Gotaq mdx hot start polymerase

The DNA of the bacterial isolates and controls were amplified with 0.3 μM primers [LPW57 5′-AGTTTGATCCTGGCTCAG-3′ (nucleotide position 10–27) and LPW58 5′-AGGCCCGGGAACGTATTCAC-3′ (nucleotide position 1370–1389)]^{19 (link)} using GoTaq® MDx Hot Start Polymerase and Colorless GoTaq® Flexi Buffer (Promega). PCR amplification was performed on a PTC-100 thermocycler (Bio-Rad) using the following conditions: an initial incubation at 94 °C for 2 min, 25 cycles of 94 °C for 30s, 58 °C for 30s, and 72 °C for 1 min, followed by a final incubation at 72 °C for 5 min. PCR products were purified using DNA Clean & Concentrator (Zymo Research). DNA sequencing was performed by the GATC company (Cologne, Germany) using the PCR primers. 16S rRNA gene sequences were subjected to BLAST analysis against the NCBI nucleotide database. The sequencing data analysis allowed assigning species when the similarity score was 99% or higher and the similarity score differences with the next closest species were equal to or greater than 0.2%, which reflected 3 or more nucleotides. The genus was assigned when the similarity score was 97% or higher. The BLAST analysis of the 16S rRNA gene sequences was verified against the curated leBiBi-QBPP database (https://umr5558-bibiserv.univ-lyon1.fr/lebibi/lebibi.cgi) not revealing any differences in identification.

A microsatellite-enriched library was conducted through magnetic bead procedure [21 (link),22 (link)]. Out of 72 SSR markers, 39 were successfully amplified to evaluate genetic variability of the populations. The development of these SSR markers was already reported by [10 (link)] under the following PCR conditions during optimization by temperature gradient: 94 °C for 2 min; 35 cycles of 94 °C for 45 sec; 50 to 60 °C for 45 sec; and 72 °C for 50 sec; followed by the final extension set at 72 °C for 7 min. The amplification was conducted using the following 20 µL mixture: 5 ng of genomic DNA, 4 µL of 5× buffer, 0.2 µM of dNTP mix, 2 mM of MgCl₂, 0.5 units of GoTaq MDx Hot Start Polymerase (Promega Corporation), 0.2 mM of both primers, and sterile ddH₂O. PCR amplification was performed using a Labnet Multigene 96-well Gradient Thermal Cycler (Labnet, Edison, NJ, USA). The amplicons were checked with 1% agarose gel electrophoresis (please see Supplementary Figure S1 for PCR amplicons of the selected 10 primers, and for more information of published primer sequences, please refer to reference [10 (link)]). The target amplicons were visualized using a digital camera (Top BIO Co., Taipei, Taiwan) after being electrophoresed in a 10% polyacrylamide gel. Amplicon sizes were scored as co-dominant using Quantity One^® software (Bio-Rad) to create a data matrix manually.