Aster savatieri Makino (Asteraceae) is a perennial herb endemic to Japan (Makino, 1898). It grows in the understory of forests on the islands of Honshu, Shikoku, and Kyushu and is distinguishable from other Japanese congeners by the lack of pappus in its achene and its spring flowering habit (flowering of other species occurs from summer to fall). Aster savatieri var. pygmaeus Makino was originally recognized as a dwarf form occurring on Mt. Asama, in Mie Prefecture, Honshu, Japan (Makino, 1913). However, the taxonomic treatment of this variety is controversial. Dwarf forms have been reported from other localities in southwestern Honshu and Shikoku, and these were sometimes considered as var. pygmaeus (Makino, 1918; Kitamura, 1936). In contrast, Iwatsuki et al. (1995) considered var. pygmaeus to be a dwarf form endemic to serpentine areas in Aichi Prefecture, Mie Prefecture (= Mt. Asama), and Shikoku. Ploidy levels may be considered in taxonomic studies because differences in ploidy can affect plant size (Kondorosi et al., 2000; Tsukaya, 2013). Although few studies have examined ploidy levels in A. savatieri, a nonserpentine population of var. pygmaeus has been reported to be diploid and polymorphisms have often been found in western Honshu populations of A. savatieri (2n = 2x = 18, 2n = 3x = 27, 2n = 4x = 36; Huziwara, 1954; N. Ishikawa, T. Fukuda, S. Sakaguchi, and M. Ito, unpublished data). Therefore, the taxonomic discrimination of A. savatieri var. savatieri from A. savatieri var. pygmaeus requires analyses of the genetic relationships among serpentine and nonserpentine populations, as well as among populations with different ploidy levels.
Although eight simple sequence repeat (SSR) markers have been reported for A. amellus L. (Mayor and Naciri, 2007), only two polymorphic markers have been successfully amplified by PCR in A. savatieri (Y. Morishita and M. Ito, unpublished data). Thus, additional markers are needed to investigate the population divergence in greater detail. We developed 22 polymorphic expressed sequence tag (EST)–SSR markers for A. savatieri and evaluated their polymorphisms in, and transferability to, multiple species of Aster L. and a related genus.
METHODS AND RESULTS
Total RNA was extracted from A. savatieri (Appendix 1; Aichi population) and A. savatieri var. pygmaeus (Appendix 1; Kochi population) using the Agilent Plant RNA Isolation Mini Kit (Agilent Technologies, Santa Clara, California, USA). Normalized cDNA libraries of shoots and roots of A. savatieri were constructed and sequenced using the HiSeq 2000 system (Illumina, San Diego, California, USA). De novo assembly of 37,253,459 cleaned 100-bp reads using Trinity (Grabherr et al., 2011) produced 162,360 contigs (N50: 1678 bp). A cDNA library of A. savatieri var. pygmaeus inflorescences was constructed and sequenced using the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, Massachusetts, USA). De novo assembly of 8,280,151 cleaned reads (≥400 bp) with CLC Genomics Workbench version 7.5.1 software (CLC bio, Aarhus, Denmark) produced 81,275 contigs (word size 43, bubble size 40, N50: 502 bp).
Microsatellite regions (≥10 dinucleotide repeats, ≥7 trinucleotide repeats) were searched using MSATCOMMANDER (Faircloth, 2008). Primer pairs with an optimal annealing temperature of 60 ± 2°C, a GC content of 30–70%, and a product size range of 100–500 bp were generated by Primer3 (Rozen and Skaletsky, 1999). We obtained 118 and 284 primer sets for A. savatieri and A. savatieri var. pygmaeus, respectively. Each of the 48 primer sets was selected from the two taxa based on the repeat numbers. For all 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 (5′-GTTTCTT-3′). A similarity search of each contig against the National Center for Biotechnology Information (NCBI) nr database was conducted using the BLASTX algorithm. PCR reactions were performed using a QIAGEN Multiplex PCR Kit (QIAGEN, Hilden, Germany) in a 10-µL volume containing 5–10 ng DNA, 5 µL 2× Multiplex PCR Master Mix, 0.01 µM forward primer, 0.2 µM reverse primer, and 0.1 µM fluorescently labeled M13 primer. The PCR protocol was as follows: 95°C for 3 min; followed by 35 cycles of 95°C for 30 s, 57°C for 3 min, 68°C for 1 min; and a 20-min extension at 68°C. The PCR product was loaded with DNA Size Standard 600 (Beckman Coulter, Brea, California, USA) onto a GenomeLab GeXP Genetic Analysis System (Beckman Coulter), and fragment size was determined with CEQ fragment analysis software (Beckman Coulter).
