Computational protocol: Characterization of polymorphic microsatellite markers in Pinus armandii (Pinaceae), an endemic conifer species to China1

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[…] Genomic DNA was extracted from a fresh needle (specimen no.: WNU-NG-SX-2013-LZH-036) of P. armandii using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and was sequenced using an Illumina MiSeq (Illumina, San Diego, California, USA) at Shanghai Genesky Biotechnologies (Shanghai, China) with 2 × 300-bp paired-end sequencing and MiSeq Reagent Kit version 3 (Illumina). A total of 6,783,777 clean reads were obtained after the adapter and low-quality sequences were removed. These clean reads were further assembled into 350,628 contigs using CLC Genomics Workbench version 7.5 (CLC bio, Aarhus, Denmark). The set of detailed parameters were: mismatch cost of 2, length fraction of 0.4, similarity fraction of 0.4, insertion cost of 2, deletion cost of 2, and a minimum contig length of 200 nucleotides. We extracted the contigs containing microsatellite markers with SciRoKo version 3.1 (), using default identification criteria used for mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, with a minimum of 14, seven, five, four, four, and four repeats, respectively. In total, 887 microsatellite-containing contigs were obtained. Then, forward and reverse primers were designed with Primer Premier version 7.0 software (). The criteria for primer design were as follows: (1) product size from 100 to 350 bp; (2) primer size from 18 to 25 bp with an optimum size of 20 bp; (3) primer melting temperate from 55°C to 63°C with an optimum temperature of 60°C; and (4) GC content of primers from 40% to 60%.Fifty pairs of primers containing microsatellite repeats were randomly selected to test amplification efficiency and polymorphism in 52 individuals from three natural populations of P. armandii (). PCR amplification was performed in a 10-μL reaction volume containing 10 ng DNA template, 5 μL 2× polymerase mixture, 0.2 μM of each primer, and 3.6 μL ddH2O. The PCR profiles were as follows: an initial denaturation of 5 min at 95°C; 35 cycles of denaturation of 30 s at 95°C, at the appropriate annealing temperature () for 30 s, and an extension of 30 s at 72°C; followed by a final extension of 5 min at 72°C. The PCR amplification products were separated in 10% nondenaturing polyacrylamide gels and were visualized by silver staining.The allele sizes for each individual were automatically determined using Quantity One (Bio-Rad, Hercules, California, USA) with pBR322 DNA/MspI as DNA molecular-weight marker. The program GenAlEx version 6.501 () was used to evaluate various population genetic parameters of microsatellite loci, including the number of alleles per locus, expected and observed heterozygosity (He and Ho), and Hardy–Weinberg equilibrium (HWE). In addition, linkage disequilibrium (LD) among loci was detected using GENEPOP version 4.2.2 (). We also detected the null allele frequencies for each primer with MICRO-CHECKER version 2.2.3 ().In total, 34 primer pairs were successfully amplified with high-quality PCR products, with 18 of them exhibiting polymorphisms (). The number of alleles of these polymorphic primers ranged from two to five with an average of 2.4. He ranged from 0.061 to 0.609 with an average of 0.384, and Ho ranged from 0.063 to 0.947 with an average of 0.436. Two pairs of primers (Pa3553 and Pa118137) were found to deviate greatly from HWE, while we did not detect any LD between loci. This deviation might have been caused by insufficient sample size, nonrandom mating between individuals, migration, and/or natural selection of these two loci. In addition, no null alleles were detected for any locus in the current study. The detailed SSR characteristics are provided in .To explore the broader utility of the SSR loci developed here, we amplified the primers in 20 individuals from five other species closely related to P. armandii (). Seventeen of the 18 primers produced robust, usually polymorphic DNA fragments across P. koraiensis, P. griffithii, P. sibirica, P. pumila, and P. bungeana. However, Pa3553 was not successfully amplified in P. pumila and P. bungeana (). […]

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