Asian Journal of Agricultural Sciences 2(4): 120-123, 2010
ISSN: 2041-3890
© M axwell Scientific Organization, 2010
Submitted Date: November 03, 2009
Accepted Date: November 26, 2009
Published Date: October 09, 2010
Seed Storage Protein Profile of Rice Varieties Commonly Grown in Pakistan
1
Inam ullah, 1 Imtiaz Ahmed K han, 2 Habib A hmad, 1 Sajid-ul-Ghafoor, 1 Sahib Gul,
1
Ikram Muhammad and 3 Muhammad Ilyas
1
Department of Genetics, Hazara University, Mansehra, N.W.F.P, Pakistan
2
Department of Botany, Hazara University, Mansehra, N.W.F.P, Pakistan
3
National Centre of Excellence in Mo lecular Biology, University of the Punjab, Lahore, Pakistan
Abstract: Present research was aimed at studying seed storage protein profiles of eleven released
varieties / genotypes of rice (Oryza sativa) in Pakistan using SDS-PAG E analysis. Lower - medium level of
genetic polymo rphism (G .D. = 0 - 0.8) w as estimated in the material. Six com parisons showed co mplete
hom ozygosity (G.D. = 0%) for seed storage protein profile while one comparison (Basmathi - 385 - Local rice
from Swat) showed maximum genetic difference (G.D. = 80%). Remaining comparisons showed varying
degree of genetic differences ranging from 20-70%.
Key w ords: Cluster analysis, genetic diversity, Oryza sativa, SDS-PA GE , seed storage protein
INTRODUCTION
Rice (Oryza sativa) is an economically important
crop and is the staple food for more than one-half of the
world’s population (Bellon et al., 1998 .). It is grown in
111 countries ranging from flood planes of Ban glade sh to
the Himalayan foothills of Nepal and from the rain forest
of Indonesia to the desse rt of plains of A ustralia
(Khakwani et al., 2006). Rice b elong to the family
“Poaceae” and genus “oryza Linn” with diploid (2n)
chromosome num bers 24. The genus oryza comprises 25
species distributed through out the world including
tropical and subtropical region of world. Out of 25
common species of oryza, tw o species i.e., O ryza sativa
and Oryza glaberrima are cultivated widely. Oryza sativa
is grown w orld wide while oryza glaberrima is only
confined to W est Africa (Grist, 1986). Species of rice,
Oryza sativa L ., are furthe r sub-d ivided into two ma in
types, indica (adapted to the tropics) and japonica
(adap ted to tem perate region s) (Kochko, 198 7).
Rice is an an nual plant, growing from 1 to 1.8 m tall,
occa sionally more, with long slender leaves 50-100 cm
long and 2-2.5 cm broad . Abo ut 27% of world rice is
cultivated under rain fed lowland and requires 15 mm
water /day (Chatterjee, 1948). About 90% of the world’s
rice is produced and consumed in Asia (Marshall and
W adsworth, 1993) where the total production is 559349
metric tones w ith an average yield of 4.11 tonn es/ha. In
Pakistan Rice is an important cash crop and ranked third
after wheat and cotton an d is grown as kharif (hot
season) crop on an area of 2.5 million ha, with an annual
production of 4.95 million tonnes giving an average yield
of 2806 kg/ha (MINFAL , 2004). Pakistan is well known
for its basmati rice with long grain and aroma as well as
for non basmati indica verities. In Pakistan 42 different
verities of rice are cultivated w ith an average yield of 2
t/ha (Ahmad and Awan, 2007). In Swat at high altitude
mou ntain valleys, temperate Japonica rices are grow n. In
the south of NWF P, Sindh and Baluchistan, mostly IRRI
type long grain heat tolerant tropical rice verities are
grown. Bey ond basm ati rice, begmi rice is mo stly grown
in Sw at.
Rice provides 20% of the world’s dietary energy
supp ly in different region of the world, in addition 100g
of rice produce 3 30 kcal energy, 79 g carbohydrates, 8.0
g proteins, 0.69 g fates and is a good source of thiamine,
riboflav in and niacin (FAO, 2009). The proteins of rice
are mainly com pose of glutilin w hile prolamin is in lower
quantity, that’s why rice kernels are consider to have high
quality protein s (M ullins, 19 99).
The technique o f Sodium D odecyl Su lphate
Polyacrylamide Gel Electrophoresis (SD S-PA GE ) is
commonly used for separa tion of se ed storage p roteins.
Seed storage protein profiles have also b een u sed to
study evolutionary relation of several crop plants
(Ravi et al., 2003 ).
During the present study, eleven released rice
varieties of Pakistan were used to study variation in total
seed storage protein using SDS-PAGE.
MATERIALS AND METHODS
National Agricultural R esearch C entre (NA RC ),
Islamabad, Pakistan, provided seeds of seven approved
verities of rice (O ryza sativa). Three verities were
provided by Mingora Agricultural Research Center and
one verity (local) was collected from a field at Fizagut,
Swat (Table 1). Studies were conducted during 2008 at
the Departm ent of G enetics, Hazara University,
Mansehra, Pakistan.
