Computational protocol: Development of microsatellite markers for the apomictic triploid fern Myriopteris lindheimeri (Pteridaceae)1

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Protocol publication

[…] Genomic DNA of a single individual of diploid M. lindheimeri (voucher: Schuettpelz 450 [DUKE], collected from the Tonto National Forest, Pinal Co., Arizona, USA) was extracted from silica gel–dried material using the DNeasy Plant Mini Kit following the manufacturer’s protocol (QIAGEN, Valencia, California, USA). Genomic DNA was run on two lanes (1/4 plate = 24 wells) using the Roche 454 GS-FLX Titanium sequencing platform (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) at the Duke University Center for Genomic and Computational Biology sequencing facility. The 454 run generated 234,428 sequence reads with a median length of 403 bp. Raw data were scanned for di-, tri-, tetra-, penta-, and hexanucleotide perfect microsatellite repeats using MSATCOMMANDER version 0.8.2 (). Of the 234,428 sequence reads searched, 25,295 sequences contained a total of 33,955 repeats. Given the surplus of repeat regions, we focused our efforts on a subset of nonplastid regions (determined by BLASTN against the M. lindheimeri chloroplast genome; ) containing di-, tri-, and tetranucleotide repeats with sufficient flanking sequence in which to develop primers (). A total of 159 unlabeled primer pairs were designed in Primer3, using default settings, implemented within MSATCOMMANDER (; ).Each microsatellite region (159 in total) was amplified by PCR from genomic DNA of the individual for which the 454 sequencing was completed (Schuettpelz 450). This amplification followed , except that the annealing temperature was set to 60°C to prevent nonspecific primer binding. Amplicons were visualized on a 1% agarose gel using SYBR Safe DNA Gel Stain (Life Technologies, Carlsbad, California, USA), run for 35 min at 75 V. Amplifications that produced a single strong band were purified and sequenced also following the protocol of . Clean sequence fragments (assumed to represent ca. single-copy markers) were assembled in Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and examined to confirm the presence of the anticipated microsatellite repeat. For regions with the repeat, new forward primers were designed with a CAG nucleotide tag (5′-CAGTCGGGCGTCATCA-3′) incorporated at the 5′ end of the primer sequence—to be used in combination with a complementary, fluorescently labeled nucleotide tag in subsequent genotyping reactions ().Genotyping reactions used 10× PCR buffer IV containing MgCl2 (ABgene, Epsom, United Kingdom) combined with 2.4 mM dNTPs, 100 μg/mL bovine serum albumin (BSA), 5 U/μL Taq polymerase, 2 μM reverse primer, 10 μM CAG-tagged forward primer, 10 μM fluorescently labeled CAG complementary primer, plus 1 μL of DNA template for a 12-μL reaction. Each reaction entailed an initial denaturation step (94°C for 7 min), followed by 10 denaturation, annealing, and elongation cycles (94°C for 30 s, 62°C [−1°C per cycle] for 30 s, 72°C for 30 s, respectively) and 27 additional denaturation, annealing, and elongation cycles (94°C for 30 s, 51°C for 30 s, 72°C for 30 s, respectively) with a final elongation step (72°C for 12 min). Fragment analyses were run using a GeneScan 500 LIZ Size Standard on a 3730xl DNA Analyzer (Applied Biosystems, Waltham, Massachusetts, USA). The resulting data were visualized using GeneMarker 2.2.0 (SoftGenetics, State College, Pennsylvania, USA). Of the 159 primer pairs tested, 138 failed to amplify, amplified multiple bands, or produced poor fragment peaks (due to stutter, multiple peaks, or inconsistent amplification) and were discarded, leaving 21 markers that amplified well in M. lindheimeri.To determine the utility of these 21 markers, we surveyed each new locus across multiple individuals spanning the northern range of M. lindheimeri (; ). Fragment analysis revealed alleles ranging from 175–453 bp in length; of the 21 markers assessed, 14 were heterozygous within or polymorphic across individuals of M. lindheimeri (number of alleles ranging from two to five; ), and eight amplified in one or more closely related taxa (). Population-level diversity measures were assessed for three populations of M. lindheimeri in Arizona, USA: Carr Canyon (n = 8 individuals; 31.4394°N, 110.2861°W), Jacobson Canyon (n = 16 individuals; 32.6834°N, 109.7632°W), and Paradise (n = 12 individuals; 31.9590°N, 109.2116°W) (). Individuals from each population were genotyped for a subset of our newly developed, polymorphic microsatellite loci (nine loci total) using a multiplex approach: for each individual, all nine loci were amplified individually and the resulting fluorescently labeled amplicons were pooled in two separate multiplex reactions (). The resulting fragment data were used to calculate percentage of polymorphic loci (P), heterozygote frequency over all loci (Het), and genotypic diversity (G = 1 − Σgi2, where gi is the frequency of the ith genotype; ); all measures were calculated manually using Microsoft Excel version 14.4.8 (Microsoft, Redmond, Washington, USA). Samples from Carr Canyon and Jacobson Canyon were polymorphic at 55.6% of loci surveyed and each had relatively low genotypic diversity (0.25); however, Jacobson Canyon had a higher heterozygote frequency compared to Carr Canyon (0.43 vs. 0.24, respectively). Samples surveyed from Paradise were polymorphic at 77.8% of loci; and, while heterozygote frequency over all loci was relatively low (0.28), genotypic diversity was high (0.52), indicating a relative abundance of unique genotypes compared to other populations sampled. […]

Pipeline specifications

Software tools msatcommander, BLASTN, Primer3, Sequencher, GeneMarker
Application qPCR
Organisms Myriopteris lindheimeri