Molecular Plant Breeding 2015, Vol.6, No.16, 1
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for genetic polymorphism studies in cultivated tobacco.
In conclusion, a set of 70 new microsatellite markers
was developed in tobacco through microsatellite-
enrichment method with high success rate, highlighting
the efficiency of this method for the isolation of SSR
motifs in tobacco as well as other crop species, for
which genome sequence is not available. These
markers were characterized in tobacco and in other
Nicotiana
species, thereby demonstrating the utility of
these markers in genetic diversity assessment. The
study also demonstrated the cross-species transferability
of these markers to other
Nicotiana
spp resulting in
the identification of species-specific markers. The
microsatellite-enrichment technique used for the capturing
of SSRs in tobacco and the markers developed in this
study will be useful for fingerprinting, genetic
diversity analysis, linkage map construction and
genetic mapping studies in tobacco.
4 Material and Methods
Genomic DNA was extracted from fresh leaves of
N.
tabacum
cv. Jayasree using CTAB method (Murray
and Thompson, 1980). Around 25 µg genomic DNA
was taken in a nebulizer and passed through inert
Nitrogen gas to fractionate into 0.1-1 kb size fragments.
The nebulized fragments were run on the gel and the
desired size range of fragments was eluted. These
fragments were polished to make blunt ends and
adapters were ligated to them. Four biotinylated SSR
probes [(TC)
15
, (GT)
15
, (GA)
15
, (AC)
15
] rich in other
plant species, were hybridized with adapter-ligated
fragments. Approximately 200 ηg of the fragments
(100 bp - 1 000 bp) were added to a single reaction
mixture containing 4.2 ×SSC (Saline-Sodium Citrate,
pH 7.0), 0.07 % SDS (sodium dodecyl sulfate), and 10
ρmol biotinylated probe. The mix was incubated at
95°C for 5 minutes and chilled quickly on ice for 2
minutes. It was then kept at proper annealing
temperatures (depending on the melting temperature
of each probe) for an hour to perform annealing [60°C
for probe (GT)
15
, 37°C for (AT)
15
and 57°C for
(GA)
15
]. Meanwhile, Dynabeads M-280 Streptavidin
(10 µg/µl) was prepared by gently shaking the vial to
obtain a homogenous slurry. About 20 µl of the beads
slurry was transferred to a 1.5 ml tube and washed
four times with 300 µl of bead washing buffer (1x TE
+ 100 mM NaCl). The beads were re-suspended with
50 µl of the same buffer, added into the fragment-probe
mix and incubated at room temperature for 30 minutes
with constant gentle agitation. After immobilization,
the supernatant was removed by applying a magnetic
field to precipitate the beads, which were attached to
the SSR containing fragments that hybridized to
biotinylated probe. The bead-probe-fragment complex
was washed three times each for 5 minutes with 400
µl non-stringency washing buffer (1 x TE + 1 M NaCl)
at room temperature. The complex was further washed
with 400 µl of stringency buffer (0.2 x SSC + 0.1%
SDS) for three times each for 5 minutes at room
temperature. After the final wash, the washing buffer
was completely removed and 40 µl of sterile water
was added, tapped gently and incubated at 95°C for 5
minutes. The eluted solution containing single strand
SSR-enriched fragments was cloned into the pGEM-T
easy vector, and the recombinants were transformed to
DH 10 B
E. coli
electro-competent cell by electroporation.
4.1 Microsatellite identification and primer design
The enriched library was screened with pooling
strategy by picking up individual clones from the agar
plate and kept into 96-well format plates for further
growth and subsequent isolation of plasmid DNA. The
DNA of all clones was isolated by using 96-well
format Qiagen DNA extraction kit (Qiagen, USA).
The isolated DNA was pooled into row and column-wise
and screened by PCR using anchored PCR. The
positive pools were screened by demerging into
individual samples. The plasmids were sequenced
using the Big Dye Terminator reaction kit on the ABI
3700 prism automated DNA sequencer (Applied
Biosystems, USA). The microsatellite motif in each
sequence was identified using the software SSRIT and
each sequence was compared against the local and
NCBI sequence databases in order to identify the
redundant clones. The unique sequences were retained
and submitted to GenBank. Primers were designed
based on the sequences flanking the microsatellite
motifs using the PRIMER 3.0 software (Rozen and
Skaletsky, 1998) using the following criteria: (1)
Primer length 18-25 nucleotide with an optimal length
of 20 nucleotides (2) primer Tm=50-60
0
C (3)
amplified PCR product size 100 - 400 bp, and (4) an
optimal 40% GC content. The presence of structures
such as hairpin or short repeat motifs was also
considered while designing the primers. All primers,
which flank the perfect repeats of the form (N1N2)X
or (N1N2N3)X; imperfect repeats of the form N1N2
