Plant materials and genomic DNA isolation
To identify the SNP markers and perform the HRM analysis suitable for selection of ‘Haryejosaeng,’ samples were collected from 7 main satsuma mandarin (C. unshiu) cultivars (‘Miyagawa wase,’ ‘Okitsu wase,’ ‘Yura wase,’ ‘Miyamoto wase,’ ‘Ueno wase,’ ‘Yonezawa wase,’ and ‘Nichinan 1 gou’) and from the original tree of ‘Haryejosaeng’ growing at the Citrus Research Institute (National Institute of Horticultural & Herbal Science, Jeju, Korea) (Table 3). For rapid and accurate selection of ‘Haryejosaeng’ at the seedling stage by multiplex PCR using the selected SNP markers, samples were collected from the following 17 cultivars: ‘Taguchi wase,’ ‘Yura,’ ‘Hiroshimakaken 7 gou’, and ‘Higo’ from the citrus nursery at the orchard of the Citrus Cooperative Society on Jeju Island; summer leaf of an outdoor cultivar ‘Miyagawa wase’; ‘Haryejosaeng-1’ from a Daejeong farm; ‘Haryejosaeng’ from a Jeoji-ri farm; ‘Haryejosaeng-2’ and ‘Imprecision wase-1’ from agricultural cooperatives; ‘Imprecision wase-2-1’ and ‘Imprecision wase-2-2’ from a farm; 5 seedling cultivars, namely ‘Haryejosaeng-S1’, ‘Miyagawa wase,’ ‘Haryejosaeng-S2’, ‘Nichinan 1 gou’, and ‘Yura wase’; breeder’s stock of ‘Haryejosaeng-BS,’ which was improved by selection of nucellar seedlings (i.e., seedlings physiologically different from the mother plant, ‘Tachima wase,’ and reproduced by apomixis; ‘Tachima wase’ was produced by crossing the satsuma mandarin ‘Tachima wase’ with pollens of C. natsudaidai ‘Hayata’; the total soluble solids to acidity rate is higher in the improved cultivar than in the common farm cultivar ‘Miyagawa wase’ and in the mother plant) (Table 3). Total genomic DNA was extracted from the samples by automatic nuclear extraction (MX 16; Promega, Madison, WI, USA) and stored at -20 °C until use.
Primer design for the SNP/HRM analysis
For designing the primers, we first performed standard genome sequencing of 4 control cultivars (‘Miyagawa wase,’ ‘Miyamoto wase,’ ‘Okitsu wase,’ and ‘Nichinan 1 gou’) on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) using the genome of Citrus × clementina ‘Clemenules’ as reference. The sequence information (approximately 6 Gb) was obtained according to a previously established method . A paired-end DNA library was constructed for genome sequencing using the TruSeq DNA Library Prep Kit (Illumina). The generated raw reads were trimmed using DynamicTrim and LengthSort in the SolexaQA package (v.1.13) . The clean reads were mapped to the standard genome of C. × clementina ‘Clemenules’ , and the consensus sequence was obtained using the Burrows-Wheeler Aligner (BWA) program .
The SNPs were identified in the consensus polymorphic SNP locus through the comparative genomic analysis of ‘Haryejosaeng’ and ‘Miyagawa wase’ (Additional file 1: Table S1) using the IGV software ( http://software.broadinstitute.org/Software/igv/). These SNPs were then compared with those in the genomes of ‘Miyamoto wase,’ ‘Okitsu wase,’ and ‘Nichinan 1 gou’ (Additional file 1: Table S1).
Using the selected SNPs, SNP-specific primers were synthesized for the identification of ‘Haryejosaeng.’ The SNPs were located at the 3′-end of each sequence, and, for specificity, an artificial SNP was introduced in the third base at the 3′-end. In addition, for the re-verification of the SNP marker, primer sets for the HRM analysis were designed on both sides of the SNP locus based on the results of genome sequencing (Table 4).
Cultivar selection using the PCR-based SNP markers
For the selection of ‘Haryejosaeng,’ multiple PCRs were carried out using the different primer sets (6 pairs) and a negative control primer set (Table 4). A multiplex PCR was performed by utilizing a combination of 2 selected SNP primers. All the PCRs were performed under the same conditions.
The PCR mixture contained 15–20 ng total genomic DNA, 0.25 μM of each primer, AccuPower® Multiplex PCR PreMix (250 μM dNTPs, 1.5 mM MgCl2, 1.0 unit Taq DNA polymerase, 10 mM Tris–HCl [pH 9.0], 40 mM KCl; Bioneer Corp., Daejeon, Korea), and the volume was adjusted to 20 μL with nuclease-free water . The PCRs were performed as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 58 °C (50 °C for P7 primer) for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The PCR products were electrophoresed on a 1.2%–1.5% agarose gel at 100 V for 30 min or were reconfirmed using a QiAxcel Advanced System electrophoresis device (Qiagen, Hilden, Germany).
For the selection of ‘Haryejosaeng,’ multiple PCRs were carried out using the different primer sets (6 pairs) and a negative control primer (one set; Table 4). A multiplex PCR was performed using a combination of 2 selected SNP primers. All the multiplex PCRs were performed under the same conditions.
The multiplex PCR mixture contained 15–20 ng total genomic DNA, 0.25 μM each primer, and AccuPower® Multiplex PCR PreMix [250 μM dNTPs, 1.5 mM MgCl2, 1.0 unit Taq DNA polymerase, 10 mM Tris–HCl (pH 9.0), 40 mM KCl; Bioneer Corp.]; the final volume was adjusted to 20 μL with nuclease-free water . The PCRs were performed as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles at 95 °C for 1 min, 58 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The multiplex PCR products were electrophoresed on a 1.2% agarose gel at 100 V for 30 min or were reconfirmed using a QiAxcel Advanced System electrophoresis device (Qiagen).
The HRM analysis was performed according to the modified method described by Park et al.  on a Roche LightCycler® 480 II (Roche Diagnostics Ltd., Rotkreuz, Switzerland). The reaction mixture included 15–20 ng total genomic DNA and 0.025 μM forward and reverse primers (Table 4), 1.8 μL of 25 mM MgCl2, and 7.5 μL LightCycler 480 High-Resolution Melting Master Mix (Roche Diagnostics GmbH, Mannheim, Germany), and the volume was adjusted to 15 μL with sterile water. The PCR conditions included pre-denaturation at 95 °C for 5 min followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. In the subsequent step, the mixture was subjected to 95 °C for 1 min and then to 40 °C for 1 min; this was followed by an increase in temperature from 65 °C to 95 °C at a rate of 0.1 °C/s, and the volume was adjusted to 20 μL using nuclease-free water.