The Cupressaceae clade has the broadest diversity of habitats and morphologies of any conifer family, and the genus Thujopsis, which belongs to the Cupressaceae, is considered to be one of the early diverging genera (Pittermann et al., 2012). Thujopsis is native to Japan and includes one species (T. dolabrata Siebold & Zucc.) and one northern variety (T. dolabrata var. hondae Makino) (Hayashi, 1960). The two varieties of Thujopsis are distinguished by variation in their cone morphology. The varieties also differ in their geographic ranges, although their distributions overlap in the central region of the Japanese archipelago (Hayashi, 1960). Thujopsis dolabrata has antimicrobial properties and demonstrates a strong antifeedant effect on termites (Inamori et al., 2006). Moreover, its essential oil is used as an antibacterial agent and as an aromatic substance. Thus, T. dolabrata is an important tree species in Japanese forestry and future forest tree breeding because of its superior wood properties. However, there have been few reports about genetic differences between the two varieties.
Recently, expressed sequence tag (EST)–based markers have been used increasingly in studies of genetic diversity and population structure in tree species (e.g., Fageria and Rajora, 2013). EST-based markers are less susceptible to null alleles than anonymous simple sequence repeats (SSRs). Moreover, because ESTs correspond to coding DNA, the flanking sequences of EST-SSRs are located in well-conserved regions across phylogenetically related species (Uchiyama et al., 2013). Nuclear SSR markers have been developed for T. dolabrata var. hondae (Mishima et al., 2012), and population genetics and phylogeographic analyses of this variety were performed using these markers (Higuchi et al., 2012). However, the nuclear SSR markers have not been tested on T. dolabrata. An analysis of the genetic diversity and population structure for Thujopsis plants in the Japanese archipelago is urgently required. Therefore, it is necessary to obtain molecular markers with high transferability within the genus Thujopsis that also exhibit a low frequency of null alleles. In this paper, we describe the development and characterization of 22 EST-SSR markers for the genus Thujopsis from expressed sequence data of T. dolabrata var. hondae.
METHODS AND RESULTS
One T. dolabrata var. hondae individual (voucher no. TF-K12-001) was used for the RNA sequencing experiment. Leaves and cambiums were sampled from a population in the Aomori Prefecture (Owani: 40°27′21″N, 140°34′08″E) and were immediately frozen in liquid nitrogen and stored at −30°C. The cetyltrimethylammonium bromide (CTAB) method was used to extract the total RNAs (Chang et al., 1993). A TruSeq RNA Sample Prep Kit (Illumina, San Diego, California, USA) was used to create the RNA sequencing library, according to the manufacturer's protocol. A HiSeq 1000 (Illumina) was used to sequence the library with 2 × 101-bp paired-end reads. More than 237.24 million paired-end raw reads were obtained. After removal of low-quality reads, 233.92 million clean reads remained (accession no.: DRA002435). Using the short reads assembly programs Velvet 1.2.08 (Zerbino et al., 2009) and Oases 0.2.08 (Schulz et al., 2012), the clean reads were assembled de novo into 76,377 contigs and 41,182 unigenes, from 100 to 14,834 bp, with a mean length of 1525 bp. MS ATCOMMANDER 1.0.8 (Faircloth, 2008) was used to screen for microsatellite loci containing di- and trinucleotides. Primer3 (Rozen and Skaletsky, 1999) was then used to design PCR primers. The minimum number of repeats was set as nine and 11 for di- and trinucleotide repeats, respectively. All forward primers were fluorescently labeled using FAM (carboxyfluorescein) with the 454A adapter sequence (5′-GCCTCCCTCGCGCCATCAG-3′) at the 5′-end (Blacket et al., 2012). Additionally, all reverse primers were attached to a PIG-tail sequence (5′-GTTTCTT-3′) at the 5′-end of the sequence (Brownstein et al., 1996).
Characteristics of 22 EST-SSR loci for use in the genus Thujopsis.
The CTAB method (Murray and Thompson, 1980) was used to extract genomic DNAs from needles sampled from 32 T. dolabrata plants in the Nagano Prefecture (Kiso: 35°43′38″N, 137°37′15″E) and 49 T. dolabrata var. hondae needles were sampled from two populations in the Aomori Prefecture (Owani: 40°27′21″N, 140°34′08″E; and Okoppe: 41°28′42″N, 140°57′10″E). These populations were located in the typical range of each variety. To confirm PCR amplification, we used eight DNA samples from four individuals per variety. PCR was performed in a final volume of 10 µL, containing 5 µL of 2× GoTaq Hot Start Colorless Master Mix (Promega Corporation, Madison, Wisconsin, USA), 1 µM of each primer, and 60 ng of template DNA. Reactions were performed with initial denaturation at 94°C for 2 min; followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and then 72°C for 5 min using an ABI 9700 (Applied Biosystems, Foster City, California, USA). The PCR products were separated electrophoretically on 1% agarose gels and stained with GelRed Nucleic Acid (Nacalai Tesque, Kyoto, Japan). To confirm the presence of SSRs, we sequenced the PCR products from two DNA samples using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). For fragment analysis, the PCR conditions were modified. The concentration of the primers were 0.15, 0.5, and 0.2 µM for the forward primer, reverse primer, and 454A-FAM primer, respectively, and the number of PCR cycles was 30. PCR products for 81 DNA samples from two varieties were electrophoresed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) and Geneious 7.0.4 software (Biomatters Ltd., Auckland, New Zealand; http://www.geneious.com/) was used to assess the fragment sizes. To characterize the microsatellite loci, CERVUS 3.0 software (Kalinowski et al., 2007) was used to calculate the number of alleles (A), the observed heterozygosity (Ho), the expected heterozygosity (He), and the frequency of null alleles (r). GenAlEx 6.501 (Peakall and Smouse, 2012) assessed the probability of identity (PID).
The results of final primer screening on samples from three populations of the genus Thujopsis.
Primer3 designed 58 primer pairs, of which 33 showed amplification for all eight samples in both varieties. For 29 of the 33 primer pairs, the SSR sequence in the PCR product was identified for both varieties. Finally, 22 of the 29 amplified primers showed clear fragment patterns, thus they were selected as the developed markers. These primer pairs show different fragment sizes and the same annealing temperature (Table 1). All 22 loci were polymorphic in both varieties. The observed number of alleles per population ranged from one to 17, Ho ranged from 0.000 to 1.000, and He ranged from 0.000 to 0.926. The PID ranged from 0.015 to 1.000 (Table 2). One of the loci (Tdest54) showed high r values relative to the other loci. These 22 EST-SSR markers had lower r and higher PID values compared with reported SSR markers developed from the genomic DNA of T. dolabrata var. hondae (Mishima et al., 2012).
In this study, we developed 22 EST-SSR markers for the two varieties of Thujopsis, using expressed sequence data of T. dolabrata var. hondae. These markers have two advantages: high ability to detect genetic polymorphisms in Thujopsis varieties, and a low null allele frequency. Accordingly, these EST-SSR markers will be valuable tools for investigating the genetic diversity and population structure of the genus Thujopsis. Moreover, these markers will help to advance breeding programs for species in the genus Thujopsis.