Molecular Plant Breeding 2015, Vol.6, No.16, 1
-
13
8
Table 5 Transferability of 70 microsatellite markers to other
Nicotiana
species
Species
No. of markers producing
atleast one amplicon
%
transferability
N. gluaca
69
98.6
N. paniculata
68
97.1
N. thrisiflora
68
97.1
N. rustica
67
95.7
N. undulate
69
98.6
N. trigo
69
98.6
N. plub
70
100.0
N. sylves
70
100.0
N. rependa
69
98.6
N. nesophila
69
98.6
N. corymbosa
70
100.0
N. acuminatae
69
98.6
N.clevlandii
69
98.6
N. nudicaulis
70
100.0
N. suav
70
100.0
N. debnyi
67
95.7
N. gossei
70
100.0
N. maritama
70
100.0
N. velutiana
68
97.1
N. occi
69
98.6
N. simulans
70
100.0
N. goodsp
69
98.6
N. rosulata
69
98.6
due to their abundance in the genome, high degree of
variability and reduced time, effort and cost etc.
However, the availability of microsatellite markers is
limited in tobacco. Although many molecular markers
were recently reported (Bindler et al., 2007; 2011,
Tong et al., 2012; Hughes et al., 2014), they are not
sufficient enough to use in inter specific hybridization
programs and to develop a core genetic map like rice
(Orjuela et al., 2010). Hence, there is an immense need
to develop additional markers for the construction of
dense genetic map, which would be the starting point
for genetic mapping and eventual cloning of important
genes from this crop. Therefore, we have employed a
genomic enrichment method to capture the SSR
motifs for the development of new microsatellite
markers. Earlier, Sethy et al., 2006, developed 74
functional sequence-tagged microsatellite sites (STMS)
primer pairs in chickpea by using genomic enrichment
method. However, no attempt has been made to isolate
microsatellite markers from the tobacco genome using
the enrichment procedure, though this provides an
attractive choice for targeted microsatellite development.
The enrichment protocol used in the present study has
resulted in the identification of clones containing the
SSR motifs with 82% success rate, which is
significantly higher than any conventional methods
exhibiting varying efficiency ranging from 0.045% to
12% (Zane et al., 2002). The genomic enrichment
method for capturing SSRs is fast and convenient to
handle compared to the conventional method involving
the genomic library preparation and sequencing.
Using the enrichment method, high efficiency of
microsatellite identification was also achieved in
several crops (Gaitan-Solis et al., 2002; Riaz et al.,
2004; Lowe et al., 2004) with varying success rates.
By employing microsatellite-enrichment procedure,
Marinoni et al., 2003, obtained 23% of clones containing
SSR motif in chestnut, while Acquadro et al., 2003,
obtained 85% in
Cynara cardunculus
. All the clones
harboring SSR motif were made publicly available
through GenBank submissions (Table 2).
More number of dinucleotide repeats (AG/TC) was
observed than any other repeats, which supported the
fact that di-nucleotide repeats are the most frequently
occurring microsatellites in plants (Lagercrantz et al.,
1993; Thomas and Scott, 1993; Wang et al., 1994).
However, a significant proportion (35.7%) of tri- and
hexa-nucleotide repeat motifs were also observed,
which could have resulted due to the chance cross
hybridization of dinucleotide probes with other repeat
region of the genome. Sequence analysis of 952
clones yielded 221 sequences with class I and II SSR
motifs (Mc Couch et al., 2002). About 70 class I
microsatellite loci were targeted for marker development.
All the identified SSR motifs showed more observed
heterozygosity than expected heterozygosity. Both
perfect and imperfect SSR motifs showed less variation of
mean H
e
and H
o
values. Whereas the mean H
o
for di-
and tri-nucleotide repeats were also found to be slight
difference. The results with more number of alleles
amplified per locus (6.4) combined with the mean
observed heterozygosity (Ho) values; suggest that
considerable polymorphism is present at these
microsatellite loci. The same results were represented
in the earlier studies, reported in chickpea (Sethy et al
2006). The highest heterozygosity detected at SSR loci,
is potentially meaningful because high heterozygosity
would indicate that the plant population likely has a
substantial amount of adaptive genetic variation to
