Apeki

Both the quality and quantity of genomic DNA were evaluated by agarose gel electrophoresis and by UV/Vis spectrophotometry (NanoDrop 2000c, Thermo Fisher Scientific, Pittsburgh, PA, USA). RAD seq was performed as reported [32 (link)] with the exception that the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA) was used. Adapter-ligated DNA fragments were pooled and sheared to a mean size of 500 bp. The RAD-Seq libraries were enriched by polymerase chain reaction amplification and sequenced on an Illumina HiSeq 2000 (BGI, Shenzhen, China) using single-ended reads (50 bp) for each peppermint variety. For SNP calling and data analysis, the Illumina sequence reads were quality-filtered by removing the adapter sequences and reads containing greater than 50% low-quality bases. These processed reads were mapped on the reference peppermint genome using BWA-MEM (version 2, GitHub, Hobro, Denmark). Then, the comparison results filtered and SNPs were called by SAMTOOLS (version 1.16, Genome Research, Cambridgeshire, UK). High quality SNPs were used for the estimate of pairwise genetic distances and the reconstruction of phylogenetic trees based on maximum parsimony using MEGA 11 (Mega Software Technologies, Philadelphia, PA, USA) [33 (link)].

DNA was isolated from approximately 100 mg of fresh leaf blade + pseudostem tissue for 577 training set mother plants, using a high-throughput method based on that described by Whitlock et al. (2008 (link)) with modifications including a final binding, washing and eluting DNA from AcroPrep™ Advance 96 Filter Plates (Pall Corporation, Ann Arbor, MI, USA). DNA quality was checked via visualisation on ethidium bromide stained 0.8% (wt/vol) agarose/TBE gels and then quantified using the Quant-iT™ PicoGreen^® dsDNA Assay Kit (Invitrogen, Carlsbad, CA). DNA concentrations were normalised to 20 ng/μl and subsequently used for GBS library preparation. GBS libraries were generated following the methodology of Elshire et al. (2011 (link)), with 100 ng of DNA digested using ApeKI (New England Biolabs, Ipswich, MA) and ligated to a unique barcoded adapter and a common adapter (99 ng). A total of six libraries were developed in 96-plex which included a blank and a common positive control sample. Each library was passed through a Pippin Prep™ DNA size selector (Sage Science, Beverly, MA, USA) to isolate fragments between 193 and 313 bp, which were then sequenced on two lanes of an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) at AgResearch Invermay, New Zealand.

Mosquitoes with CHIKV-positive saliva were selected from mosquitoes inoculated with CHIK_IC, CHIK_IC^P398A, CHIK_IC^PPR401AAA, CHIK_IC-FG_N or CHIK_IC-FG_C. A selection of mosquitoes was made to represent each replicate experiment. Thirty μl of mosquito sample supernatant was used to inoculate a well of a 96-wells plate pre-seeded with C6/36 cells. At 3 days post infection RNA was isolated with TRIzol reagent (Invitrogen) and 300 ng total RNA was subjected to one-step RT-PCR with primers 3,4 using SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) following the manufacturer’s protocol. RT-PCR products were observed on agarose gel to confirm the infection of mosquitoes with CHIKV. To investigate the presence and preservation of the expected mutations, RT-PCR products were subjected to digestion with SacII (NEB), NotI (NEB) or ApeKI (NEB) according to the manufacturer’s protocol. Digestions were observed on agarose gel.

Genomic DNA of the 57 olive cultivars was extracted with the cetyl-trimethyl-ammonium-bromide (CTAB) method as described by Murray and Thompson (1980) (link). Qualified DNA samples, after checking on agarose gel, were digested with ApeKI (New England Biolabs, USA) and then ligated to either barcoded adaptors or common adaptors. Only short samples featuring both barcode and common adaptor were enriched by PCR amplification and then purified by magnetic beads with a range of 250–300 bp. Finally, paired-end sequencing was performed on an Illumina HiSeq 2000 platform at Beijing Genomics Institute (BGI) in Hong Kong.

Extracted DNA samples were digested for 3 h at 75 °C with the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, U.S.A), and genotyping-by-sequencing (GBS) was performed based on the methods outlined by Vigueira et al. [26 (link)] using the Illumina Hiseq 2000 platform (Illumina Inc., San Diego, CA). Raw sequence reads in a FASTQ format were processed using a standard TASSEL-GBS pipeline version 5.0 (http://www.maizegenetics.net/) [27 (link)]. Briefly, filtered reads were aligned to the rice genome MSU 6.0 (http://rice.plantbiology.msu.edu) using the Burrows–Wheeler alignment (BWA) tool [28 (link)]. This allowed a maximum of four mismatches and no gap within 5 bp at the end of each read. Loci with more than 30% of missing data and monomorphic data were discarded. Individuals with more than 95% of missing data were removed and not included in this study. After filtering, a total of 32,053 SNPs were retained for GWAS analysis. The mixed linear model (MLM) in the TASSEL-GBS program was used to generate a Manhattan plot for association analysis.

Preliminary testing with three restriction enzymes (PstI, MspI and ApeK1) for fragment size range led to selection of ApeKI for GBS library construction. Three GBS libraries were constructed at the USDA-ARS National Clonal Germplasm Repository (NCGR) and one was constructed at Clemson University according to the procedure previously described (Elshire et al., 2011 (link)) for 96 samples using DNA (100 ng per sample) digested with 4 U of ApeKI (New England Biolabs, Ipswich, MA, USA). The annealed and normalized unique and four-nucleotide-barcoded adaptors were obtained from Clemson University Genomics Institute (CUGI) and from the Oregon State University (OSU) Center for Genome Research and Biocomputing (CGRB) core facility. Two libraries were sequenced at the CGRB, one at CUGI, and one at the North Carolina State University Genomic Sciences Laboratory (Table S1). At each of these labs, libraries were quantitated with a Qubit^® fluorometer (Invitrogen, Carlsbad, CA, USA), checked for adequate size distribution (150–350 bp) with the Bioanalyzer 2100 HS-DNA chip (Agilent Technologies, Santa Clara, CA, USA), and sequenced with the Illumina HiSeq2000 (101 bp, single-end).

RADseq libraries were prepared from the isolated DNA as described following Grewe et al. (2017 (link)). In summary, DNA isolations were pooled with sequence adapters (Rubin & Moreau, 2016 (link)), digested with the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, USA), and ligated using T4 ligase (New England Biolabs). All samples with compatible barcodes were pooled and selected for fragment sizes between 300 and 500 bp using the BluePippin DNA size selection system (Sage Science, Beverly, MA, USA). The pooled libraries were amplified using the REDTaq ReadyMix (Sigma‐Aldrich, St. Louis, MO, USA) prior to sequencing on an Illumina MiSeq using the MiSeq Reagent Kit v3 for 150 cycles (Illumina, San Diego, CA, USA) to produce single‐end sequences with a length of 150 bp.