2.2 | Genotyping
A double-digest restriction-site-associated DNA (ddRAD) library was prepared using a modified protocol of the standard method (Peterson et al. 2012) described in our previous study (Nagamitsu et al. 2020). Extracted DNA was digested with Pst I and Sau 3AI restriction enzymes, ligated with Y-shaped adaptors, and amplified by polymerase chain reaction (PCR) with the KAPA HiFi polymerase (Kapa Biosystems, Woburn, MA, USA). After PCR amplification with adapter-specific primer pairs (Access Array Barcode Library for Illumina, Fluidigm, South San Francisco, CA, USA), an equal amount of DNA from each tree was mixed and size-selected with the BluePippin agarose gel (Sage Science, Beverly, MA, USA). Approximately 450-bp library fragments were retrieved. The library quality was checked using a 2100 Bioanalyzer with a high sensitivity DNA chip (Agilent Technologies, Waldbronn, Germany). The ddRAD library was sequenced using Illumina MiSeq and HiSeq to generate paired-end reads with a 150-bp length.
Obtained reads were mapped to the reference sequences consisting of 12 pseudomolecules (chromosomes) of the Q. robur genome assembly PM1N (Plomion et al. 2018). Although some reference sequences of white oak species including Q. mongolica var. mongolica andQd are available (Sork et al. 2022; Ai et al. 2022; Wang et al. 2023), we selected PM1N because Q. robur is a species closely-related to the common ancestor of Qc and Qd . The read mapping and variant calling were conducted using dDocent 2.7.8 (Puritz et al. 2014). The subsequent filtering procedures in detail are explained in our previous study (Nagamitsu et al. 2020). From the variant loci with various types of polymorphism, we selected loci that were biallelic without indels and polymorphic with high sequencing quality, < 5% missing genotypes, and > 5% minor allele frequency, using VCFtools 0.1.14 (Danecek et al. 2011). We removed loci mapped to the regions of transposable elements determined in Q. robur (Plomion et al. 2018). In addition, we removed loci that extremely deviated from the Hardy–Weinberg equilibrium (P< 0.01) probably due to null alleles within a population of each taxon, Qc , Qa , or Qd , using VCFtools.