Materials and Methods
Total RNA was extracted from an individual of T. standishii collected from the Forestry and Forest Products Research Institute Arboretum using a plant RNA isolation mini kit (Agilent Technologies, USA). An RNA-seq data set was constructed by the Beijing Genomics Institute on an Illumina HiSeq 4000 platform. The T. standishii RNA-seq data consisted of 38,076,160 paired-end reads of 100 bp length. De novo assembly was undertaken in CLC Genomics Workbench 8.5.1 and the 53,614 resultant contigs (N50 = 1,503 bp) were mined for microsatellite regions. Primers were developed bordering these regions with default settings using PrimerPro (http://webdocs.cs.ualberta.ca/∼yifeng/primerpro/). Microsatellites were selected if the number of tandem repeat units was greater than eight and if the microsatellite was located less than 25 bp from the beginning or end of the contig. These criteria resulted in 64 microsatellite primer pairs which were trialled for amplification in four samples. A total of 36 primer pairs successfully amplified and were subsequently tested for size heterogeneity in eight samples representative of the species range. For all loci, the forward primer was synthesized with one of three different M13 sequences (5’ GCCTCCCTCGCGCCA 3’, 5’ GCCTTGCCAGCCCGC 3’, and 5’ CAGGACCAGGCTACCGTG 3’), and the reverse was tagged with pig-tail (5’ GTTTCTT 3’; (Brownstein et al., 1996)). The PCR reactions were performed following the standard protocol of the Qiagen Multiplex PCR Kit (Qiagen, Hilden, Germany), and consisted of a 10 uL reaction volume, containing approximately 5 ng of DNA, 5 uL of 2x Multiplex PCR Master Mix, and 0.06 uM of forward primer, 0.1 uM of reverse primer, and 0.08 uM of ﬂuorescently labelled M13 primer. The PCR thermocycle consisted of an initial denaturation at 95°C for 3 min; followed by 35 cycles of 95°C for 30 sec, 60°C for 3 min, 68°C for 1 min; and a 20 min extension at 68°C. The PCR products were separated by capillary electrophoresis on an ABI3130 Genetic Analyzer (Life Technologies, Waltham, MA, USA) with the GeneScan 600 LIZ Size Standard (Life Technologies) and genotyping was done in GeneMarker (SoftGenetics, LLC, PA, USA). Overall, 15 loci were found to amplify reliably, display polymorphism and were readily scorable. The genetic variability of these 15 markers were tested in three populations from Atebi Daira Small Bird Forest, Mt Chausu Nature Park, in Nagano Prefecture (35.2286° N, 137.6673° E), Mt Torigata in Kouchi Prefecture (33.4936° N, 133.0638° E) and Mt Yamizo in Fukushima Prefecture (36.9343° N, 140.2679° E). The 15 primer pairs were also tested in 13 samples of T. sutchuenensis and four of T. koraeinsis (Table S1). Genetic analyses were undertaken in GenAlEx 6.5 (Peakall and Smouse, 2006) and Genepop 4.2 (Raymond and Rousset 1995). In addition, a similarity search of the contigs containing the 15 loci was conducted by the BLASTX algorithm (Altschul et al. 1990) against the National Center for Biotechnology Information (NCBI) non-redundant protein sequences (nr) database.
The multi locus probability of identity (PID) for the 15 markers, that is, the probability that two individuals drawn at random from a population will have the same genotype (Waits, et al. 2001), was calculated in Gimlet version 1.3.3 (Valiere 2002). Three PID estimates outlined by Waits et al. (2001) were estimated: biased PID which assumes individuals mate randomly; unbiased PID which corrects for sampling a small number of individuals and, sibs PID which assumes the population is composed of siblings