Veratrum maackii Regel (Melanthiaceae) is distributed widely in Japan, Korea, northern China, and eastern Siberia. The species is a polymorphic perennial, and many infraspecific taxa have been described. In Japan, three varieties, namely, var. maackii, var. japonicum (Baker) T. Shimizu, and var. parviflorum (Miq.) Hara & Mizushima, and two forms, var. japonicum f. atropurpureum (Honda) Kitam. and var. parviflora f. alpinum (Nakai) Kitam., are recognized (Satake, 1982). These infraspecific taxa are distinguished based on perianth color, leaf shape, and capsule size, although these characteristics are continuous, and delimitation among taxa is difficult. Takada and Kawanobe (1996) examined variations in perianth colors and leaf shapes in populations of V. maackii in the Japanese Archipelago, reporting clinal variations in leaf shape. Individuals with green perianths were found in the northern part of Honshu Island, those with dark purple perianths were observed in other areas, and those with intermediate color perianths were observed in the zones where green and dark purple perianth individuals were sympatric. They inferred that these character variations reflect the distribution contraction and expansion of the species in the past. To obtain phylogeographic inferences regarding the species, molecular genetic data are indispensable but have not been available to date. In this study, we developed microsatellite markers for V. maackii to investigate its genetic diversity and population structure in Japan and surrounding areas. Furthermore, we explored cross-amplification of the developed markers in closely related taxa.
METHODS AND RESULTS
The genomic DNA of one individual of V. maackii from a population located in Sendai, Japan (Appendix 1), was used for the development of microsatellites. Genomic DNA was extracted from frozen leaf tissues using a modified cetyltrimethylammonium bromide (CTAB) method (Maki and Horie, 1999). The GS Junior Titanium Series Kit (Roche 454 Life Sciences, Branford, Connecticut, USA) was used to generate a shotgun library of genomic DNA. A 500-ng aliquot of genomic DNA was nebulized at 0.21 MPa for 1 min. The DNA fragments were purified using the MinElute PCR Purification Kit (QIAGEN, Hilden, Germany). The purified fragments were end-repaired, A-tailed, ligated to the Rapid Library Adapter, and suitably sized by removing small fragments (<350 bp) using the GS FLX Titanium Rapid Library Preparation Kit (Roche 454 Life Sciences). The fragments were then mixed with capture beads and amplified through emulsion PCR (emPCR) using the GS Junior Titanium emPCR Kit (Roche 454 Life Sciences). After emPCR, beads capturing the DNA library were enriched with sufficient amounts of template DNA for sequencing. The enriched beads were annealed with sequencing primers, and the amplified fragments were pyrosequenced on the GS Junior 454 System (Roche 454 Life Sciences).
A total of 33,542 sequences with an average read length of 450 bp were obtained and further screened for putative microsatellites using the MISA Perl script (Thiel et al., 2003). A total of 1158 microsatellite repeats were identified, including 1064 di-, 40 tri-, 27 tetra-, seven penta-, and 15 hexanucleotide repeat motifs, with minimum contiguous repeat units of eight, eight, five, five, and five, respectively. The most common motifs were AG/CT, AT, AC/GT, and ACC/GGT. We additionally developed and sequenced a microsatellite-enriched library using a modified biotin-capture method (Fischer and Bachmann, 1998). The detailed library construction procedure was presented in Li and Maki (2013). We obtained 30 sequences containing microsatellite repeats using the biotin-capture method.
