Extraction of DNA and genotyping
The extraction of DNA, microsatellite genotyping, and a mitochondrial
DNA haplotype analysis were conducted using the procedure described in
previous studies (Shimozuru, et al. , 2019; Shirane, et
al. , 2018). Briefly, DNA was extracted using the DNeasy Blood & Tissue
Mini Kit (Qiagen Inc., Tokyo, Japan) for blood and tissue samples, the
DNA Extractor FM Kit (Wako, Osaka, Japan) or Isohair Easy (Nippon Gene,
Inc., Tokyo, Japan) for hair samples, and the QIAamp DNA Stool Mini Kit
(Qiagen Inc.) for feces samples. Twenty-one microsatellite markers and
one sex marker, amelogenin (Yamamoto, et al. , 2002), were
analyzed by a multiplex PCR assay (Shimozuru, et al. , 2020;
Shimozuru, et al. , 2019; Shimozuru, et al. , 2020). Allele
size was determined using an ABI PRISM 310 genetic analyzer or SeqStudio
Genetic Analyzer (Thermo Fisher Scientific K.K., Tokyo, Japan). In
addition, eight Y-linked microsatellite alleles (Y318.1, Y318.2, Y318.4,
Y318.6, Y318.9, Y369.1, Y369.4, and 15020.1; Hirata, et al. ,
2017) were determined by a multiplex PCR assay, using the same primer
sets as those used in previous studies (Bidon, et al. , 2014;
Hirata, et al. , 2017). The mitochondrial and Y chromosome
haplotype information were used to select candidate mothers for
offspring, and candidate fathers for male offspring, respectively.