Genemarker v 4

We extracted genomic DNA from the dried leaf tissue using a modified cetyl trimethyl ammonium (CTAB) protocol. Quantification of DNA was carried out with a SmartSpecTM Plus Spectrophotometer (Bio-Rad). To select candidate diagnostic microsatellites to identify parental species and hybrids, 219 SSR primers were designed based on sequences previously obtained on a MiSeq Benchtop Sequencer (Illumina, Inc., San Diego, CA, USA) for the close relative P. chungensis [24 (link)]. Detailed protocols for PCR amplification and condition regarding these primers followed Zhou et al. [24 (link)]. PCR products were directly analyzed on a 3730xl Sequence Analyzer (Applied Biosystems, Foster City, CA, USA), using a LIZ GeneScan-500 size standard. Resulting chromatograms were visualized and converted to diploid genotypes using automated allele-calling implemented in GENEMARKER v.4.0 (SoftGenetics LLC, State College, PA, USA). All automated genotyping was re-checked manually. All genotypes for each locus and individual were entered into an Excel file following the format of GenALEx 6.5 [25 (link)].

Total DNA was extracted from ca. 20 mg of dry leaves according to a modified CTAB method [59 , 60 ]. DNA concentration and quality were measured on 1% TAE agarose gels and using a NanoDrop®ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA); subsequently, all samples were diluted to 30–50 ng/mL for PCR reactions. Previous studies [46 (link), 61 (link)] described a multiplex of 31 pairs was used for genotyping the 508 walnut trees (Tables 1, S2). PCR amplification and cycling conditions were performed according to Magige et al. [46 (link)]. Briefly, pre-denaturation at 98 °C for 2 min, 35 cycles of denaturation at 98 °C for 10 s, primer annealing at 53–61 °C for 15 s, extension at 72 °C for 10 s, and a final extension at 72 °C for 5 min, with a holding temperature of 4 °C. The fragment sizes of PCR products were separated with an ABI 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). GENEMARKER v4.0 (SoftGenetics, State College, PA, USA) was applied to score the SSR data as diploid genotypes.

Genotyping of the 280 tea accessions was carried out with a total of 23 highly polymorphic nSSRs employed in our previous study (Wambulwa et al., 2016 (link)). Visualization and sizing of the SSR fragments was performed using GENEMARKER v.4.0 (SoftGenetics LLC, State College, PA, USA). The genotyping procedure (amplification and fragment size determination) for all samples followed our previous study (Wambulwa et al., 2016 (link)). The nSSR data has been deposited at http://dx.doi.org/10.5061/dryad.3g02j in the Dryad Digital Repository.