Microsatellite markers for Corybas (Orchidaceae) species in New Zealand

Premise of the Study Microsatellite markers were developed for New Zealand species of Corybas (Orchidaceae) to investigate population genetics and species delimitation. Methods and Results From sequencing a total genomic DNA library (using Illumina MiSeq), we developed 22 microsatellite markers for C. obscurus. The di‐ and trinucleotide repeat loci were initially trialed on individuals representing seven Corybas taxa (C. “rimutaka,” C. confusus, C. hypogaeus, C. macranthus, C. obscurus, C. trilobus, and C. walliae) and had one to eight alleles per locus. Twelve polymorphic markers were further tested on six Corybas populations from three of the seven taxa (C. obscurus, C. “rimutaka,” and C. trilobus). Observed and expected heterozygosities ranged from 0–1 and 0–0.859, respectively. The utility of these 12 loci was further validated in five related Corybas species (C. hypogaeus, C. obscurus, C. vitreus, C. walliae, and C. “rimutaka”; 38 individuals) representing populations from across the North and South Islands. The average value for genetic diversity among populations (FST) of 0.439 shows differentiation among species. Conclusions These markers will be useful for future studies aimed at delimiting species boundaries and examining the genetic diversity of the New Zealand Corybas species.

Germany) with slight modifications of the manufacturer's protocol (0.5% β-mercaptoethanol [BME] added to Buffer AP1; incubated at 65°C for 15 min; chilled on ice for 10 min). A DNA library was prepared using the Illumina TruSeq Library Preparation Kit (Illumina, San Diego, California, USA) following manufacturer's protocols. The indexed library was pooled with three other libraries (Fuchsia excorticata [Onagraceae;Van Etten et al., 2013], Sophora microphylla [Fabaceae; Van Etten et al., 2014], and Korthalsella salicornioides [Salicaceae; S. M. Pearson et al., unpublished]) in equal concentration and sequenced via Illumina MiSeq (Illumina) using 250-bp paired-end chemistry (New Zealand Genomics Limited, Palmerston North, New Zealand). The resulting 2.6 million sequences (991 million base pairs) were trimmed of low-quality results using a 0.01 quality cut-off in DynamicTrim in SolexaQA (Cox et al., 2010), and the remaining sequences were assembled using Velvet version 1.1 (Zerbino and Birney, 2008). The raw data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA; accession SRP150798).
Plastid and mitochondrial sequences were removed by performing BLAST searches against related organellar sequences in GenBank ( NC_008362). Phobos (version 3.3.12; Mayer, 2010) was used to identify di-to hexanucleotide repeats with a length of ≥6 repeat units, resulting in 111,813 repeat regions. Primers were designed from regions containing a single uninterrupted repeat type in Geneious (Biomatters Ltd., Auckland, New Zealand) using Primer3 (Rozen and Skaletsky, 1999) with default settings except: product size = 100-300 bp; primer size = 17 (minimum)-19 (optimal)-21 (maximum); melting temperature (T m ) = 52-55-58°C; GC content = 40-50-60%; maximum T m difference = 5°C; GC clamp = 1; maximum poly x = 4. Forty-eight primer pairs were chosen to sample the range of repeat types and lengths and product sizes. An M13 tag (Boutin-Ganache et al., 2001) was added to the 5′ end of the forward primer (CACGACGTTGTAAAACGAC) and a PIG-tail was added to the 5′ end of the reverse primer (GTTTCTT) to promote non-template (A) addition (Brownstein et al., 1996).
Primers were tested initially on seven individuals from a range of named species and one tag-named entity from New Zealand (C. "rimutaka, " C. confusus Lehnebach, C. hypogaeus, C. macranthus (Hook. f.) Rchb. f., C. obscurus, C. trilobus, and C. walliae Lehnebach; Appendix 1). DNA was extracted from silica-dried leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). The 10-μL PCR cocktail contained 5-50 ng of DNA, 0.02 μM of forward primer, 0.45 μM of reverse primer, 0.45 μM of M13 primer (labeled with FAM, NED, or VIC), 1.5 mM of MgCl 2 , 1× buffer BD (Solis BioDyne, Tartu, Estonia), 250 μM of each dNTP, and 0.5 units of Firepol Taq polymerase (Solis BioDyne). The PCR cycling program had an initial denaturation of 95°C for 3 min; 35 cycles of 95°C for 30 s, 53°C for 40 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products (0.14-1.25 μL) for two to three loci of distinguishable sizes and labeled with different fluorophores were co-loaded and added to 9 μL of Hi-Di formamide (Applied Biosystems, Carlsbad, California, USA) and 1 μL of CASS ladder (Symonds and Lloyd, 2004) for subsequent fragment sizing on an ABI 3730 Genetic Analyzer (Applied Biosystems) (Massey Genome Service at Massey University, Palmerston North, New Zealand). Alleles were visualized and scored using GeneMapper version 3.7 (Applied Biosystems).