For SDS-PA GE ana lysis, five seeds from each v erity
was grounded to a fine powder with mortar and pestle.
Corresponding Author: Dr. Imtiaz Ahmad Khan, HEC Faculty Member, Department of Genetics, Hazara University Pakistan.
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Asian J. Agric. Sci., 2(4): 120-123, 2010
Table1: List of eleven rice verities used during present study
S. No.
Name
Source
1
BAS -385
NA RC, Islamabad
2
KSK-133
NA RC, Islamabad
3
Shaheen basmathi
NA RC, Islamabad
4
IR6 N A R C , Islamabad
5
JP-5
NA RC, Islamabad
6
BAS-370
NA RC, Islamabad
7
KS-282
NA RC, Islamabad
8
JP-5
MA RC(N ), Swat
9
Fakhre Malakand
MA RC(N ), Swat
10
Basmathi -385
MA RC(N ), Swat
11
Local
Fizagut, Swat
Tab le 2: G enetic distance estimates (G.D) among 11 rice v arietie s
usin g S DS -PA GE ana lysis
1
2
3
4
5
6
7
8
9
10
1
2
0.2
3
0.0
0.2
4
0.2
0.0
0.2
5
0.4
0.3
0.4
0.3
6
0.4
0.3
0.4
0.3
0.0
7
0.4
0.3
0.4
0.3
0.0
0.0
8
0.2
0.0
0.2
0.4
0.3
0.3 0.3
9
0.3
0.4
0.3
0.4
0.6
0.6 0.6
0.4
10
0.6
0.5
0.6
0.5
0.4
0.4 0.4
0.5
0.7
11
0.6
0.5
0.6
0.5
0.7
0.7 0.7
0.7
0.3 0.8
1 = BAS -385, 2 = KSK-133, 3 = Shaheen basmathi, 4 = IR-6, 5 = JP-5,
6 = BAS-370, 7 = KS-282, 8 = JP-5, 9 = Fakhre malakand, 10 =
Ba sma thi -38 5, 11 = L oca l rice fro m S wa t
Five hundred mL of protein extraction buffer (PEB) was
added to 0.01 g of seed flour and vortexed thorou ghly to
homogenize. The proteins were extracted at room
temperature for 20 m in. In order to purify, the
hom ogenate samples were centrifuged at 6,000 rpm for 10
min at room temperature. The extracted crude proteins
were recovered as clear supernatant and were transferred
to a new 1.5 mL eppend orf tubes and stored at 4ºC until
they w ere run on the polya crylam ide gel.
The electrophoretic procedure was carried out using
slab type S DS -PA GE with 12.5% polya crylam ide gel. A
12.5% resolving gel (3.0M Tris-HCL, pH9, 0.4% SDS
and 4.5% stackin g gel (0.4M Tris-H CL pH 7.0, 0.4%
SDS) was prepared and polymerized chemically by
addition of 17 mL of N, N’, N’, N’ tetramethylenediamine
(Wako) and 1 0% Am mon ium p ersulphate. E lectrode
buffer solution was poured into the bottom pool of the
apparatus. Gel plates were placed in the apparatus
carefu lly so as to prevent bubbles formation at the bottom
of gel plates. Then electrode buffer (0.025 M Tris, 1.29 M
Glycine, 0.125% SDS) was added to the top pool of the
apparatus. Four ml of the extracted protein was loaded
with the micropipette into each wells of the gel. The
apparatus was conn ected with constant electric supp ly
(75 V) till the tracking dye “bromophenol blue” (BPB)
reaches the bottom of the gel. Gels were stained with
staining solution comprising 0.2 % (W /V) C oma ssie
Brilliant Blue (CBB) R 250 dissolv ed in 10% (V/V ) acetic
acid, 40 % (V/V) methanol for about an hour at room
temperature. Gels w ere distained in a solution containing
5 % (V/V) acetic acid and 20% (V/V) methano l. Gels
were shacked using Double Shaker Mixer model DH -10
gently until the background of the ge l became clear and
protein bands were clearly visible. After destaining the
gels were photographed using gel documentation system.
