Molecular characterization of bacterial leaf blight resistant

which occur as interspersed repetitive elements in all

modified CTAB method (Zidani et al ., 2005). All of the

eukaryotic genomes (Tautz and Renz, 1984). Variation in

required reagents were prepared as per Sambrook et al .,

the number of tandemly repeated units is mainly due to

(1989). Fresh leaf tissues (0.3 g) was ground in liquid

strand slippage during DNA replication. Microsatellites

nitrogen and taken into a 2 ml microcentrifuge tube.

are highly popular genetic markers because of their co-

The ground sample was extracted with 0.8 ml of CTAB

dominant inheritance, high abundance, locus-specificity,

extraction buffer. [To prepare 10 ml of CTAB extraction

high reproducibility, multi-allelic nature and the ease

buffer, 1 M Tris HCL (1ml), 0.5 M EDTA (1ml), 5 M NaCl

of assessing SSR size variation by PCR with pairs of

(2.8ml) were mixed with 4 ml of distilled water. Then,

flanking primers.

4% CTAB (0.4g) and 1% PVP (0.1g) were added to this

mixture. Dissolved PVP and CTAB properly and heated

Materials and Methods

this mixture at 60°C (about 20-30 minute). Two percentage

of β-mercaptoethanol (200µl) was added, just before

The seeds of 30 rice genotypes (Table 1) which included,

use.] Sample was mixed well with extraction buffer by

19 NILs, six recurrent parents and five susceptible check

inversion. The sample was incubated for one hour at 65°C

cultivars used in the study were obtained from the Main

in water bath (allowed it to cool down). Equal amount of

Rice Research Station, Anand Agricultural University,

800 µl of chloroform: isoamylalcohol (24:1) was added

Nawagam, Gujarat, India.

to centrifuge tube and mixed by inversion. Centrifugation

was carried out it for 20 minutes at 10,000 rpm at 4°C.

Plant material and DNA extraction

Supernatant was transferred into a new tube and further

The seeds of 30 rice genotypes (Table-1) were grown in

extracted with chloroform: isoamyl alcohol (24:1), and

pots, 15 days old seedlings were collected and used for

the DNA was precipitated with 80% ethanol. The pellet

isolating genomic DNA. DNA was extracted using a

was air dried and resuspended in 100µl of Tris–EDTA

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