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.