Nucleospin plant 2

Genomic DNA was extracted from fully expanded leaves of various Rutaceae species (Table 2) using NucleoSpin Plant II (TaKaRa Bio Inc. Kusatsu, Japan). The LOB1 promoter regions containing the PthA4 EBE were amplified from genomic DNA using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA) and fragments were cloned into pGEM-T vector (Promega, Madison, WI). DNA sequence was determined for 3–5 clones. Amplified LOB1 promoter sequences, along with LOB1 promoter regions of other Rutaceae species available at the citrus genome database (https://www.citrusgenomedb.org/) were analyzed using the Clustal Omega multiple sequence alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) and separated into allelic variants.
TALE protein sequences of X. citri were extracted from NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/?term=) and the compositions of RVDs in repeat arrays were manually determined. Binding affinity was analyzed against the promoter region of LOB1 using target finder feature of “TAL Effector Nucleotide Targeter 2.0” [56 (link)] (parameters were set to score cutoff of 4.0, T only upstream base, and Doyle scoring matrix). All TALEs that were predicted to bind to LOB1 according to score cutoff of 4.0 were considered as putative LOB1 targeting TALEs.

Genomic DNA was extracted from fully expanded leaves of Meiwa kumquat using NucleoSpin Plant II (TaKaRa Bio Inc. Kusatsu, Japan). Four fragments, including one fragment covering a 3,456 bp (NCBI accession num’ MT247386) containing the LOB1 promoter, coding region and the PthA4 EBE sequences; one fragment covering 1,264 bp (NCBI accession num’ MT247387) containing the LOB2 promoter and coding regions; and one fragment covering 1,515 bp (NCBI accession num’ MT655137) containing the LOB3 promoter and coding regions, were amplified (primers are shown in S2 Table) from genomic DNA using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA). The LOB1, LOB2 and LOB3 fragments were cloned into pGEM-T (Promega, Madison, WI) or pHSG298 (TaKaRa Bio Inc. Kusatsu, Japan) vectors. Sequences were determined using Sanger sequencing (Eton Bioscience, Inc., San Diego, CA).

Genotype analysis using next-generation genome sequencers was performed using confirm whether mutations were induced in the genomes of the mutants. Leaves were sampled from mutants and stored at −20 °C. The sampled leaves were frozen in liquid nitrogen and ground, DNA was extracted using NucleoSpin Plant II (Takara Bio Inc., Shiga, Japan), and the yield was confirmed to be >600 ng using a biospectrometer (Eppendorf Inc., Tokyo, Japan). We confirmed that there was no degradation of DNA by electrophoresis using 2% agarose. The obtained DNA was used as a template and the entire genome was amplified and sequenced using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) technology using a next-generation genome sequencer (Illumina, Eurofins Genomics Inc., Tokyo, Japan). In contrast to conventional genotyping by sequencing, GRAS-Di technology has fewer biased analysis regions, genome-uniform genotyping, and genomic mutation detection and marketization, even in species without reference sequences such as gerbera [51 (link),52 (link)]. The sequences obtained from the wild type and mutant were compared and sequences that differing and determined to be highly accurate were selected to determine DNA markers specific to the mutant.