In the family Pinaceae, Abies is the genus with the second highest number of species. Approximately 40 species are widely distributed in the northern hemisphere in regions ranging from temperate to subarctic zones. Four of the five species that grow in the Japanese archipelago are endemic to Japan. Abies firma Siebold & Zucc. is a major tree species occurring only in warmtemperate forests in Japan. This species is frequently found in mixed forest along with species such as Tsuga sieboldii Carrière and Fagus crenata Blume, but it sporadically forms pure stands at the late succession stage (Farjon, 1990). In recent years, the area covered by A. firma forest has been significantly reduced by logging and exploitation. Moreover, since the early 1960s, forest decline and tree dieback in A. firma forests in many areas of Japan have been observed as a consequence of environmental stress factors such as air pollution (Suzuki, 1992). For effective genetic conservation of these forests, it is necessary to understand the phylogeographic pattern and the genetic diversity within and among A. firma populations. Population genetic studies to date have relied on allozyme markers (Saito et al., 2005) and mitochondrial DNA markers (Tsumura and Suyama, 1998), and have not made use of microsatellites. Microsatellite markers are recognized as versatile molecular tools for inferring genetic structure and gene flow. In recent years, expressed sequence tag (EST)-based markers have been increasingly used in studies of genetic variation because large numbers of polymorphic markers can be developed with relative ease using EST data and markers of this type are less susceptible to null alleles than are anonymous simple sequence repeats (SSRs). Moreover, because ESTs correspond to coding DNA, the flanking sequences of EST-SSRs are located in wellconserved regions across phylogenetically related species, making them markers of choice for comparative mapping and relevant functional and positional candidate genes to study their colocation with quantitative trait loci. In the work described here, we developed EST-SSR markers for A. firma from published expressed sequence data, and evaluated the extent of the polymorphism that they exhibit and their potential for transfer to two other closely related Japanese Abies species (A. homolepis Siebold & Zucc. and A. veitchii Lindl.).
METHODS AND RESULTS
A total of 486 322 A. sachalinensis F. Schmidt (a species related to A. firma) ESTs were downloaded from the National Center for Biotechnology Information (NCBI) database and used for PCR primer design. First, poly A and adapter sequences were removed from the cDNA sequences using the program Cross_ match ( http://bozeman.mbt.washington.edu/phrap.docs/phrap.html) and the TIGR SeqClean sequence trimming pipeline ( http://compbio.dfci.harvard.edu/tgi/software/). EST sequences were then assembled de novo using MIRA (Chevreux et al., 2004), resulting in a total of 38 953 contigs (hereafter referred to as unigenes). Using the resultant unigene library, PCR amplicon primers were designed using MISA (Thiel et al., 2003) and Primer3 (Rozen and Skaletsky, 2000), after trimming low quality regions using the quality Trimmer command in the Euler-SR package (Chaisson and Pevzner, 2008). The criteria applied to identify microsatellite loci were at least six dinucleotide repeat units, or five tri- to hexanucleotide repeat units. To eliminate redundancy (i.e., multiple sets of primers for the same locus), all assembled sequences containing microsatellites were subjected to a BLAST search against the NCBI nonredundant (nr) protein database using the BLASTX algorithm with an E-value cutoff of 1.0E-3.A total of 153 EST-SSR primer pairs bordering sequence regions with more than four di- to hexanucleotide repeats were designed. Ninety-six of the 153 primers, for nonredundant loci with large numbers of repeats, were selected for further evaluation. For each primer pair, genomic DNA from one individual of A. firma was used to check PCR amplification. The PCR reaction was carried out following the standard protocol supplied with 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 0.2 µM of each primer. The PCR thermal profile involved denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 55°C for 1 min, 72°C for 1 min, and a final 7-min extension step at 72°C. PCR products were labeled with ChromaTide Alexa Fluor 488-5-dUTP (Invitrogen, Carlsbad, California, USA) according to Kondo et al. (2000), and loaded onto an automated sequencer (ABI Prism 3100 Genetic Analyzer; Applied Biosystems, Carlsbad, California, USA) to determine fragment lengths, which were analyzed using GENOTYPER software (Applied Biosystems). Thirty-two loci exhibited clear PCR amplification with fragment sizes ranging from 50 to 500 bp (Table 1). The polymorphism of these fragments was evaluated using eight individuals of each of three Abies species (A. firma, A. homolepis, and A. veitchii) sampled across the species' geographical range. Fourteen of the 32 loci were polymorphic and provided clear fragment patterns. The genetic variation at these 14 loci was evaluated using 20 individuals from the A. firma population. Information about the populations sampled is provided in Appendix 1, and specimen vouchers were deposited in the Forestry and Forest Products Research Institute herbarium. To characterize each EST-SSR marker, the following four genetic diversity statistics were calculated using FSTAT 2.9.3 (Goudet, 2001): number of alleles per locus (A), observed heterozygosity (Ho), expected heterozygosity (He), and fixation index (FIS). In addition, the significance of Hardy—Weinberg equilibrium and genotypic equilibrium were tested by 1000 randomizations with adjustment of the resulting P values by sequential Bonferroni correction, using FSTAT 2.9.3. Cross-amplification was conducted on one population each for two Abies species (Table 2, Appendix 1) following the protocol described above. Of the 14 polymorphic loci, As_rep_c4656, As_rep_c32446, As_c14394, As_rep_c11017, and As_rep_c17556 were not polymorphic in this population, but they were polymorphic in other populations (data not shown). As_c14606 was also monomorphic in A. firma but polymorphic in A. veitchii. As_rep_ c7912 was monomorphic in all three species but polymorphic in other populations of A. veitchii.
Characteristics of the 14 polymorphic EST-SSR markers used for three Abies species.
A ranged from one to three and He ranged from 0 to 0.284. The results of cross-species amplification showed that all 14 loci were amplified successfully in A. homolepis and A. veitchii. The total number of alleles ranged from one to six. Analysis of the 14 polymorphic loci indicated no significant deviation in FIS or genotype disequilibrium among locus pairs for any of the three species.
The EST-SSR markers described here will be useful for future genetic studies of A. firma. Interspecific amplification of these markers also shows their potential for use in closely related species. These markers may therefore provide a tool for understanding population demography, population structure, gene flow, and mating systems in Abies species.