Molecular Plant Breeding 2015, Vol.6, No.16, 1
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Table 1 List of tobacco genotypes and other
Nicotiana
species
used in this study
Species / Cultivar
Features/types
N. gluaca
Resistance to PM
N. paniculata
-
N. thrisiflora
-
N. rustica
-
N. undulate
Resistance to RV, TE, TMV, WF
N. trigonophylla
Resistance to An, PM
N. plumbagenifolia
Resistance to BS, PM, WF
N. sylvestris
-
N. repanda
Resistance to An, CN, PM, RK, TMV
N. nesophila
Resistance to An
N. corymbosa
-
N. acuminatae
-
N. clevlandii
-
N. nudicaulis
Resistance to An, PM, RK, WF
N. suav
-
N. debneyi
Resistance to An, PM, BM, PM, RV
N. gossei
Resistance to RR, PM, TMV, TS
N. maritama
-
N. velutina
-
N. occidentals
Resistance to An, SP, PM
N. simulans
-
N. goodspeedi
-
N. rosulata
-
N.
tabacum
cv.
Jayashree
-
Hema
FCV
Kanchan
FCV
Abhirami
Chewing
Lichchavi
Chewing
GC-2
Chewing -
N. rustica
Dharla
Chewing and hookah
Banket A1
Burley
HDBRG
Burley
Sendarpatty Special
Cheroot
Bhagyasri
Bidi
S-5
Cigar wrapper
Lanka Special
Natu
An: Anthracnose, BM: Blue mold, BS: Black shank, FCV: Flue
Cured Virginia; SP: Brown spot, CN: Cyst nematode, TMV:
Tobacco mosaic virus, TE: Tobacco Etch Virus, PM: Powdery
mildew, RR: Black Root-Rot, RK: Root-Knot nematode, RV:
Rattle virus, WF: Wild fire
N1N2N2N1N2N2N2N1N2; interrupted repeats of the
form (N1N2)X(N)Y(N1N2)Z; and compound repeats
of the form (N1N2)X(N3N4)Y were synthesized from
IDT, Canada and they were standardized with gradient
PCR to obtain the perfect annealing temperature to
avoid non-specific amplification.
4.2 Testing of microsatellite markers
Each primer pair was tested by the amplification of
genomic DNA isolated from the tobacco cultivar,
Jayasree. PCR amplification was done in 25 µl
reaction volume containing 200 mM Tris-HCl (pH
8.0), 500 mM KCl, 2 mM MgCl
2
, 0.25 mM of each
dNTPs, 0.5 µM of each primer, 25 ng of genomic
DNA and 1.0 U of
Taq
DNA polymerase (MBI
Fermentas, Lithuania). Reactions were carried out in
thermal cycler following the thermo-profile of an
initial denaturation at 94ºC for 5 min followed by 35
cycles of 94ºC for 1 min, 55-57ºC for 1min, 72ºC for
2 min and a final extension of 7 min at 72ºC.
Amplified products were resolved on 6% polyacrylamide
gels and stained with ethidium bromide. Initially, all
the 70 primer pairs were used to amplify the DNA
from 12 different tobacco types (Table 1) and one wild
species of
Nicotiana
in order to establish their
usefulness in the detection of intra- as well as
inter-species-specific polymorphism. In order to
check the transferability of these markers to other
Nicotiana
species, these primer pairs were further
checked with 24 wild species of
Nicotiana
(Table 1).
4.3 Data analysis
The size of the alleles for each microsatellite locus
was estimated by comparison with standard size DNA
markers and scored across all the 24
Nioctiana
species
as well as genotypes from different types of cultivated
tobacco using the gel documentation system (Alpha
Innotech Corp., USA). A binary matrix was developed,
in which 1 (one) represents the presence of an
amplicon and 0 (zero) represents the absence of an
amplicon. Genetic parameters like alleles per locus,
the expected heterozygosity (H
e
) were calculated as
H
e
= 1 – ∑
pi
2
(Nei 1973), where
pi
represents the
frequency of allele
i
among the varietal set.
The
observed heterozygosity (H
o
) was obtained by direct
calculation. The null allele frequency was calculated
as
r
= (H
e
– H
o
)/(1 +
H
e
) (Brookfield, 1996). The
probability of identity was calculated as PI = 1 – ∑
pi
4
+ ∑∑ (2
pipj
)
2
(Paetkau et al., 1995), where
pi
and
pj
represent the frequency of alleles
i
and
j
, respectively.
Pair-wise genetic similarity was calculated among the
24 accessions of wild species as well as 12 genotypes
belonging to different types of tobacco using Jaccard’s
similarity coefficient. The similarity matrix was used
