2.3 | Genotyping-by-Sequencing Library and SNPs
Selection
We collected leaf tissue samples from 140 individuals of J.
chilensis (juveniles and adults) spaced within 150-500 m, between
February and December 2015. Samples were stored in silica. For molecular
analysis, DNA was extracted using the DNeasy Plant Mini Kit (Qiagen,
EUA). DNA concentration was quantified using the Qubit High Sensitivity
Assay kit (Invitrogen) and DNA integrity was assessed through
visualization of bands in a 1.2% agarose gel electrophoresis. Prior to
library construction, DNA amount per sample was normalized at 100 ng/μL.
The Genotyping-by-Sequencing (GBS) library was constructed using the
standard protocol described by Elshire et al. (2011) and employing theApeK I restriction enzyme. Single-end 100-bp sequencing was
conducted on the Illumina HiSeq 2500 platform. Samples were
demultiplexed according to their respective barcodes. The generated
sequences were filtered to remove low-quality sequences and contaminated
reads using SeqyClean 1.9.9 (https://github.com/ibest/seqyclean);
only high-quality paired-end sequences (with average PhredScore over 24
and over 65bp) were used for further analysis. The SNPs prospection was
performed using the software pipeline PyRAD v1.2 (Eaton, 2014). The data
was filtered with PLINK. To conduct population genetic analysis, we
identified putative loci under selection using software BayeScan 2.1
(Foll & Gaggiotti 2008) using the default values (Q-value
<0,05). The LOSITAN software was used to identify loci under
selection (Antao et al. 2008) and removed them from the SNPs dataset.
The R package r2vcftools (https://github.com/nspope/r2vcftools) - a
wrapper for VCFtools (Danecek et al., 2011) - was used to perform final
quality control on the genotype data. Filtering criteria included
biallelic SNPs, linkage disequilibrium (r2 < 0.8, (Xuereb et
al., 2018)), Hardy-Weinberg Equilibrium (HWE, p > 0.0001),
and loci with less than 20% missing data.