Computational protocol: Development of 12 polymorphic microsatellite loci for the endangered Seychelles palm Lodoicea maldivica (Arecaceae)1

Similar protocols

Protocol publication

[…] Size-selected fragments from genomic DNA were enriched for simple sequence repeat (SSR) content using magnetic streptavidin beads and biotin-labeled CT and GT repeat oligonucleotides. The SSR-enriched library was made by the company ecogenics (Balgach, Switzerland) and analyzed on a Roche 454 platform using the GS FLX Titanium reagents (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). The 6607 reads had an average length of 143 base pairs. Of these, 617 contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least 10 repeat units. Primer design was done using the Primer3 core (). Suitable primer design was possible in 212 reads. Seventy-eight primer pairs were tested, and the most reliable polymorphic candidates were optimized. Genomic DNA was extracted from silica gel–dried L. maldivica leaf or flower tissue (n = 1252) following the DNeasy 96 Plant Kit (QIAGEN, Hombrechtikon, Switzerland) manufacturer’s protocol, except that grinding was carried out at four cycles of 30 s at 30 Hz, and the first incubation step was extended to 1 h at 65°C. Leaf tissue samples from L. maldivica individuals from each population are located at the Tissue Collection of the Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom ().Two methods were used for PCR reactions: two multiplex PCRs were used to amplify six primers, and the remainder of the primers were amplified in singleplex. Multiplex PCRs (MP1 and MP2) were carried out using primers labeled with either FAM, ATTO565, ATTO550, or Yakima Yellow (YY) (Microsynth, Balgach, Switzerland) (). PCR amplifications were carried out in 10.3-μL reactions containing 1× PCR Buffer (colorless Flexi GoTaq PCR buffer), 0.2 mM dNTPs, 3.1 mM MgCl2, 0.05 U/μL Taq Polymerase (all Promega Corporation, Zürich, Switzerland), 0.18 μg/μL bovine serum albumin (BSA; BioConcept, Allschwil, Switzerland), 1.3 μL DNA, labeled forward primers, and unlabeled forward and reverse primers (for primer concentrations see ).Touchdown PCRs were carried out on a Bio-Rad Dyad Cycler (Bio-Rad Laboratories, Hercules, California, USA) with the following conditions: initial denaturation 95°C/4 min; 12× (denaturation 95°C/30 s, starting annealing temperature 62°C/30 s, decreasing by 0.5°C/cycle, extension 72°C/30 s); 29× (MP1)/28× (MP2) (denaturation 95°C/30 s, annealing 56°C/45 s, extension 72°C/30 s); and final extension 72°C/30 min and storage at 10°C. PCR product (2.5 μL) was added to 10 μL of HIDI formamide and 0.25 μL GeneScan 500 LIZ Size Standard (Applied Biosystems, Waltham, Massachusetts, USA).The singleplex PCRs used forward primers labeled with M13 tails (5′-TGTAAAACGACGGCCAGT-3′) at the 5′ ends (as described by ) (). PCRs occurred in 11-μL reaction volumes containing 1× PCR Buffer, 0.2 mM dNTPs, 2.5 mM MgCl2, 0.025 U/μL Taq Polymerase, 0.18 μg/μL BSA, 1.0 μL DNA, forward primers with M13 tails, reverse primers and M13-primer universal tails labeled with either FAM, ATTO565, ATTO550, or YY (Microsynth) (for primer concentrations see ). Cycling for singleplex PCRs was as follows: initial denaturation 95°C/5 min; 12× (denaturation 95°C/30 s, starting annealing temperature 62°C/30 s, decreasing by 0.5°C/cycle, extension 72°C/30 s); 25× (denaturation 95°C/30 s, annealing 56°C/45 s, extension 72°C/30 s); 8× (denaturation 95°C/30 s, annealing 53°C/45 s, extension 72°C/45 s); and final extension 72°C/30 min and storage at 10°C. PCR products were combined to create two pseudo-multiplex mixes (). For each PCR product (Lm4293, Lm2407, Lm6026, and Lm0144 were diluted 20× first), 1.2 μL were added to 10 μL of HIDI formamide and 0.15 μL of GeneScan 500 LIZ Size Standard (Applied Biosystems). Singleplex and multiplex products were denatured for 3 min at 92°C and run on an ABI 3730xl automatic capillary sequencer (Applied Biosystems). Electropherograms were scored with GeneMarker 2.6.0 (SoftGenetics, State College, Pennsylvania, USA).The number of alleles, deviations from Hardy–Weinberg equilibrium (HWE), and observed and expected heterozygosity values were calculated () using GenAlEx 6.5 (). Linkage disequilibrium was tested in GENEPOP (). The 12 polymorphic loci revealed between five and 21 alleles, with a total of 158 alleles across all L. maldivica individuals (). Significant deviation from HWE was seen in the majority of loci in all populations (). Expected heterozygosity values ranged from 0.399–0.896 (mean ± SE: 0.687 ± 0.048) for the polymorphic markers. No significant linkage disequilibrium was detected between loci pairs after sequential Bonferroni correction (α = 0.05) (). The putative presence of null alleles in 11 loci (all except the monomorphic loci and Lm4716) was detected using MICRO-CHECKER 2.2.3 (); however, these are unlikely to affect HWE at such low frequencies (). There was no evidence for large allele dropout. […]

Pipeline specifications

Software tools Primer3, GeneMarker, GenAlEx, Genepop
Applications Population genetic analysis, qPCR
Organisms Lodoicea maldivica, Homo sapiens