Mishra and Fougat
EST SSR (Microsatellite) PCR Amplification
0.465411. Molecular marker JESPR 29 was proved to be
We used 35 primer pairs to investigate genetic variation
the least informative marker with a PIC 0.165289, while,
and phylogeny in our 24 cotton cultivars. Thirty
on the contrary, NAU923 was proved to be the most
one of 35 primer pairs easily produced detectable
informative with a PIC of 0.730489. Markers with higher
products. Amplifica tions were carried out in 200-μL
PIC values have the potential to reveal the variation
microtubes containing a 25 μL reaction mix consisting
between alleles and could be used more effectively for
of 20 ng template, 0.2 μM of each primer, 1 U Taq DNA
molecular mapping and analysis of genetic variation in
polymerase (Finzymes), 0.2 mM of each dNTP, 0.2 to
a population .
0.325 mM MgCl 2 , and 1X reaction buffer (10 mM Tris-
The 31 polymorphic molecular markers amplified from
HCl and 50 mM KCl, pH 8.3). The amplification was
1-4 alleles for each locus with BNL4030 found to amplify
carried out in a Thermal Cycler (Whatman Biometra
5 alleles. This marker is an informative marker with a PIC
T-Gradient, Germany) using a program consisting of
of 0.553469 while a similar PIC has been reported by Liu
a denaturation step of 5 min at 94°C followed by 40
et al. (2000b). An SSR profile was constructed for each
cycles of 60 s at 94°C, 60 s at 55°C, and 1.5 min at
cultivar using the 31 primer pairs, which were able to
72°C. The program ended with an extension step at
discriminate between 24 cultivars studied. Phylogenetic
72°C for 7 min. Agarose gel of 2.5% concentration was
analysis revealed that the 24 cotton cultivars that were
prepared in 1X TBE (2.5 g agarose in 100 ml 1X TBE
examined belong to 2 main groups. The finding of
and 2.5 µl Ethidium bromide from 10 mg/ml stock).
genetic distance between the cotton cultivars using SSR
PCR amplified products (9 µl and 1 µl 6X loading
molecular markers can be useful in marker-assisted
dye) were loaded into the wells. The 1000 bp standard
selection (MAS). This acquires greater importance for
DNA ladder (1 µl) (marker) was also run along with the
cotton where the genetic base of improved cultivars is
samples. The electrophoresis was conducted at 100 V
limited enough, because EST SSR mo lecular markers
current (constant) for 2.5 hrs. to separate the amplified
have higher analytical force, and hence, they reveal
bands. The separated bands were visualized under UV
higher variation between genotypes compared to other
transilluminator (Biometra,Germany) and photographed
molecular markers.
using gel documentation system (Bio-rad, California).
In the present SSR analysis with 35 microsatellite markers
Scoring and analysis of data
only 31 markers gave the results. All thirty one markers
produced 83 alleles. The average number of alleles per
Clear and distinct bands amplified by 35 SSR primers were
locus was found to be 2.47. A maximum of 5 alleles were
scored for the presence and absence of the corresponding
recorded for marker BNL 4030, JESPR 208 and NAU
band among the all 24 cotton genotypes. The scores 1 and
923 was followed by BNL 2920, JESPR 307, NAU 4024
0 indicates the presence or absence of bands respectively.
AND NAU 1531 which produced 4 alleles. BNL 1317,
The softwares used for the analysis of the scored data
BNL 580, CIR30, NAU 1200 and NAU3393 produced
were NTSTSpc version 2.02 (Rholf 1998), GeneAlEx
three alleles each and BNL1531, CIR 246, CIR 307,
(Peakall et al., 2010) and SPSS. The major part of the
JESPR65, NAU 1369, NAU2035 which was lowest in
analysis was carried out using NTSYSpc version 2.02
the present investigation.
(Rholf, 1998) except for the calculation of the Shannon
index, observed and effective number of alleles which
The highest PIC value was recorded for NAU 923
were calculated using GeneAlEx6.4.
(0.730489) and lowest for JESPR 29 (0.165289); whereas
mean PIC value from all tested microsatellite was
Results and Discussion
0.465411 The average heterozygosity for 31 EST-SSR
markers was 0.60. Dendrogram Based on Nei’s (1978)
The 31 pairs of SSR primers amplified a total of 83 alleles
unbiased measures of genetic distance by UPGMA
with an average of 2.48 per SSR locus and PIC values
method formed two major clusters which grouped all 24
varying from 0.165289 to 0.730489 with an average of
cotton genotypes (Fig. ). Cluster 1(B) which was smallest