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

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