bands were observed (Table 2). Nei and Li (1979)
procedure of “Un Paired Group of Arithmetic mean”
(UPGMA) was used to estimate Genetic Distances (GD)
among all the possible combinations. Genetic distance
estimates are presented in Table 2. Genetic distance
estimates range d from 0 and 80% . Maxim um genetic
distance (80%) was estimated between Basmathi-385 and
local rice (collected fro m Swat). Six comparisons viz;
BAS-385 - Shaheen basmathi, KSK-133-IR-6, KSK-133JP-5, JP-5-BAS-370, JP-5-KS-282 and BAS-370-KS-282
showed complete homozygosity for seed storage protein
profile. Six, twelve, thirteen, five, seven and five
comparisons showed 20, 30, 40, 50, 60 an d 70% g enetic
distance, respectively. The comparisons showed 20% GD
included BAS-385 - KSK-133, KSK -133-Shaheen
Basmathi, BAS-385-IR-6, Shaheen Basmathi-IR-6,
BAS-385-JP-5, and Shaheen Basmathi-JP-5. The
comparisons showed 30% G D were BAS-3 85-Fak hre
Malakand, BAS-385-Fakhre Malakand, KSK-133-JP-5,
KSK-133-BA S-370, K SK - 1 3 3 -K S -282, Shaheen
Basm athi-Fakhre Malakand, IR-6-JP-5, IR-6-BAS370,
IR-6-KS282, JP-5-JP-5, B AS-3 70-JP-5, KSK-282-JP-5,
Fakhre Ma lakand-L ocal rice. The comparisons showed
40% GD were BAS-385-JP-5, BAS-385-BAS-370,
BAS-385-KS-282, KSK -133-Fak hre Malakand, Shaheen
Basmathi - JP-5, Shaheen Basmathi - BAS-370, Shaheen
Basmathi - KS-282, IR-6-JP-5, IR-6-JP-5, IR-6-Fak hre
Malakand, JP-5-B asmathi-38 5, BAS-370-Basmathi385,KS-282-Basmathi-385 and JP-5-Fakhre Malakand.
The comparisons showed 50% GD was KSK-133Basmathi-385, KSK-133-L ocal, IR-6-Basmathi-385, IR6-Local and JP-5-Basmathi-385. The comparisons showed
60% GD were BAS-385-Basmathi-385, BAS-385-Local
rice, Sha heen Bas math i-Bas math i-385 , Shaheen
Basmathi-local rice, JP-5-Fakhre Malakand, BAS-370Fakhre Malakand and KS-282- Fakhre Malakand. The
com parisons showed 70% GD were JP-5-Local,
BA S-370-Lo cal, KS-282-Local rice, JP-5-Local rice and
Fakhre Malakand - Basmathi-385. It is evident that most
of the comparisons (25 comparisons) showed medium
genetic distances. Various workers were reported similar
results (low-medium Genetic diversity) including
RESULTS AND DISCUSSION
Total seed storage proteins extracted from 11 rice
varieties were separated using Sodium Dodecy l Sulph ate
Polyacrylamide Gel Electrophoresis (SDS -PAG E).
Results of protein profile are presented in Fig. 1. Each
single band was con sidered as a locus / allele. Loci we re
scored as present (1) or absent (0). Bivariate (1 -0) data
matrix is presented in Table 2. A total of 45 scoreab le
121
Asian J. Agric. Sci., 2(4): 120-123, 2010
Fig. 1: Total seed storage protein profile of 11 rice varieties using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
(SDS-PAGE). 1 = BAS -385, 2 = KSK-133, 3 = Shaheen basmathi, 4 = IR-6, 5 = JP-5, 6 = BAS-370, 7 = KS-282, 8 = JP-5,
9 = Fakhre malakand, 10 = Basmathi -385, 11 = Local
Fig. 2: Dendrogram constructed for 11 rice varieties using 1-0 Bivariate data matrix generated from SDS-PAGE analysis
(Muehlbauer and Singh, 1987; Murphy et al., 1990;
Ahmad and Slinkard, 1992; Das and Mukharjee, 1995;
De Vries et al., 1996; Ferreira et al., 2000;
Sultana et al., 2005).
Biva riate data matrix was also used to construct
dendrogram using computer package “Popgene 323”.
Eleven rice varieties were grouped in 3 clusters “A”, “B”,
and “C” (Fig. 2). Group “B” was largest group comprised
5 varieties (JP-5, BAS-370, KS-282, JP-5, Basm athi-385),
while group “C” was smallest comprised 2 varieties
(Fakhre Malakand and Local rice from Swat) and group
“A” com prised 4 varieties (BA S-385, Shahee n Basmathi,
KSK-133 and IR-6). In general, results of dandrogram
(Fig. 2) analysis were similar to those based upon
U PG M A procedure (Table 2). It is evident from the
dendrogram that genotypes BAS-385 and “local rice from
swat” are most distantly related, hence it is recommended
that these 2 varieties sho uld be used in future breeding
programs to create higher amount of gene tic variab ility in
Pakistani germplasm of rice.
122
Asian J. Agric. Sci., 2(4): 120-123, 2010
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CONCLUSION
It is concluded that medium amou nt of genetic
diversity (estimated as g enetic distanc e G.D. ranging from
0-80%) at seed storage protein level is present in Pakistani
rice varieties / genotype s wh ich can be u tilized in
Pakistani breeding programs aimed at increasing level of
genetic diversity wh ich ultimately will be useful for the
development of new improved genotypes of rice. Also to
increase chances of developing more diverse germplasm
of rice, the genotypes showing maximum genetic distance
(e.g., BA S 385 a nd local rice from Swa t) wil be utilized
in further breed ing pro gram s.
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