Computational protocol: Analysis of microsatellites in the vulnerable orchid Gastrodia flavilabella: the development of microsatellite markers, and cross-species amplification in Gastrodia

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Protocol publication

[…] In order to develop the molecular markers for evaluating the genetic variation of populations and testing transferability in Gastrodia species, we selected one individual of G. flabilabella to build (AG)n, (AC)n, (TTG)n, (TCC)n, (ACG), (CCA)n, (AACT)n, and (AGAT)n enrich DNA library. Microsatellite loci were isolated following the magnetic bead enrichment method (Liao et al. []; Hsu et al. []), modified from the method proposed by Zane et al. ([]) based on AFLP, magnetic bead enrichment, and TA cloning protocol. Genomic DNA of G. flabilabella was digested using the restriction enzyme Mse I (Promega, Madison, Wisconsin, USA) and DNA fragments from 400 to 1000 bps were isolated from agarose gels using the HiYieldTM Gel PCR DNA Fragments Extraction Kit (RBC Bioscience). The purified partial genomic library was ligated to adaptors (complementary oligo A: 5′-TACTCAGGACTCAT-3′ and 5′ phosphorylated oligo B: 5′-GACGATGAGTCCTGAG-3′). The partial genomic library was enriched using 15 cycles of prehybridization polymerase chain reaction (PCR) using adaptor specific primers (5′-GATGAGTCCTGAGTAAN-3′, hereafter referred to as Mse I-N). The enriched partial genomic library was denatured and hybridized to eight different biotinylated probes [Biotin-(AG)15, Biotin-(AC)15, Biotin-(TTG)10, Biotin-(TCC)10, Biotin-(ACG)10, Biotin-(CCA)10, Biotin-(AACT)8, and Biotin-(AGAT)8] at 68°C for 1 hour for enrichment. The DNA fragments hybridized to probes was incubated and captured using Streptavidin MagneSphere Paramagnetic Particles (Promega) at 42°C for 2 hours. The microsatellite enriched DNA fragments were eluted with high- and low-salt solutions and used as template DNAs for 25 cycles of PCR amplification. The microsatellite enriched DNA fragments were then used as templates for 25 cycles of PCR amplification using Mse I-N. The PCR products were purified using the HiYieldTM Gel PCR DNA Fragments Extraction Kit (RBC Bioscience) and then cloned directly into the p GEM®-T Easy Vector System (Promega). Plasmids containing the PCR product were isolated using an alkaline lysis protocol (Birnboim and Doly []), screened using PCR with primer pairs: (AG)10 or (AC)10/SP6 or T7), and purified with a PureYieldTM Plasmid Miniprep System (Promega). The selected plasmids were subsequently sequenced in both directions using an ABI BigDye3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, USA) with the ABI PRISM® 3700 DNA Automated Sequencer. Sequences enclosing tandem repeat sequences were recognized using Tandem Repeats Finder version 4.07b (Benson []) by general setting on 2, 3, and 5 of match, mismatch, and indel for alignment parameters and 20 for minimum alignment score to report repeat. The pair of specific primers for each microsatellite locus detected by Tandem Repeats Finder was designed using FastPCR software version 6.4.18 (Kalendar et al. []) based on the setting of parameters at a PCR product size ranging from 100 to 400 bp, an optimum annealing temperature of 55°C, and a GC content ranging from 35% to 70%. [...] Several genetic variation parameters were calculated using GenAlEx version 6.5 (Peakall and Smouse []), including the number of alleles (Na), the number of effective alleles (Ne), the observed and expected heterozygosity (Ho and He), Shannon’s information index (H), fixation index (FIS). Hardy–Weinberg equilibrium (HWE) was evaluated using Arlequin software version (Excoffier and Lischer []). […]

Pipeline specifications

Software tools TRF, FastPCR, GenAlEx, Arlequin
Applications Population genetic analysis, qPCR
Diseases Pulmonary Fibrosis
Chemicals Carbohydrates