Fifty sequences containing microsatellite repeats with sufficient flanking regions were chosen for primer design. Of the 50 sequences, 44 came from the 454 sequencing and six came from the modified biotin-capture method. Primer pairs were designed with an expected product size ranging from 120 to 350 bp using Primer3 version 2.0 (Rozen and Skaletsky, 2000). For the loci, the forward primer was ligated to a 454A adapter primer sequence (5′-GCCTCCCTCGCGCCATCAG-3′; Margulies et al., 2005), and the reverse was tagged with a PIG-tail (5′-GTTTCTT-3′; Brownstein et al., 1996). To evaluate amplifications of the primer pairs and polymorphisms at each locus, a panel of six individuals (two individuals from each population) collected from three natural populations located across the V. maackii range distribution in Japan (see Appendix 1; a northern population: Tanesashi; a central population: Mitsuke; and a southern population: Kirishima-dake) were used. PCR reactions were performed separately for each locus in 3-µL volumes containing approximately 60 ng of genomic DNA, 1× Type-it Multiplex PCR Master Mix (QIAGEN), 0.075 µM tailed forward primer, 0.1 µM fluorescently labeled 454A primer, and 0.25 µM reverse primer. The reaction parameters involved an initial denaturation step at 95°C for 5 min; followed by 32 cycles at 95°C for 30 s, 60°C for 90 s, and 72°C for 30 s; and a final step at 60°C for 30 min. PCR products were run with a GeneScan 600 LIZ internal size standard (Applied Biosystems, Foster City, California, USA) on an ABI 3100 Genetic Analyzer (Applied Biosystems).
Sequence and multiplex characteristics of the 10 microsatellites developed for Veratrum maackii.a
After excluding loci that showed unclear amplification patterns (no monomorphic markers were detected), we selected 10 polymorphic primer pairs, which were combined into three multiplex reactions using Multiplex Manager 1.0 (Holleley and Geerts, 2009). Of the 10 primer pairs, eight came from the 454 sequencing and two came from the modified biotincapture method. The forward primer was labeled using one of the fluorochromes 6-FAM, VIC, NED, or PET (Life Technologies, Tokyo, Japan; Table 1). Preliminary population genetic analyses were performed on individuals from Tanesashi (n = 20), Mitsuke n = 14), and Kirishima-dake (n = 19). Cross-species amplification was performed in V. album L. subsp. oxysepalum (Turcz.) Hultén, V. stamineum Maxim. var. stamineum, and V. stamineum var. micranthum Satake (Appendix 1). Multiplex PCRs were performed in 3-µL volumes containing approximately 30 ng of genomic DNA, 1× Type-it Multiplex PCR Master Mix (QIAGEN), and 0.2 µM forward and reverse primer, respectively. The reaction parameters were an initial denaturation step at 95°C for 5 min; followed by 28 cycles at 95°C for 30 s, 60°C for 90 s, and 72°C for 60 s; and a final step at 60°C for 30 min. PCR products were run with a GeneScan 600 LIZ internal size standard (Applied Biosystems) on an ABI 3100 Genetic Analyzer (Applied Biosystems). Allele sizes were scored using GeneMapper version 3.7 software (Applied Biosystems).
Characteristics of the 10 polymorphic microsatellite markers for Veratrum maackii.
The number of alleles per locus ranged from three to 19. The observed heterozygosity and expected heterozygosity ranged from 0.143 to 0.929 and 0.269 to 0.904, respectively (Table 2). The tests for deviation from Hardy—Weinberg equilibrium (HWE) at each locus and the linkage disequilibrium (LD) of all combinations of loci were conducted using GENEPOP 4.2 (Rousset, 2008). No significant departures from HWE were detected in loci among populations Tanesashi and Mitsuke of V. maackii (P > 0.01) (Table 2). One significant deviation (P < 0.01) from HWE was detected in the population Kirishima-dake (maackii-056) (Table 2). No combination of loci was in LD after Bonferroni correction (P > 0.05) (Rice, 1989). Of the 10 microsatellite markers developed for V. maackii, three, two, and three were successfully amplified for samples in each of three congeneric taxa, namely, V. album subsp. oxysepalum, V. stamineum var. stamineum, and V. stamineum var. micranthum, respectively (Table 3).
Cross-species amplifications of 10 microsatellite markers in three congeneric species of Veratrum maackii.