The Solidago virgaurea L. complex (Asteraceae), a deciduous herbaceous perennial (2n = 18), is proposed to comprise three species (S. virgaurea L., S. minutissima (Makino) Kitam., and S. yokusaiana Makino) in the Japanese archipelago (Iwatsuki et al., 1995). However, high levels of morphological variation among populations due to its polymorphic nature and plasticity make it difficult to clearly delimit taxonomic boundaries (Hayashi, 1978; Takasu, 1978; Kawano, 1988), and thus the taxonomic treatment of this species complex is still in contention (Kadota, 2008; Semple, 2013). In particular, S. virgaurea is the most ecologically and morphologically diverse taxon, and includes five entities inhabiting alpine grassland (subsp. leiocarpa var. leiocarpa (Benth.) A. Gray), lowland forest and grassland (subsp. asiatica var. asiatica Nakai ex H. Hara), seashores and lowland hills in northern Japan (subsp. gigantea (Nakai) Kitam.), and southern island chains (subsp. leiocarpa var. praeflorens Nakai and subsp. asiatica var. insularis (Kitam.) Hara). Despite the apparently differentiated ecological niches, these taxa sometimes occur sympatrically or parapatrically, and in such circumstances intermediate individuals are often found, indicating probable hybridization (gene flow) among taxa (S. Sakaguchi, personal observation). Therefore, there is a great need for molecular studies that can provide new insights into phylogenetic relationships, population genetic structure, and gene flow between taxa and populations of the S. virgaurea complex in this region.
EST-SSR (simple sequence repeats in expressed sequence tags) markers are useful in these studies, because highly polymorphic markers can be developed with relative ease using the EST database (e.g., Sakaguchi et al., 2011) and they are less susceptible to null alleles than anonymous SSRs (Ellis and Burke, 2007). Here we developed 15 polymorphic EST-SSR markers for the S. virgaurea complex and evaluated their polymorphisms and transferability to other species of Solidago native to Eurasia and North America.
METHODS AND RESULTS
Assembled RNA sequencing (RNA-seq) data of S. canadensis L. (71,433 contigs) was obtained from the Plant OneKP Project repository ( https://sites.google.com/a/ualberta.ca/onekp/home), and a similarity search of the contigs against the National Center for Biotechnology Information (NCBI) nr database was conducted using the BLASTX algorithm (Altschul et al., 1990) with an E-value cutoff of 1.0E-5. We screened the sequences including microsatellite regions for ≥6 dinucleotide repeats and ≥4 tri- to hexanucleotide repeats using MSATCOMMANDER (Faircloth, 2008) and designed primers using Primer3 software (Rozen and Skaletsky, 2000). A total of 6471 primer pairs bordering microsatellites were designed, and 96 pairs were selected for PCR amplification trials using eight individuals representing the seven taxa collected from a broad range of the Japanese archipelago (Appendix 1). For all the loci, the forward primer was synthesized with one of three different M13 sequences (5′-CACGACGTTGTAAAACGAC-3′, 5′-TGTGGAATTGTGAGCGG-3′, or 5′-CTATAGGGCACGCGTGGT-3′), and the reverse primer was tagged with a PIG-tail sequence (5′-GTTTCTT-3′) to promote full adenylation (Brownstein et al., 1996). Plant DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). The PCR reaction was carried out following the standard protocol of the QIAGEN Multiplex PCR Kit (QIAGEN, Hilden, Germany), in a final volume of 10 µL, which contained approximately 5 ng of DNA, 5 µL of 2× Multiplex PCR Master Mix, and 0.01 µM of forward primer, 0.2 µM of reverse primer, and 0.1 µM of M13 primer (fluorescently labeled with Beckman Dye, Beckman Coulter, Brea, California, USA). The PCR thermal profile involved denaturation at 95°C for 3 min; followed by 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min; and a final 7-min extension step at 72°C. PCR products were loaded onto an auto sequencer (GenomeLab GeXP, Beckman Coulter) to assess fragment lengths using Fragment Analysis Software version 8.0 (Beckman Coulter). For the 34 primer pairs that exhibited clear microsatellite peaks, extracted DNA of 93 individuals of the S. virgaurea complex (from four populations in Fukushima [37°41′02″N, 140°27′09″E], Nagano [36°19′59″N, 137°39′34″E], Tokyo [34°13′18″N, 139°09′28″E], and Hyogo [34°51′28″N, 135°18′53″E]; see also Table 1) was used to evaluate allelic polymorphism. In addition, transferability among the other Solidago species (S. minutissima [N = 2] from Yakushima Island, Japan [30°20′07″N, 130°30′17″E]; S. altissima L. [N = 2], diploid individuals from Minnesota, USA [46°51′12″N, 92°01′52″W]; S. canadensis [N = 1], diploid individual from Jena, Germany [50°54′40″N, 11°34′02″E]; S. hispida Muhl. ex Willd. [N = 1], diploid individual from Minnesota, USA [46°47′52″N, 92°04′43″W]) was tested using the same PCR conditions described above. To characterize each EST-SSR marker, three summary statistics were calculated using GenAlEx 6.5 (Peakall and Smouse, 2012): number of alleles per locus (A), expected heterozygosity (He), and observed heterozygosity (Ho). In addition, the significance of Hardy–Weinberg equilibrium and genotypic equilibrium were tested by 1000 randomizations with adjustment of resulting P values by sequential Bonferroni correction, using FSTAT 2.9.3 (Goudet, 1995).
Characteristics of the 15 polymorphic EST-SSR markers for the Solidago virgaurea complex.a
Fifteen primer pairs (Table 1) were shown to be polymorphic, with A ranging from three to 14 alleles, while He and Ho ranged from 0.053 to 0.874 and 0.054 to 0.634, respectively (Table 2). Significant departures from Hardy–Weinberg equilibrium were detected in eight loci in the four populations, but most are specific to one or two populations (Table 2). No significant genotypic equilibrium for any pair of loci was detected. Of the 34 EST-SSR primer pairs tested, 33 were successfully PCR amplified for S. minutissima and 30 for each North American species of S. altissima, S. canadensis, and S. hispida (Table 3).
Transferability of the 34 EST-SSR markers for the Eurasian and North American Solidago species.a
We developed 15 polymorphic EST-SSR markers for the S. virgaurea complex, most of which are transferable in different Solidago species. These markers may be useful for evaluating the population structure and taxonomic delimitation of the S. virgaurea complex, as well as providing useful markers to investigate the population genetics and reproductive ecology of Solidago species.
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 This research was by supported by a Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JSPS KAKENHI; grant no. 25291085). The authors thank Dr. M. Deyholos for preparing the A-seq library of Solidago canadensis and Plant OneKP Project for kindly allowing us to access the sequence data. We are grateful to O. Kurashima for helping with primer design, and Dr. S. Kaneko, Y. Matsuura, Y. Sakata, and K. Ishida for collecting plant materials.