Genemapper version 4

The genomic DNA was extracted from dried leaves of C. communis using the DNA extraction Kit (Qiagen, Hilden, Germany). The quality of extracted DNA was measured by NanoDrop spectrophotometer (Thermo Scientific, USA).
Polymorphism was assayed on each DNA sample with 12 microsatellite markers developed in our previous study (Supplementary Table S1; Li et al., 2015b (link)). The amplification reaction was carried out in 20 μL reaction mixture, containing 30–50 ng genomic DNA, 7.5 μL of 2×Taq PCR MasterMix (Tiangen, Beijing, China), 0.6 μM of each primer. Three fluorescent compounds (HEX, ROX or 6-FAM, Invitrogen, Carlsbad, CA, USA) were used to label the forward primers for the automated sequencers analysis (Supplementary Table S1). Amplification were carried out with an Eppendorf Mastercycler pro vapo protect thermocycler (AG, Hamburg, Germany), using the following thermocycling condition: initial denaturation at 95°C for 3 min, followed by 30 cycles of 30 s denaturation at 94°C, 30 s annealing at the optimal temperature (depending on each locus) and 1 min extension at 72°C, then 7 min at 72°C for the final extension. The ABI PRISM 3730XL DNA Sequencer (Applied Biosystems) and GeneMapper version 4.0 (Applied Biosystems) were used to separate the amplified products and determine the allele sizes, respectively.

The loci that generated PCR products with expected sizes on agarose gel were assessed for polymorphisms by high-resolution capillary electrophoresis. PCR products were generated by Touchdown PCR with fluorescently labeled M13 (–21) (5′-TGTAAAACGACGGCCAGT-3′) sequence-tag method [42] (link). Touchdown PCR was carried out using the following program: 95°C for 5 min; 30 cycles of 30 s at 94°C, 45 s at 56°C and 45 s at 72°C; 10 cycles of 30 s at 94°C, 45 s at 53°C and 45 s at 72°C; and a final extension of 5 min at 72°C. Fluorescently labeled PCR products were initially evaluated by 2% agarose gel electrophoresis and then analyzed by capillary electrophoresis with the GeneScan-500 LIZ Size Standard on an ABI 3730XL sequencer and their sizes were determined with GeneMapper version 4.0 (Applied Biosystems).

DNA was extracted from 120 mg of leaf material per plant using a scaled‐down version of the CTAB method (Elliott and Byrne 2005) and genotyped at seven microsatellite loci (HoA102, HoB103, HoB126, HoB010, HoB105, HoA116, and HoB125) using primers and conditions developed for H. oldfieldii by Byrne and Hankinson (2009).
Amplification products were separated on a 3730 capillary sequencer (Applied Biosystems, Foster City, CA) using a LIZ 500 (‐250) size standard. Bins were set and genotypes were scored using GENEMAPPER version 4.0 (Applied Biosystems) and checked manually. Samples were repeated for loci that failed to amplify or where seed genotypes were inconsistent with maternal genotypes. In addition, approximately 5% of samples were reamplified and rescored to check scoring accuracy.
To validate the microsatellite data, genotypes were tested for large allele dropout and stuttering artifacts, and the occurrence of null alleles using MICROCHECKER V. 2.2.3 (van Oosterhout et al. 2004). Tests for linkage disequilibrium were performed with global and exact tests for each locus/population combination using GENEPOP 4.2 (Rousset 2008).

Five aphids from each iso‐female line from the Osborne population were genotyped across ten microsatellite loci: M35, M37, M40, M49, M55, M63, M86, myz2, myz9 and myz25¹⁶ following the DNA extraction and genotyping methods outlined in Umina et al.⁹ Loci were labelled with unique fluorophores and co‐amplified in three separate multiplex polymerase chain reactions (PCR) using a Qiagen multiplex kit and an Eppendorf Mastercycler S gradient PCR machine as described in Blacket et al.¹⁷ PCR products were analysed using a 3730 capillary analyser (Applied Biosystems) and genotyping was conducted using GeneMapper version 4.0 (Applied Biosystems).
Additionally, using the same approach described above, we identified the clonal type of aphids from all other M. persicae populations phenotypically screened for resistance via laboratory bioassays.

The significant sites in the Mozambique sex QTL interval were validated in another population. One hundred individuals were randomly collected from our breeding population, which is an admixture population. The DNA was extracted using a HiPure Tissue and Blood DNA Kit (Magen, Guangzhou, China), and the quality was tested with 1% agarose gel electrophoresis. Primers were designed using Primer-BLAST to cover the SNPs. PCRs were performed using a PCR amplification Kit (PrimeSTAR^®HS, Takara, Dalian, China) with a program of 5 min at 94°C, 35 cycles of 45 s at 94°C, 45 s at 60°C, 45 s at 72°C, and 10 min at 72°C. Finally, PCR products were genotyped and sequenced on a 3130xl capillary DNA analyzer (Applied Biosystems, Foster City, CA, United States), and the allele sizes were analyzed using GeneMapper version 4.0 (Applied Biosystems, Foster City, CA, United States) and sequences were viewed using the Seqman software package (Lasergene Version 7.1; DNA Star Inc., Madison, WI, United States). The sites that contained the target SNPs in the mapping population were genotyped in the validation population as SNP or Indel (insertion or deletion). A genotypic (2 df) test was performed to test the relationship between genotype and sex (Purcell et al., 2007 (link)).

Methods for DNA extraction and microsatellite typing follow Garg et al., [21 (link)]. We used Ampli-Taq Gold (Applied Biosystems) for PCR amplification for samples from seasons one to six and PCR Multiplex mastermix (MM; Qiagen) for the rest of the samples. We scored all microsatellite alleles twice with GENEMAPPER version 4.0 (Applied Biosystems) and standardized the entire dataset using TANDEM [23 (link)]. We determined the error rate for microsatellite typing by regenotyping 5% of samples from seasons one to six (for Ampli-Taq Gold) and seasons seven to ten (for MM) respectively. We also regenotyped 5% of samples from season one to six with MM to test for consistency between Ampli-Taq Gold and MM. Overall 12% of the samples were regenotyped.