Genomic DNA was obtained using Invisorb Spin Plant Mini Kit (STRATEC Molecular GmbH, Berlin, Germany) and was extracted from three individuals of C. grandiflorum subsp. boissieri belonging to three different populations of Andalucía Province in Spain (“Universidad Pablo de Olavide”, Seville [37º21'18.04''N − 5º56'16.56''O]; “El Bosque”, Cadiz [36º45'55.14''N − 5º29'48.05''O]; and “Río Frío”, Granada [37º09'36.77''N − 41º11'07.93''O], whose vouchers are located in SEV herbarium). Microsatellite libraries were developed by Ecogenics Company.
The design of potential microsatellites loci was outsourced to the company Ecogenics (Schlieren-Zürich, Switzerland, https://www.ecogenics.ch), which combined enrichment for microsatellite motifs in a C. g. boissieri genomic DNA library with 454-sequencing on a Illumina Miseq system (Illumina, San Diego, California, USA) with an average read length of 80–400 bp. Using a pipeline property of Ecogenics, a total of 640 microsatellites loci were found, including 263 di-, 352 tri- and 25 tetranucleotides (Table 1).
Table 1
Characteristics of 10 microsatellite loci for Centaurium g. boissieri based upon genotyping three populations.
Locus | Primer sequences (5’-3’) | Repeat motif | Allele size range (bp) |
Boi8 | F: GAGATGCAACGAGTCGAACC R: TCGTAGCCTGAGCCATCTTC | (GAA) | 161–167 |
Boi9 | F: GCTACCCGAAGTTTTCCGAC R: CGAGTTTGACCGAGCCATTC | (AGG) | 238–277 |
Boi16 | F: ACATGTACGTGCCTCCTAGC R: TTGGGAGCCAAAAACGCATC | (TA) | 215–233 |
Boi19 | F: AATAATCATGGTGGCGCACG R: TGCATACAAGAATTCGCAAAAGC | (GTAT) | 156–252 |
Boi23 | F: TGTGTTGGAAACCGCTAATATCC R: GTGCAAGGCTCACAATCTCC | (ATGT) | 230–282 |
Boi32 | F: GTTAAGATCACACAGCCCGC R: GTATGGCTCGTTTCACCTGC | (AAT) | 199–238 |
Boi38 | F: TGTTCCTACATATACGAGTAAAAGC R: AATAGGTTCTCAAGAGCCATAAAC | (AT) | 238–272 |
Boi39 | F: AATGCAAGGCAAGTTCTCGG R: TCACGAGAATGGATTGGGGC | (GA) | 225–253 |
We selected 40 primer pairs within a size range from 100 to 250 bp to be tested on 59 individuals of C. grandiflorum ssp. boissieri collected from three different populations. Also, we tested another eight microsatellites designed for Sabatia campestris from the same Tribe Chironieae (Gentianaceae) [10]. The two loci that amplified for C. g. boissieri were also tested for cross-amplification Centaurium quadrifolium, using 7 individuals per subspecies (Table 1). A total of 80 individuals were used. PCR’s were performed in 20µL of reaction mixture containing 60 ng/µl of template genomic DNA, 0.5 U taq polymerase, 1 x My Taq Red Reaction Buffer (Bioline, London, UK), 0.01% bovine serum albumin (BSA) (Promega, Madison, WI, USA), 0.04 µM M13-tailed forward primer, 0.40 µM PIG-tailed reverse primer and 0.40 dye-labelled M13 primer (FAM, VIC, NED or PET dyes; Invitrogen, Madrid, Spain), following the methods of Boutin-Ganache et al.[11]. PCR were undertaken using a touchdown PCR protocol on a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Foster city, CA, USA). The thermal profile consisted of initial denaturalization at 94°C for 2 min; 17 cycles with denaturalization at 92°C for 30 s, annealing at 60–44°C for 30 s (1°C decrease in each cycle), and extension at 72°C for 30 s; 25 cycles at 92°C for 30 s, 44°C for 30 s, 72°C for 30 s; and a final extension of 5 min. at 72°C. PCR products were analyzed in ABI prism 3130 and 3730 systems (STAB VIDA, Portugal) and sized using Gene Marker 2.4 (SoftGenetics, State College, PA, USA) and Gene Scan 500 LIZ size standard.
We estimated: 1) number of observed alleles (A), 2) observed (Ho) and expected (He) heterozygosity, and 3) deviations from Hardy-Weinberg equilibrium (PHWEq) using Marcov chain with 100,000 permutations. All analyses were done using Arlequin software v.3.5.2.2 [12].