For PCR amplification trials, we used two individuals from each of the two A. savatieri populations (Appendix 1; Aichi and Nagano populations) and the two A. savatieri var. pygmaeus populations (Appendix 1; Mie and Kochi populations). For the 22 primer pairs that showed clear peaks (Table 1), 24 individuals from each population (Aichi, Kyoto, and Mie) were evaluated for polymorphisms. All of the 24 individuals were considered to be diploid because no more than two alleles were found in any loci. We also confirmed the diploid status of these samples by microscopic chromosome counting of one individual from the Mie population, which showed that it was diploid (2n = 2x = 18). Flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) analyses of 10 individuals from each population revealed that all were diploid. Summary statistics were generated using GenAlEx 6.5 software (Peakall and Smouse, 2012), i.e., number of alleles per locus (A), expected heterozygosity (He), and observed heterozygosity (Ho). The significance of Hardy–Weinberg equilibrium and genotypic equilibrium was tested by 1000 randomizations with adjustment of the resulting P values through the Bonferroni correction using FSTAT 2.9.3 software (Goudet, 1995).
Twenty-two primer pairs were polymorphic; A ranged from four to 15 alleles, while H e and H o ranged from 0.417 to 0.870 and 0.174 to 0.690, respectively (Table 2). No significant departures from Hardy–Weinberg equilibrium were detected for any of the populations or loci after correcting for multiple tests (nominal level of significance: 0.05). No significant genotypic equilibrium was detected for any pair of loci. We examined the transferability of these primers to six representative Japanese Aster species and Solidago virgaurea L. subsp. asiatica Kitam. ex H. Hara var. asiatica Nakai ex H. Hara, a member of the tribe Astereae (Asteraceae). The Aster species were selected to cover the main lineages of Japanese Aster (Table 3; Appendix 1; Ito et al., 1998). The Solidago L. species was included to assess the general applicability of the primers. The PCR protocol was as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 57.5°C for 3 min (with reductions of 0.1°C per cycle), 68°C for 1 min; with a 20-min extension at 68°C. Of the 22 EST-SSR primer pairs tested, 14–20 and 16 loci were successfully amplified in the six Aster species and S. virgaurea subsp. asiatica var. asiatica, respectively (Table 3, Appendix 1). Thus, most of the loci were transferable to the examined species.
The 22 EST-SSR markers developed were substantially polymorphic within and between populations. Thus, these markers will be useful for investigations of intraspecific relationships among A. savatieri var. savatieri and A. savatieri var. pygmaeus populations occurring at serpentine and nonserpentine sites. Transferability analyses were conducted with six representative species of Japanese Aster and S. virgaurea subsp. asiatica var. asiatica, a member of the tribe Astereae (Asteraceae). Of the 32 Japanese Aster species, 20 are endemic to Japan and 11 are regarded as endangered (Iwatsuki et al., 1995; Ministry of the Environment, 2012). Thus, our markers should also prove useful in conservation-directed investigations of genetic variation in endangered Aster species that occur in Japan.
Characteristics of the 22 polymorphic EST-SSR markers for Aster savatieri and A. savatieri var. pygmaeus.
Transferability of the 22 EST-SSR markers for Japanese Aster and Solidago species.
The authors thank Dr. Y. Watanabe (Chiba University, Japan), Dr. S. Nakagawa (Kyoto University, Japan), and T. Nishino (Osaka Prefecture University, Japan) for providing plant material. This research was partly supported by the Environment Research and Technology Development Fund (no. 4-1403) from the Ministry of the Environment, Grants-in-Aid for Scientific Research (no. 25291085) from the Japan Society for the Promotion of Science, and the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology.