Genetic Diversity of Seed Storage Proteins of rice (Oryza sativa L.

) varieties
in Sri Lanka

L.Vithyashini* and H.A.M.Wickremasinghe1

Postgraduate Institute of Agriculture

University of Peradeniya
Sri Lanka

ABSTRACT

Owing a long history of rice cultivation and the rice improvement programmes, Sri
Lanka enriched with plentiful rice germplasm. Characterization of germplasm is
essential to provide information on the traits of genotypes assuring the maximum
utilization of the germplasm collection to the final users. Therefore this study was
carried out assess the genetic diversity of seed storage proteins of rice (Oryza sativa
L.) varieties in Sri Lanka. A total of thirty two rice varieties including both traditional
and new improved varieties subjected for seed protein extraction followed by
denatured- sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and silver staining. Resultant gel was showed a total twelve bands consisting of four
monomorphic and eight polymorphic bands. 46 kDa unique band was identified in
BW 272-6B indicated the usage of protein profiling for its fingerprinting. On gel
quantification using Gel Quant.NET 1.6.6 software revealed highest and lowest
protein levels in BW 272-6b and BG 360 respectively. Dendrogram obtained from
both on gel quantification and band binary scoring resulted correlation in displaying
of seed storage protein rich rice genotypes as one group viz BW 272-6B,
Pachchaperumal, Hondarawalu and Mawee. Therefore this study was suggested to
use protein profiling as a molecular tool for diversity exploration of rice genotypes
using seed storage protein finger prints.

Key words: Cluster analysis, new improved rice varieties, SDS-PAGE, seed
storage protein, traditional rice varieties.

1
Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Sri Lanka
* Corresponding author: vithya_6@yahoo.com
 INTRODUCTION

Rice is a cereal grain and it is the most widely consumed staple food for a large part
of the world's human population, especially in Asia. Asian countries contributes
more than 90% of rice production and rice consumption. Considering Sri Lanka, rice
is the single most important crop occupying 34 % of the total cultivated area in Sri
Lanka. On average 651,000 ha are cultivated during Maha and 313,000 ha during
Yala making the average annual extent sown with rice to about 964,000 ha. About
1.8 million farm families are engaged in paddy cultivation island-wide. Sri Lanka
currently produces 3.3 million tonnes of rough rice annually and satisfies around
95% of the domestic requirement (Central Bank of Sri Lanka, 2014; Department of
Census and Statistics, 2015). Of the total value added in agriculture, it accounts for
12.26% and 1.23% to GDP (Central Bank of Sri Lanka, 2014).

Rice production in Sri Lanka is acutely dependent on rainfall thus government

supply necessity of water through irrigation channels throughout the cultivation
seasons. However, for the last few decades, drastic changes in climatic conditions
highly affected the cultivation and quality of rice varieties (Department of Census
and Statistics, 2015). Thus selection of suitable rice varieties for farming, breeding
programmes and utilization are the most crucial steps in current Srilankan rice
industry to counteract biotic and abiotic stresses of rice cultivation as well as socio
economic concerns on rice industry.

Characterization of germplasm is essential to provide information on the traits of

accessions assuring the maximum utilization of the germplasm collection to the final
users (Ayres et al., 1997). Several strategies have been adopted for germplasm
characterization including morphological markers (Kancherla and Bhalla, 2003),
cytological analysis (Bennici et al., 2004), biochemical analysis, physiological
supervision and molecular markers (Aggarwal et al., 1999; Rout et al., 2009).

Among these numerous techniques available for assessing the genetic variability
and relatedness among crop germplasm, seed storage protein analysis signifies a
valid alternative improved approach for identification of germ plasm (Mennella et al.,
1999) via exploring genetic diversity using sodium dodecylsulphate-polyacrylamide
gel electrophoresis (SDS-PAGE) (Sadia et al., 2009) with in a short period of time
(Netra and Prasad, 2007) since seed storage protein markers are highly
polymorphic and environmental influence on their electrophoretic pattern is low
(Gepts et al., 1986; Sadia et al., 2009 and Galani et al., 2011).

So far, the polymorphism of seed storage proteins has already widely applied in
plant classification (Portyanko et al., 1998), screening of mutants for seed storage
proteins (Kumamaru et al., 1988), germplasm resource analysis (Yu and Chen,
2003), variety identification (Song et al., 1996) , genetic diversity analysis (Yang et
al., 2005), and so on.

Considering the above facts the present study was carried out to investigate the
genetic diversity of sri Lankan rice varieties including both traditional and new
improved varieties using seed storage protein polymorphism.

MATERIALS AND METHODS

The present study was conducted in Molecular laboratory, Department of

Agricultural Biology, Faculty of Agriculture, University of Peradeniya, Sri Lanka.

Plant Material

Total of 30 rice varieties including both traditional and improved varieties with two
standard varieties viz cultivar 93-11, Nippon bare seeds which were grown at the
Rice Research and Development Institute (RRDI) Bathalagoda in Maha 2013/2014
under similar management practices were subjected for this study. Seeds were
collected at the harvest maturity and shade dried to 12-14% of moisture content.
The details of rice varieties used in this study are given in Table 1 below.

Table 1: Details of Rice varieties

ID Name of rice variety Remarks
1 BG 360 New Improved Variety, White pericarp
2 BG 366 New Improved Variety, White pericarp
3 BW 272-6B New Improved Variety, Red pericarp
4 Pachaperumal Traditional Variety, Red pericarp
5 Hondarawalu Traditional Variety, Red pericarp
6 BG 358 New Improved Variety, White pericarp
7 Pokkali Traditional Variety, Red pericarp, saline tolerant
8 Gonabaru Traditional Variety, White pericarp
 9 Suwadal Traditional Variety, White pericarp, fragrant
10 Wanni Dahanalu Traditional Variety, Red pericarp
11 Kaluheenati Traditional Variety, Red pericarp
12 Masuran Traditional Variety, Red pericarp
13 Kuruluthudu Traditional Variety, Red pericarp
14 Mavee Traditional Variety, Red pericarp
15 BG 352 New Improved Variety, White pericarp
16 BW 267-3 New Improved Variety, White pericarp
17 Inginimitiya Traditional Variety, Red pericarp
18 Madathawalu Traditional Variety, Red pericarp
19 Rathal Traditional Variety, Red pericarp
20 Kahata wee Traditional Variety, Red pericarp
21 Sudu heenati Traditional Variety, Red pericarp
22 Suduru samba Traditional Variety, White pericarp
23 AT 306 New Improved Variety, White pericarp, long grain
24 BG 94-1 New Improved Variety, White pericarp
25 Dular Traditional Variety, Red pericarp
26 Dik wee Traditional Variety, Red pericarp
27 Herath banda Traditional Variety, Red pericarp
28 Sulai Traditional Variety, Red pericarp
29 Dewaraddiri Traditional Variety, Red pericarp
30 AT 405 New Improved Variety, White pericarp
31 Cultivar 93-11 New Improved Variety, White pericarp
32 Nipon bare Traditional Variety, White pericarp

Extraction of Rice Seed Storage Protein

All extraction procedures were carried out from powdered de-hulled mature grains.
To extract total proteins, 3 mg of flour and 100 L of extraction buffer (0.055M Tris-
HCl 2.3% pH-6.8, SDS, 5% -merceptoetanol, 10% Glycerol and 0.1% Bromo
phenol blue) was added and allowed to boil for 10 minutes. Then the samples were
kept for 4-5 hours aside followed by centrifugation at 12,000 rpm for 10 minutes at
4C in order to collect total proteins which were available in supernatant.

Protein Profiling

Protein profiling of extracted samples was analyzed through SDS-PAGE in a

discontinuous buffer system with 5% stacking gel and 15% resolving gel. 10 l of
extracted proteins were loaded along with a protein marker (Promega- V849a) as
standard and the gel was subjected to electrophoresis at 30 mA.

Silver Staining

Gel was kept in fixing solution (12.5% acetic acid, 37.5% ethanol) for 15 minutes to
overnight, followed by 20% ethanol wash and a distilled water wash. Then the gel
was subjected for sensitization using 0.02% Sodium thiosulfate for 10 minutes
following three washes of distilled water.

For silver incubation, 0.2% silver nitrate was added and incubated for 45 minutes.
Then the gel was placed in developing solution (3% K 2CO3, 0.001% Na2S2O3,
0.024% formaldehyde). Once the bands were prominently appeared; the gel was
immediately placed in fixation solution (5% Tris base, 2.5% acetic acid) with mild
shaking. Finally the gel was rinsed with distilled water and visualized.

Protein Imaging and Data Analysis

Silver stained gels were visualized under UV and photographed. Obtained image
were subjected for on-gel quantification of extracted protein Gel Quant.NET 1.6.6
software in volume values. Also the banding profiles were viewed and compared
with protein ladder. Clear and unambiguous bands were identified by using My
Image Analysis v2.0 Software.

Cluster analysis done for both on-gel quantified seed protein in quantity basis as
well as for the clear and unambiguous banding profile in binary score basis using
SAS 9.1.3 portable software.

RESULTS AND DISSCUSSION

In this study, SDS-PAGE of seed proteins of thirty two rice genotypes were carried
out to investigate the genetic diversity. Initially cluster analysis was done for on-gel
quantified extracted seed storage protein. Obtained dendrogram was given in
figure1.

Three prominent groups of rice varieties at 1.0 of average distance between clusters
were observed based on the quantity of grain reserve protein. Here one smaller
group consisting four of rice varieties viz BW 272-6B, Pachchaperumal,
Hondarawalu, Mawee and the larger group containing seventeen of rice varieties
viz BG 360, BG 358, Inginimitiya, BG 352, BG366, Nipon bare, Gonabaru, Suwadal,
AT 405, At 306, BW 267-3, Dik wee, Rathal, Suduru samba, Sulai, Cultivar 93-11,
BG 94-1 and the third group having eleven of rice varieties viz Pokkali, Wanni
dahanala, Masuran, Kaluheenati, Dular, Madathawalu, Kuruluthudu, Kahata wee,
Sudu heenati, Dewaraddiri, Herath banda were clearly seen.
Average genetic distance between
clusters

Figure 1: Dendrogram derived in cluster analysis for the quantity of seed storage protein of rice.
1-32 numbers representing different varieties which were already given in Table:1

During the gel analysis, both major and minor band were considered and twelve of
clear and unambiguous bands were elected consisting of four monomorphic and
eight of polymorphic bands. The resultant gel pictures are given in Figure: 2 and
figure: 3.

46 kDa

Figure 2: SDS-PAGE analysis of total seed storage protein of 16 rice species. (Genotype Id 1-16;BG 360,
BG 366, BW 272-6B, Pachchaperumal, Hondarawalu, BG 358, Pokkali, Gonabaru, Suwadal, Wanni
Dahanala, Kaluheenati, Masuran, Kuruluthudu, Mawee, BG 352, BW 267-3, L- protein marker)
Figure 3: SDS-PAGE analysis of total seed protein of 16 rice species.(genotype Id 17-32; Inginimitiya,
Madathawalu, Rathal, Kahata wee, Sudu heenati, Suduru samba, AT 306, BG 94-1, Dular, Dik wee, Herath
banda, Sulai, Dewaraddiri, AT 405, Cultivar 93-11, Nipon bare, L- protein marker)

Most of the time, bands appeared were looks alike in all the genotypes. The intensity
of bands appeared to be not uniform. So they were subjected to analysis using My
Image Analysis v2.0 Software. With the aid of software and the utility of protein
marker, bands were identified as well as their sizes were determined.

All 32 rice varieties were showed approximate sizes of 32 kDa, 30 kDa, 14 kDa and
10 kDa of monomorphic bands and 100 kDa, 75 kDa, 50 kda, 26 kDa, 21 kDa, 13
kDa of polymorphic bands.

10 kDa, 13 kDa , 22-23 kDa band was already identified by Jugran et al., 2010 as
albumin, prolamin, glutelin -subunit and they were successfully used in
characterization of agro-diversity of forty eight rice Germplasm from Uttarakhand
Himalaya, India.

Amazingly a 46 kDa of unique band was identified which is produced from variety
BW 272-6B. Also another 133kDa band was detected only in BW 272-6B,
Pachchaperumal and Hondarawalu. Above observations indicating the possible
usage of 133kDa and 46kDa protein as an indicator for BW 272-6B genotype which
was showed higher amount of seed storage protein among all 32 rice varieties.

Galani et al., 2011 have found this 46 kDa unique band from Kanwal-95 out of ten
elite rice genotypes of Sindh. Indicating that BW 272-6B may have some
relationship with Kanwal-95 genotype from Sindh.
In order to find out the diversity among rice varieties, the cluster analysis was
performed. Binary scored data matrix based on band profile subjected for cluster
analysis resultant dendrogram( Figure:4) calculated from the similarity matrix
revealed three types of group containing 22% 66% and 12% of total varieties.

Figure 4: Dendrogram showing relationship of 32 rice varieties based on grain reserve protein bands.

Rice varieties showing higher grain storage protein viz BW 272-6B,

Pachchaperumal, Hondarawalu, Mawee were grouped together in both dendrogram
reinforcing the results obtained.

Reports indicate that the glutelin and prolamin content is a good indicator of high
protein content in rice and account for 80%85% of the total seed protein (Takaiwa
et al., 1999). Where glutelin is identified as more easily digestible and comprises
high amount of essential amino acids (Ogawa et al., 1987; Resurreccion et al.,
1993).

In this present study, the presence of glutelin and prolamin observed in all in 32 rice
varieties could be further exploited for high protein content and amino acids together
with carbohydrates.
By analyzing the seed storage protein profile, can accomplish the task of grouping
of similar storage protein, in other words grouping of similar gene products.
According to the theory of central dogma, these groupings are indirectly linked with
their genetic makeup thus can interpret their genetic relationship or diversity.

The presence or absence of these proteins could be characteristic markers for

identification of these different rice varieties. Finally, it is concluded that the seed
storage protein profiles could be useful markers in varietal identification and genetic
diversity analyses

CONCLUSIONS

In this study on protein profile comparative analysis, considerable polymorphism

were observed. Thus this technique can be effectively used as a tool for
characterization or identification of Srilankan rice varieties.

BW 272-6B specific unique protein of 46 kDa can be utilized as molecular tool for
identification of this variety. Further it can be confirm with the presence of 133 kDa
protein band which is also present in Pachchaperumal and Hondarawalu. However
this result should be confirmed with further studies.

ACKNOWLEDGEMENTS

Gratitude is expressed to the Faculty of Agriculture, University of Peradeniya for

providing the laboratory facilities to conduct this research and NRC grand for
providing chemicals required for research work.

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