Rice Genomics and Genetics 2015, Vol.6, No.2, 1-5
2
variation and relatedness using seed protein
polymorphism.
1 Materials and Methods
1.1 Plant materials
A total of twenty four drought tolerant donor accessions
collected from Central Rice Research Institute (CRRI)
gene bank and International rice research institute (IRRI)
were used in the present study (Table 1). Seeds of all the
rice accessions were germinated in individual pots in the
net house of Central Rice Research Institute, Cuttack
(India) during 2011-2012. Further, the seeds were
harvested and collected from individual genotypes
separately.
Table 1 List of genotypes used for SDS PAGE
Serial No.
Genotypes
1000 seed (g)
Serial No.
Genotypes
1000 seed (g)
1
Naveen*
19.7
13
Mahulata**#
19.5
2
CR 143-2-2*#
23.5
14
IR 20*
19.0
3
Swarna*
19.5
15
Sarjoo50*
28.6
4
T 1*
18.0
16
Kalamkati*
24.9
5
PSBRC 80**
22.7
17
Tam Cau 9 A**#
23.8
6
Dhagad deshi**
20.0
18
Koshihikari*
25.4
7
Vandana*#
19.7
19
Saita**
23.6
8
Black Gora (NCS 12) **
26.7
20
Kalakeri**
18.6
9
Jhona 349**#
23.5
21
BR 21**
24.0
10
Samba Mahsuri*
16.0
22
Nan Te Hao*#
23.4
11
Binnatoha*
26.8
23
Dhalasaita **#
21.7
12
Basmati 370*
19.3
24
T 136*
16.8
Note: *Collected from gene bank, Central Rice Research Institute, Cuttack; **Collected from gene bank, International Rice Research
Institute; Philippines; #drought tolerant donors
1.2 SDS-PAGE electrophoresis
Study on seed protein profile by SDS-PAGE (sodium
dodecyl sulphate polyacrylamide gel electrophoresis)
was conducted at rice biotechnology laboratory, Crop
Improvement Division, CRRI as described by
Laemmli (1990) with necessary modifications.
De-husked seeds of each accession were finely ground
by sterilized mortar and pestle. Seed flour of 0.01g
was taken and mixed with 400μl of extraction buffer
(0.05M Tris-HCl pH 8.0, 0.2% SDS, 5M Urea, 1% β
mercaptoethanol and 0.05% bromophenol blue as
tracking dye). Centrifugation was done at 15,000rpm
for 5 min at room temperature. Extracted samples
were analyzed through 15% SDS-polyacrylamide gel
electrophoresis (Laemmli, 1990) with known
molecular weight (GeNei) of protein marker.
1.3 Staining and data analysis
Gels were stained with 0.2% (w/v) coomassie brilliant
blue R250 for about 1 hour and de-stained overnight.
Gels were preserved by using gel dryer and visualized
under Typhoon FLA 7000 fluorescent image analyzer
(GE Healthcare Bio-Sciences AB, Uppasala, Sweden).
Bands were scored for their presence (1) and absence
(0) and a scoring matrix was generated. The pairwise
genetic distance (Nei, 1973) was calculated by
POPGENE v 1.32
). The
dendrogram was constructed based on similarity
co-efficient, using the un-weighted pair group
method of arithmetic averages (UPGMA)
employing sequential, agglomerative hierarchic and
non-overlapping clustering (SAHN) (Sneath and
Sokal, 1979) in NTSYSpc, version 2.1 (Applied
Biostatistics Inc., USA).
2 Results
In the present investigation, SDS-PAGE banding
pattern of seed proteins of twenty four rice genotypes
were investigated to assess the genetic diversity
(Figure 1). The storage protein profiling banding
pattern isolated from seed sample showed distinct
variability among the tested samples. A total of 90
poly peptide bands were harvested from twenty four
individuals ranging from 14.3 kD to 99.0 kD. The
number of bands ranged from two (lowest), in
Kalakeri, T136 and T1 to seven (highest), in Jhona
349 with an average of 3.75. Moreover, in the present
study, protein banding profile were grouped into two
zones i.e. Zone I (having band size more than 30 kD)
and Zone II (with protein bands of less than 30 kD)
based on molecular weight of peptides. Diversity in