Of the 48 primer pairs trialed, seven did not amplify, three were unscorable, four were monomorphic, 31 were polymorphic within an individual, and three were polymorphic among species. Twentytwo loci (Table 1) successfully amplified across all taxa. The number of alleles per locus ranged from one to two in the C. obscurus sample and from two to eight in the six other taxa. From these, the 12 most polymorphic loci were used for preliminary population genetic analyses on three populations of C. obscurus, one population of C. "rimutaka, " and two populations of C. trilobus (Table 2, Appendix 1); DNA extraction and locus amplification were as described above. We aimed to sample 15-20 individuals per population, but because of the small and precarious nature of the C. obscurus populations, only five individuals per population were included of that species. The total number of alleles, observed heterozygosity (H o ), and expected heterozygosity (H e ) were determined using GenAlEx 6.501 (Peakall and Smouse, 2006). Deviation from Hardy-Weinberg equilibrium (HWE) was determined using the Markov chain method provided by Web version 4.2 of GENEPOP software (Rousset, 2008). The number of alleles ranged from 3-22 (average of 8.8) per locus, H o from 0-1 (average of 0.452), and H e from 0-0.859 (average of 0.390) ( Table 2). All loci except Corybas-19 deviated significantly from HWE in at least one population. These deviations were usually a lower than expected H o , suggesting population substructure due to inbreeding or clonality, both of which should be examined more closely in future studies to inform conservation efforts. The average genetic diversity among populations (F ST ) of 0.439 shows the markers are detecting substantial population structure in Corybas (Hartl and Clark, 1997).
To test the transferability of the markers for use in species delimitation, 38 individuals from both the North and South Island were chosen, representing four species and one tag-named entity (C. hypogaeus, C. obscurus, C. vitreus Lehnebach, C. walliae, and C. "rimutaka"; 3-16 individuals per taxon; Appendix 1). For these, we amplified the 12 novel microsatellite markers and genotyped them as described above. GenAlEx 6.501 was used to determine the percentage of successful amplifications per locus and F ST . Amplification success rate was 95.8% on average, ranging from 81.58% to 100% amplification across all taxa (Table 3). Alleles ranged from 2.8-5 per species, with an average of 8.4 across all loci and species.

CONCLUSIONS
We developed 22 polymorphic microsatellite markers from C. obscurus that amplified to varying degrees in seven congeneric species and one undescribed entity. Twelve markers amplified reliably across seven species and were further tested on multiple populations and species to test their amplification across species and potential utility for population genetics. Due to the high success rate of amplification and the number of polymorphic loci, these markers will be informative for population genetics, mating system analysis, species delimitation, and determining the extent of hybridization within populations of mixed species. As such, these markers will facilitate the development of a conservation strategy for these species in New Zealand, as well as Australia.

ACKNOWLEDGMENTS
This project was supported by Marsden Fund MNZ1001 to C.A.L. and A.W.R. Samples were collected under New Zealand Department of Conservation and Greater Wellington City Council permits (BP-30352-FLO, TW-29987-FLO, 35026-FAU, WE-33250-FLO, and CA-29892-FLO). We thank Prashant Joshi for technical assistance.

DATA ACCESSIBILITY
The raw data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (accession SRP150798); primer sequences were uploaded to GenBank, and accession numbers are provided in Table 1.