Taq pcr starmix

Total genomic DNA was extracted from young seedling leaves by means of the cetyltrimethylammonium bromide (CTAB) method [30 (link)]. DNA samples were quantified using a NanoDrop One spectrophotometer instrument (Nanodrop Technologies, Wilmington, DE, USA) and diluted to a concentration of 30 ng/µL.
The Pm60 alleles were tested via amplification with M-Pm60 primers [11 (link)]. The flanking markers of Pm60 used in the experiments were all from Zou et al. [11 (link)] (Table 1). Polymerase chain reaction (PCR) amplification was performed in a 20 µL reaction volume, containing 10 µL of 2 × Taq PCR StarMix (Genstar, Beijing, China) and loading dye, 4 µL of 30 ng/mL DNA, 2 µL of primers (mixture of forward and reverse primers, 2 mM), and 4 µL of ddH₂O. PCR amplification was performed using the marker M-Pm60 with a protocol of 5 min at 94 °C; 35 s at 94 °C, 35 s at 55 °C, 1 min 40 s at 72 °C, and termination after 10 min of extension at 72 °C for a total of 35 cycles. For the flanking markers, the primer extension time was 30 s at 72 °C and the other PCR conditions were the same as those described above. PCR products were separated in 1% agarose gels or via 10% non-denaturing polyacrylamide gel electrophoresis (acrylamide: bisacrylamide = 39:1), and gels were visualized with silver nitrate staining [31 (link)].

Rhizobial cultures in VMM medium containing 0.37 or 37 μM FeCl₃ were collected at an OD₅₉₀ of 0.6. Total RNA from these free-living cells was extracted by using an RNAprep pure cell/bacteria kit (Tiangen). Extraction of total RNA of both host and bacteroid in nodules was performed as described previously (48 (link)). cDNA was synthesized by using a FastKing RT kit with gDNase (Tiangen). RT-qPCR was performed by using SYBR Green real-time PCR mix (GenStar) and an ABI QuantStudio 6 Flex System real-time PCR system. The 16S rRNA gene was used as an internal control to normalize the relative transcription levels of target genes. Three biological replicates were performed. To determine the cotranscription of adjacent genes, reverse transcription-PCR (RT-PCR) was conducted by using the 2×Taq PCR StarMix (GenStar). cDNA synthesized from total RNA sample extracted from the ΔrirA culture grown in VMM medium containing 37 μΜ FeCl₃ was used as the template. Primers used in RT-PCR and RT-qPCR are listed in Table S4.

High-quality genomic DNA of ND3331 and Zang1817 was extracted and sequenced with an average 6 × coverage of the assembled genome using the Illumina NovaSeq 6000 platform as 2 × 150 bp reads. High-quality reads were aligned to the Chinese Spring IWGSC RefSeq v1.0 using the Burrows-Wheeler Aligner 0.7.15 program with default parameters (Li and Durbin 2009 (link)). Insertion/deletion (InDel) calling was performed using the HaplotypeCaller module, and InDels between Nongda3331 and Zang1817 were used to develop molecular markers for QTL analysis (Dataset S1). Primer 3 was used to design the sequences of InDel primers.
DNA amplification was programmed for an initial 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and finally 5 min at 72 °C. A 10 µL PCR reaction system was used, containing 5 µL of 2 × Taq PCR StarMix (GenStar, Beijing, China), 1.5 µL of DNA template (about 50–100 ng), 1.5 µL of each InDel primer, and double-distilled H₂O. The PCR products were analyzed on 8% non-denaturing polyacrylamide gels with silver staining.