The second evaluation procedure was the molecular DNA fingerprint analysis, which was realized in the years 2012 to 2014 and 2017 to 2019. The microsatellite primers were selected according to the guidelines of the ECPGR Prunus Working Group, which recommended a standard set of 16 SSR markers [10 (link)]: BPPCT037 [39 (link)], CPPCT006, and 022 [40 (link)], EMPA002, 003, 017, and 026 [10 (link)], EMPaS01, 02, 06, 10, 12, and 14 [22 (link)], PceGA34 [41 (link)], PS05C03 [42 (link)], and UDP98-412 [43 (link)] (Table S1 ). These markers were developed mainly from P. avium and provided good coverage of the cherry genome (two unlinked markers per linkage group).
The first sample set, collected in 2012–2014, was genotyped by the Competence Centre for Fruit Production–Lake Constance (KOB, Ravensburg, Germany). Leaf material of the single trees was collected from the different collection sites in 2 mL reaction tubes and dried using silica gel according to a modified protocol by Slotta, et al. [44 (link)]. The leaf material was stored at room temperature until DNA isolation. The DNA isolation was performed using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Multiplex PCR reactions were carried out using the Taq DNA Polymerase (cloned) (GE Healthcare Life Science, Freiburg, Germany) according to the manufacturer’s instructions, with an initial denaturation at 94 °C for 2 min, followed by 37 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 1 min, and elongation at 72 °C for 2 min. The PCR reaction was completed with a final elongation step at 72 °C for 10 min. Fragment lengths analysis was done on a CEQ 8000 capillary sequencer (Beckman Coulter GmbH, Krefeld, Germany) using the GenomeLab™ GeXP software (Beckman Coulter GmbH, Krefeld, Germany).
The second sample set, collected in 2017–2019, was genotyped by Ecogenics GmbH c/o Microsynth AG (Balgach, Switzerland). Leaf material of each single tree was collected from the different collection sites in a sample bag and was immediately stored on dry ice. Until DNA isolation, the samples were stored at −80 °C. For DNA isolation, the Hotshot method, according to Truett, et al. [45 (link)], was used. In order to remove any PCR inhibitors, an additional DNA purification step was performed using the OneStep PCR Inhibitor Removal Kit (Zymo Research, Freiburg, Germany). Multiplex PCR was performed using the Type-It kit (Qiagen, Germany) according to the manufacturer’s instructions, with an initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 94 °C for 0.5 min, annealing at 55 °C (48 °C for EMPaS01 und PS05C03) for 1.5 min, and elongation at 72 °C for 1 min. The PCR reaction was completed with a final elongation step at 72 °C for 30 min.
Fragment lengths analysis was done on a 3730XL DNA-Analyzer (Applied Biosystems, Waltham, MA, USA) using the Software GeneMarker V2.6.4 (SoftGenetics LLC., State College, PA, USA). In addition, the fragments were visually checked for appropriate quality. The eight genotypes P. avium F12/1, P. avium ‘Goodnestone Black’, P. avium ‘Napoleon’, P. avium ‘Noble’, P. avium ‘Noir de Meched’, P. incisa E621, P. mahaleb SL64 and P. nipponica F1292 (also recommended by the ECPGR), were used as reference cherry genotypes [10 (link)]. These reference genotypes allowed the harmonization of the SSR fingerprints originating from the two different laboratories that used different fragment length analysis protocols and devices for the genotyping.
The first sample set, collected in 2012–2014, was genotyped by the Competence Centre for Fruit Production–Lake Constance (KOB, Ravensburg, Germany). Leaf material of the single trees was collected from the different collection sites in 2 mL reaction tubes and dried using silica gel according to a modified protocol by Slotta, et al. [44 (link)]. The leaf material was stored at room temperature until DNA isolation. The DNA isolation was performed using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Multiplex PCR reactions were carried out using the Taq DNA Polymerase (cloned) (GE Healthcare Life Science, Freiburg, Germany) according to the manufacturer’s instructions, with an initial denaturation at 94 °C for 2 min, followed by 37 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 1 min, and elongation at 72 °C for 2 min. The PCR reaction was completed with a final elongation step at 72 °C for 10 min. Fragment lengths analysis was done on a CEQ 8000 capillary sequencer (Beckman Coulter GmbH, Krefeld, Germany) using the GenomeLab™ GeXP software (Beckman Coulter GmbH, Krefeld, Germany).
The second sample set, collected in 2017–2019, was genotyped by Ecogenics GmbH c/o Microsynth AG (Balgach, Switzerland). Leaf material of each single tree was collected from the different collection sites in a sample bag and was immediately stored on dry ice. Until DNA isolation, the samples were stored at −80 °C. For DNA isolation, the Hotshot method, according to Truett, et al. [45 (link)], was used. In order to remove any PCR inhibitors, an additional DNA purification step was performed using the OneStep PCR Inhibitor Removal Kit (Zymo Research, Freiburg, Germany). Multiplex PCR was performed using the Type-It kit (Qiagen, Germany) according to the manufacturer’s instructions, with an initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 94 °C for 0.5 min, annealing at 55 °C (48 °C for EMPaS01 und PS05C03) for 1.5 min, and elongation at 72 °C for 1 min. The PCR reaction was completed with a final elongation step at 72 °C for 30 min.
Fragment lengths analysis was done on a 3730XL DNA-Analyzer (Applied Biosystems, Waltham, MA, USA) using the Software GeneMarker V2.6.4 (SoftGenetics LLC., State College, PA, USA). In addition, the fragments were visually checked for appropriate quality. The eight genotypes P. avium F12/1, P. avium ‘Goodnestone Black’, P. avium ‘Napoleon’, P. avium ‘Noble’, P. avium ‘Noir de Meched’, P. incisa E621, P. mahaleb SL64 and P. nipponica F1292 (also recommended by the ECPGR), were used as reference cherry genotypes [10 (link)]. These reference genotypes allowed the harmonization of the SSR fingerprints originating from the two different laboratories that used different fragment length analysis protocols and devices for the genotyping.
Free full text: Click here
