Gotag

The rp5H gene library was generated using error-prone PCR as described previously [29] (link), [30] (link), with the following modifications: The PCR cycling parameters were 95°C for 5 min; 35 cycles at 95°C for 45 s, 55°C for 55 s, 72°C for 40 s, followed by 72°C for 10 min. The PCR mixture contained 1.0 mM MgCl₂, 1.0 mM MnCl₂, 0.35 mM dATP, 1.35 mM dTTP, 0.4 mM dCTP, 0.2 mM dGTP, 1 µM of each primer (5′rpb5_EcoRI and 3′rpoH_NotI), 5 U DNA polymerase (GoTag, Promega, Mannheim, Germany), and 25 ng of plasmid pRS423_rp5H as template. The resulting gene repertoire was ligated into pRS423 and used to transform electrocompetent E. coli XL1 Blue MRF' cells (Life Technologies GmbH, former Stratagene, Darmstadt, Germany). Colonies grown on SOB rich medium agar containing 20 mM glucose and 150 µg/ml ampicillin were swept off the plates, followed by the isolation of the plasmid library containing the rp5H mutants. As estimated from the number of grown colonies and the ligation efficiency tested by colony PCR, the library contained approximately 4.5×10⁵ independent mutants. Sequencing of 10 clones revealed an average number of 11+/−7 nucleotide exchanges per gene, with a minimum number of two and a maximum number of 22 base substitutions. A bias for AT (71%) over GC (29%) exchanges and a ratio of transitions over transversion of 0.63 were observed.

Genomic DNA was isolated using the modified CTAB method from young leaves of F₂ plants and HIF plants for QTL analysis (Murray and Thompson 1980 (link)). InDel markers were developed based on P. hallii var. HAL2 genome v2.0 and P. hallii var. FIL2 genome v3.0 assembly (https://phytozome.jgi.doe.gov/pz/portal.html). Primers were designed using software Primer 3.0 (http://bioinfo.ut.ee/primer3-0.4.0/) and were then amplified from F₂ individuals and HIF individuals. A total volume of 10 µl reaction mixture was used for PCR amplification, which is composed 1 µl of template DNA, 0.3 µl of each primer (10 mM), 2 µl 5 × Buffer, 1.2 µl MgCl₂, 1 µl dNTP, and 0.1 µl GoTag (Promega). Amplification was performed on program for the initial denaturing step with 96°C for 3 min, followed by 38 cycles for 30 s at 96°C, 30 s at 58°C, 30 s at 72°C, with a final extension at 72°C for 5 min. The PCR products can be well separated using 2.5% agarose gel electrophoresis.

Plasmid DNA from bacterial cells was isolated using E.Z.N.A. Plasmid Mini Kit I (Omega Bio-tek). Genomic DNA from mammalian cells was isolated using E.Z.N.A. Tissue DNA Kit (Omega Bio-tek). Amplification of the DNA fragments for cloning was performed using Pfu-Ultra polymerase (Agilent). PCR analysis of the genomic DNA was performed using Q5 (New England Biolabs) and GoTag (Promega) master mixes. The ClustalW program (https://www.genome.jp/tools-bin/clustalw) was used for sequence alignments. Golden Gate TALEN kit was used to assemble the DNA binding domains of TAL^{28 (link)}. General genetic engineering experiments were performed as described in Molecular Cloning Manual⁵¹ . 3D structures of the proteins were analyzed using Swiss-PdbViewer^{52 (link)}.