Introduction
The family Orchidaceae is one of the largest families of flowering plants, comprising approximately 20,000–30,000 species belonging to 800 genera that are distributed worldwide, although most species are distributed in the tropics and subtropics (Cronquist, Reference Cronquist1981). Numerous orchid species exhibit similar characteristics, and many species are endangered and diminishing in number. Hence, they are ideal species for investigating the patterns of genetic diversity and differentiation within the scope of conservation genetics. Spathoglottis Blume belongs to a group of over 40 species and also has a long history of cultivation in Southeast Asia (Teoh, Reference Teoh2005). Spathoglottis plicata Blume is a tropical–subtropical terrestrial orchid occurring from India, throughout Southeast Asia, Taiwan, Malaysia and the Philippines, to the Pacific Islands and Australia (Lavarack, Reference Lavarack1984). Taiwan is the northern boundary of its natural distribution, and this species is only majorly distributed on the Orchid and Green Islands of Taiwan. Spathoglottis plicata has become vulnerable because of its small population size and scattered distribution. The main potential threats for wild populations are overcollection, illegal collection and habitat loss. Therefore, this species is listed as critically endangered in Taiwan (Wang et al., Reference Wang, Chiou and Chang2012) and vulnerable in Australia (Backhouse, Reference Backhouse2007). The species has considerable horticultural value and numerous varieties or hybrids have been created, differing largely in colour of flowers which ranges from purple, yellow to white (Teoh, Reference Teoh2005). Microsatellite markers are widely employed for evaluating genetic diversity of endangered species and have become essential tools for cultivars identification and hybrid analysis for horticultural industry. In this study, we developed polymorphic microsatellite markers for S. plicata to facilitate future studies on the conservation genetics of S. plicata and identification for varieties or hybrids of S. plicata.
Experimental
We performed polymerase chain reaction (PCR)-based isolation of microsatellite arrays (PIMA) to isolate microsatellite markers (Lunt et al., Reference Lunt, Hutchinson and Carvalho1999; Hung et al., Reference Hung, Lin, Huang, Hwang, Hsu, Kuo, Wang, Hung and Chiang2012). Random amplified polymorphic DNA (RAPD)-PCR enrichment was used to construct a PCR library for PIMA, and then these RAPD fragments were used for screening microsatellite markers. We dried leaves samples in silica gel and extracted genomic DNA following CTAB method (Doyle and Doyle, Reference Doyle and Doyle1987). RAPD-PCR was performed in a thermal cycler with a reaction mixture containing 10 ng DNA, 0.2 mM dNTP, 0.5 U Taq and 5 pmol of one RAPD primer. The PCR cycling conditions were as follows: 40 cycles of 1 min at 95°C, 1 min at 40°C and 2 min at 72°C, followed by an additional extension step for 10 min at 72°C. We used several RAPD primers to amplify DNA fragments from the genome of S. plicata in separate reactions. The PCR products were selected on the basis of size to obtain small fragments ranging from 300 to 800 bp, which were ligated into pGEM-T EasyVector Systems, and then transformed into Escherichia coli. We screened clones of DNA fragments using microsatellite and vector primers (T7 primer or SP6 primer) to mine microsatellites. In positive clones, PCR electrophoresis would show a DNA fragment containing a microsatellite signal, whereas no amplification signal would be detected in negative clones. Finally, plasmid DNA from 150 positive clones was extracted, and then sequenced in an ABI 377 Sequencer (Applied Biosystems, Foster City, CA, USA). The DNA sequences were screened for microsatellites using Tandem Repeats Finder version 4.04 (Benson, Reference Benson1999), and 12 clones were detected with longer microsatellite repeats. The primer pairs were designed using Primer 3 (Untergrasser et al., Reference Untergrasser, Cutcutache, Koressaar, Ye, Faircloth, Remm and Rozen2012).
We tested the designed microsatellite primers with 11 and 33 samples of S. plicata collected from Orchid and Green Islands of Taiwan. We performed PCR amplification of microsatellites in a 25 µL reaction solution containing 10 ng DNA, 0.2 mM dNTP, 0.5 U Taq and 5 pmol of each primer. The PCR cycling conditions were as follows: 3 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at the primer-specific annealing temperature (T a) (Table 1), 30 s at 72°C and a final extension step for 5 min at 72°C. Electrophoresis was performed in denaturing 6% polyacrylamide gels with a 10 bp DNA ladder (Invitrogen, Carlsbad, CA, USA) to estimate the size of alleles stained with ethidium bromide. The number of alleles per locus (A), and observed (H O) and expected (H E) levels of heterozygosity were calculated using GenAlEx version 6.5 (Peakall and Smouse, Reference Peakall and Smouse2012). The polymorphic information content (PIC) and Hardy–Weinberg equilibrium (HWE) tests were also assessed with PowerMarker version 3.25 (Liu and Muse, Reference Liu and Muse2005) and GENEPOP version 3.4 (Raymond and Rousset, Reference Raymond and Rousset1995).
Characterization of 12 polymorphic microsatellite markers for Spathoglottis plicata
Discussion
The 12 novel primer sets were successfully amplified with size matching expectations on the basis of the initially repetitive DNA fragment. Overall, the number of alleles per locus ranged from 2.000 to 8.000 in total populations, 2.000 to 4.000 in the Orchid Island population and 1.000 to 6.000 in the Green Island population. The observed and expected heterozygosity ranged from 0.000 to 0.756 and 0.208 to 0.813 in total populations, 0.000 to 0.900 and 0.314 to 0.617 in the Orchid Island population and 0.000 to 0.710 and 0.000 to 0.770 in the Green Island population, respectively (Table 2). We found the higher average number of alleles, but lower average observed heterozygosity in Green Island than Orchid Island. Heterozygote deficiency in most loci was expected to result from the higher level of selfing in S. plicata. All loci in S. plicata deviated from the HWE in total populations. The deviations of all loci in S. plicata may have resulted from the smaller population size, which was caused by overcollection and habitat loss. We also determined PIC values of the loci for S. plicata. The PIC values of the 12 polymorphic microsatellites ranged from 0.405 to 0.805. Of all microsatellites, eight exhibited medium polymorphism (0.50 > PIC > 0.25), and four exhibited high polymorphism (PIC > 0.50) (Table 2). The 12 polymorphic loci developed can be used to study the genetic diversity and population structure of S. plicata, which may provide essential conservation information for this threatened species. Similarly, we also expect that these microsatellite loci could be useful for variety/hybrid identification of S. plicata in horticultural industry.
The detailed genetic diversity parameters for Spathoglottis plicata determined using the 12 newly developed microsatellite markers
For each locus, the number of alleles (A), observed heterozygosity (H O), expected heterozygosity (H E), polymorphism information content (PIC) and P values of deviation from the Hardy–Weinberg equilibrium (HWE) were evaluated.
