on a 3130 XL DNA Analyzer (Applied Biosystems). The size of the amplified products was determined on internal standard DNA (GeneScan 500 Liz, Thermo Fischer Scientific) and the scorable peaks were assigned by GeneMapper software (Applied Biosystems).

Ten microliters of GeneScan –500 LIZ or GeneScan –1200 LIZ Size Standard (Thermo Fisher Scientific, US-MA) were added to 1 mL HiDi formamide (HD-LIZ). An aliquot of 0.5 μL of the reaction mixture was added to 12.5 μL of the HD-LIZ solution, vortexed, heated for 3 min at 96 °C, and then analyzed using a 3130xl Genetic Analyzer (Thermo Fisher Scientific, US-MA), a G5 dye set, the POP-7 polymer, and 50-cm capillaries. Sequencing data was analyzed using TraceViewer (http://www.ige.tohoku.ac.jp/joho/traceviewer/), and two LIZ bands were used to calibrate electrophoresis. To determine the G-tailing products of DNA molecules with 207, 560, 1,039, and 2,000 bp, the tailing reaction mixture was purified by phenol/chloroform/isoamylalcohol extraction and ethanol-precipitation, digested with DraII, and purified again. Double-stranded FAM-labeled DNA with an overhang complementary to DraII cleavage ends was obtained by annealing primers SA201 and SA202 and purifying using polyacrylamide gel electrophoresis. The resulting adapter molecule was ligated to DraII-digested products using a DNA ligation kit (TAKARA) and then analyzed by capillary sequencing as described above.

Genomic DNA was extracted from dried leaves using the CTAB method⁴⁵ . A set of six nSSR markers originally developed for J. thurifera that were highly polymorphic according to Teixeira et al.^{34 (link)} were used: JT_01, JT_04, JT_30, JT_33, JT_40 and JT_46 (Supplementary Table S1). Two multiplex PCRs were performed in a final volume of 10 µL containing approximately 60 ng of template DNA, 1 U of Silver Hot Start DNA Polymerase (Syngen Biotech, Poland), 1 µM of each primer pair, 1x reaction buffer, 2.5 mM MgCl₂, and water. The first multiplex reaction involving loci JT_01, JT_30 and JT_33 and the second one with loci JT_04, JT_40 and JT_46 were amplified in a Labcycler Basic thermocycler (SensoQuest, GmbH) with the following conditions: an initial denaturation step of 15 min at 95 °C, followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at a temperature specific to each multiplex for 90 sec (57 °C for multiplex I and 55 °C for multiplex II), and extension at 72 °C for 60 sec, with a final extension step at 72 °C for 10 min. PCR products were analysed using an AB 3130 Genetic Analyzer (Thermo Fisher Scientific, USA) capillary electrophoresis system with an internal size standard, GeneScan™ 500 LIZ^®. Genotypes were scored using GeneMapper 4.0 software (Thermo Fisher Scientific, USA).

A total of 21 natural populations of C. sativa from the South Caucasus (17 in Georgia and 4 in Azerbaijan) were analysed (Fig. 1, Supporting Information—Table S1). In addition, a single population from North Macedonia was included to understand the genetic distinctiveness between European and Caucasian gene pools. A total of 653 mature trees were analysed. DNA was extracted from dry leaves using the CTAB protocol. Nine nuclear microsatellite markers (nSSR)—CsCAT1, CsCAT6, CsCAT14, CsCAT15, CsCAT41 (Marinoni et al. 2003 ) and EMCs2, EMCs13, EMCs15, EMCs22 (Buck et al. 2003 )—were employed in genetic analyses. PCR conditions were according to Marinoni et al. (2003) and Buck et al. (2003) . PCR (Polymerase Chain Reaction) products were run on Applied Biosystems ABI PRISM 3130 XL genetic analyser (Thermo Fisher Scientific, USA) using an internal standard GeneScan 500LIZ® (Thermo Fisher Scientific, USA). Then, fragment sizes were scored using GeneMapper 4.0 software (Thermo Fisher Scientific, USA).