Publication protocol
"The total genomic DNA was extracted from the silica
gel dried leaves using DNeasyTM Tissue Kit (Qiagen). The
microsatellite regions were isolated following Zhang et al.
(2007). About 500 ng genomic DNA was digested into
approximately 500 bp fragments with a restriction enzyme
RsaI (NEB) and XmnI (NEB), then ligated to SuperSNX24
double-stranded adaptors (mixation of equal volumes of
equal molar amounts of SuperSNX24-F: 50
-GTTTAA
GGCCTAGCTAGCAGAATC-30 + SuperSNX24 + 4P-R:
50
-GATTCTGCTAG-CTAGGCCTTAAACAAAA-30
). For
enrichment, the ligation products were hybridized with an
oligonucleotide combination of 50
-biotinylated probes,
(AG)15, (CT)15, (AC)15, (GT)15, (CG)15, (GTG)12, and
(CCA)12. The hybridization in the 50 ll solution (2· SSC,
1 lmol/l probe and 10 ll ligation products) was as follows:
an initial 5 min at 95C, then a rapid cooling to 70C
followed by 0.2C incremental decreases every 5 s for 99
cycles, and maintenance at 50C for 10 min; then decreases of 0.5C every 5 s for 20 cycles, and finally rapid
cooling to 15C. The DNA hybridized to the probe was
captured by streptavidin-coated magnetic beads at 37C for
1 h and then washed by the solution I (2· SSC, 0.1% SDS)
and solution II (1· SSC, 0.1% SDS). The captured DNA
was recovered by polymerase chain reactions (PCR) with
SuperSNX-F (50
-GTTTAAGGCCTAGCTAGCAGAATC30
) and PCR product was purified with TIANquick Midi
Purification Kit (TIANGEN). These fragments enriched
with microsatellite loci were cloned using pMD 18-T
vector (Takara) and transformed into the E. coli competent
cell (JM109, Takara). Positive colonies were amplified
using BcaBESTTM Sequencing Primers RV-M and BcaBESTTM Sequencing Primers M13-47. Only those PCR
products, sized between 300 and 600 bp were sequenced
on ABI3130 Automated DNA Analysis System using the
BigDye Ready Reaction Terminator Kit following the
manufacture’s protocols. The sequences containing motifs
repeating more than 5 times were regarded as microsatellites. A total of 26 sequences were identified among the
sequenced 200 sequences and primer pairs for amplification of the microsatellite regions were designed using the
Primer 5.0 (Clarke and Gorley 2001)."
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