Computational protocol: Development of microsatellite markers in Ilex kaushue (Aquifoliaceae), a medicinal plant species1

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Protocol publication

[…] We sampled 12 I. kaushue trees in a natural population (Baisha County/Hainan Province, China [QS]: 19°08′50.38″N, 109°16′14.9″E) and 10 trees in a cultivated population (Dapu County/Guangdong Province, China [DM]: 24°16′40.31″N, 116°28′02.83″E). Voucher specimens of each population were deposited in the Guangxi Institute of Traditional Medical and Pharmaceutical Sciences herbarium (GXMI; accession numbers Ik-012-ZQW and Ik-008-HYF, respectively; ). Genomic DNA (gDNA) was extracted from silica gel–dried leaves using the cetyltrimethylammonium bromide (CTAB) method (). We mixed gDNA of all wild-collected individuals to be shotgun sequenced by Sangon Biotech (Shanghai, China) using 454 GS FLX Titanium (Roche Applied Science, Branford, Connecticut, USA). The 454 sequencing technique is described in detail in .We obtained 29,247 reads ranging from 32 to 691 bp with an average read length of 401 bp, for a total of 11,736,223 bases. All reads were further screened for microsatellite motifs implemented in the program SSRHunter 1.3 with the default parameters (). A total of 1109 sequences containing 1104 dinucleotide, 259 trinucleotide, and nine tetranucleotide repeats were obtained. Of these sequences, those containing at least six dinucleotide or trinucleotide repeats and sufficient lengths at either end of the repeat motif were chosen for primer design using Primer Premier 5.0 (); a total of 631 sequences, containing 691 dinucleotide and 82 trinucleotide repeats, were subjected to primer design. The settings for Primer Premier were as follows: (i) each search range of sense primer and antisense primer was at each end of the repeat motif; (ii) the primer length was between 17 and 25 bp; (iii) the PCR product size was between 100 and 400 bp long; (iv) the annealing temperature of primers was between 50°C and 64°C, and the difference in annealing temperature between the forward and reverse primers was <4°C; (v) the GC content was between 40% and 60%; (vi) there was not obvious hairpin structure within the primer; and (vii) other parameters followed the default settings of “High” stringency in the search criteria. A total of 78 primer pairs were successfully designed for a total of 99 repeats including 65 dinucleotide, 18 trinucleotide, and 16 compound repeats. These primers were tested for polymorphism in 22 individuals from the two populations.PCR reactions were performed in a 20-μL reaction volume containing 50–100 ng of gDNA, 0.5 μM of each primer, and 10 μL of 2× Taq PCR MasterMix (0.1 U/μL Taq polymerase, 0.5 mM dNTP each, 20 mM Tris-HCl [pH 8.3], 100 mM KCl, and 3 mM MgCl2 [Tiangen Biotech, Beijing, China]). PCR amplifications were conducted under the following conditions: 95°C for 5 min; followed by 35 cycles at 94°C for 45 s, at the annealing temperature for each specific primer (optimized for each locus, ) for 45 s, 72°C for 45 s; and a final extension step at 72°C for 5 min. PCR products were resolved on 6% polyacrylamide denaturing gel using a 10-bp or 25-bp DNA ladder (Invitrogen, Carlsbad, California, USA) as a reference and were visualized by silver staining.Sixteen primer pairs were successfully amplified; these products exhibited the expected sizes and showed clearly defined banding patterns with a maximum of two alleles in each locus per individual. The number of alleles per locus (A) and the observed and expected heterozygosity (Ho and He) of the two populations were estimated by GenAlEx version 6 (). Linkage disequilibrium (LD) and deviation from Hardy–Weinberg equilibrium (HWE) were calculated by GENEPOP version 4.2 ().Across the cultivated and wild populations, A varied from one to nine, and a total of 73 alleles were scored in 22 individuals (). Ho and He in the natural and cultivated populations ranged from 0.000 to 1.000 and from 0.000 to 0.785, respectively. No pairs of loci showed significant LD. The P value of tests for HWE ranged from 0.024 to 1.000 (). Only locus KDC66 in population DM significantly deviated from HWE (P < 0.05), which may be due to overdominant selection or admixture from different resources given the high level of heterozygosity for this locus. […]

Pipeline specifications

Software tools Primer Premier, GenAlEx, Genepop
Application Population genetic analysis
Chemicals Titanium