Isolation and characterization of 13 microsatellite loci in

Journal of Biogeography
Geographical pattern of isolation and diversification in karst habitat islands: a
case study in the Primulina eburnea complex
Yong Gao, Bin Ai, Hanghui Kong, Ming Kang and Hongwen Huang
Appendix S2 Supplementary methods.
The development of nuclear microsatellite loci from Primulina eburnean
Total genomic DNA was extracted from fresh leaf tissue of P. eburnea using a
modified CTAB method (Doyle & Doyle, 1987). The microsatellites were isolated
according to a modified genomic DNA enrichment protocol of FIASCO (Fast
Isolation by AFLP of Sequences Containing repeats) (Zane et al., 2002). AC and GA
repeats were enriched for by hybridizing with a 5’- biotinylated (AC)8 and (GA)15
probe, respectively, and hybridization products were selectively captured with
Streptavidin MagneSphere® Paragmagnetic Particles (Promega, Madison, WI, USA).
The enriched DNA fragments were ligated into pMD 18-T plasmid vector (Takara)
and transformed into E. coli strain (JM109, Promega). Recombinant clones were
identified by PCR with M13 universal primer. PCR products were validated by
agarose gel electrophoresis.
Of the 126 positive clones sequenced, 47 primer pairs were successfully
designed using PRIMER PREMIER 5.0 (Lalitha, 2000) and used for further
characterization. PCR amplifications were performed in a final volume of 10 µL
containing 10 mM Tris–HCl (pH 8.4), 50 mM (NH4)2SO4, 1.5 mM MgCl2, 0.2 mM
dNTPs, 0.2 µM each primer, 20 ng of genomic DNA, and 1 unit Taq polymerase
(Fermentas, Lithuania). The amplification profiles included an initial denaturing at
94°C for 5 min, followed by 35 cycles of 45 s at 94 °C, 50 s at 49-64 °C depending
on the primer pairs and 1.5 min at 72 °C, followed by a final extension step for 5 min
at 72 °C. Amplified products were visualized on a 6% denaturing polyacrylamide gel
using silver staining. A 10 bp DNA ladder (Promega) was used as a size standard for
allele scoring.
Polymorphism of these loci was evaluated using 48 individuals from two
populations of P. eburnea and 40 individuals of two populations of P. lutea. Thirteen
polymorphic loci could be successfully amplified in both P. eburnean and P. lutea.
The number of alleles per locus (A), observed heterozygosity (HO), expected
heterozygosity (HE) and Hardy–Weinberg equilibrium were estimated for each locus
and population using GENEPOP version 4.0 (Rousset, 2008). Substantial genetic
diversity was found in the investigated populations.
Doyle, J. & Doyle, J. (1987) A rapid DNA isolation procedure for small quantities of
fresh leaf tissue. Phytochemical Bulletin, 19, 11-15.
Lalitha, S. (2000) Primer Premier 5. Biotech Software and Internet Report, 1,
Rousset, F. (2008) GENEPOP’007: a complete re-implementation of the genepop
software for Windows and Linux. Molecular Ecology Resources, 8, 103-106.
Zane, L., Bargelloni, L. & Patarnello, T. (2002) Strategies for microsatellite isolation:
a review. Molecular Ecology, 11, 1-16.