Solutions For Electrophoresis And Protein Extraction Buffers Biology Essay


Plant material for the study comprised of 174 landraces of Rice (Oryza sativa L.) collected from Gene Bank, Institute of Agricultural Biotechnology and Genetic Resources (IABGR), National Agricultural Research Center (NARC) Islamabad, which were acquired from various parts of the country that represent a wide ecological variation from dry mountains to irrigated plains.. The field work was carried out during May, 2006 to January, 2007 and May, 2007 to January, 2008 under field conditions at Institute of Agricultural Biotechnology and Genetic Resources (IABGR), National Agricultural Research Center (NARC) Islamabad.

All the experimental accessions (Appendix 1) of Oryza sativa were first planted in a small field for nursery growing (Fig 2.1). The seeds were sown in the pots for raising nursery at the end of May during both years and seedlings were transplanted into the field in an augmented design after one month of growth. Each cultivar as well as germplasm accession was planted in a three-row plot of four meters length with a spacing of 20cm x 20cm. One seedling was transplanted per hill and the inter-plot spacing was kept 40cm. Each experimental unit consisted of 60 plants, while five plants were selected at random from the central row for recording observations as reported by Satoh et al. (1990c, d, e, and f). The mean values of each character for each entry were used for statistical analysis according to Adair et al. (1973). Recommended cultural practices for rice evaluation were carried out from transplanting till harvest of the crop to get healthy and vigorous crop. Proper water treatment was applied to avoid water stress, flooded irrigation was continued after every 15 days till maturity of crop. Experimental field received two hoeings, one during nursery transplantation and other after one month. Fungicide Capton was sprayed twice to save the crop from fungal infections.

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Figure 2.1: Nursery for field plantation

2.1.1: Agro-morphological Characterization

All the cultivars were characterized for 18 quantitative and 9 qualitative traits from flowering till maturity and harvest of the crop during both years. Traits selection and measurement techniques were based on IRRI Standard Evaluation System of Rice (Table 2.1). Quantitative traits included days to 50% flowering, days to maturity, leaf length, leaf width, total and productive tillers per plant, plant height, panicle length, number of branches per panicle, seed setting (%), grain yield per plant, straw yield per plant, harvest index, 100-seed weight, grain length, grain width and grain length/width ratio, while qualitative data was observed for flag leaf angle, flag leaf shape, leaf appearance, lodging incidence, panicle type, panicle exertion, awning, awn color and seed coat color.

2.1.2: Data Analysis

Data were subjected to simple statistical analysis like mean, minimum, maximum, standard deviation, coefficient of variation, etc. for all the quantitative traits to assess the amount of genetic diversity present in the local germplasm as well as cultivars. Qualitative traits were categorized into different classes and frequency percentage was calculated. Simple correlation coefficients between all pairs of quantitative characters were also calculated according to Steel and Torrie (1981) using plot mean values.

All recorded morphological traits were also analyzed by numerical taxonomic techniques using two complementary procedures: cluster and principal component analyses (Sneath and Sokal, 1973). To avoid effects due to scaling differences, means of each character were standardized prior to cluster and principal component analyses using Z-scores. Estimates of Euclidean distance coefficients were made for all pairs of varieties. The resulting Euclidean dissimilarity coefficient matrices were used to evaluate the relationships between the entries with a cluster analysis using complete linkage method (NTSys, version 2.1). Principal component analysis was also performed with the same data matrix. Scatter plots of first three principal components were produced to provide a graphical representation of the pattern of variation among all the traditional varieties and improved cultivars, and landrace genotypes of rice (Statistica, version 6.0).

Table 2.1: Agro-morphological traits recorded for the cultivars and landrace genotypes



Description of the Trait

Quantitative Traits:

Days to 50% flowering (DF)


Number of days from transplanting to heading (50% of plants are starting heading).

Days to maturity (DM)


Number of days from transplanting to grain ripening (85% of grains on panicle are mature).

Leaf length (LL)


Actual measurements (cm) of the leaf just below flag leaf.

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Leaf width (LW)


Actual measurements (cm) of the widest portion of leaf blade just below the flag leaf.

Leaf area (LA)


Leaf length x Leaf width x 0.76

Productive tillers plant-1 (PT/P)


Total number of tillers bearing panicle with filled grains.

Plant height (PH)


Actual measurement (cm) from soil surface to tip of the tallest panicle (awns excluded). Record in whole numbers.

Panicle length (PL)


Actual measurements (cm) from panicle base to tip.

Spikelets panicle-1 (S/P)


Total number of spikelets counted on main panicle.

Seed setting percentage (SS)


Identify the fertile spikelets by pressing spikelets with fingers and noting those that do not have grains.

Grain yield plant-1 (GY/P)


Grain weight of individual plant at 13% moisture content.

Straw yield plant-1 (GY/P)


Straw weight of individual plant after drying in sunlight.

100-seed weight (100-SW)


Enter measurements in grams of 100 well-developed whole grains, dried to 13% moisture content.

Paddy grain length (PGL)


Enter the actual measurement of length in millimeters as the distance from the base to the tip of the paddy grain.

Paddy grain width (PGW)


Actual measurement of width (mm) as the distance across fertile lemma and the palea at the widest point.

Qualitative Traits:

Flag leaf angle (FLA)


1 = Erect, 3 = Intermediate, 5 = Horizontal, 7 = Descending

Leaf shape (LS)


1 = Erect, 2 = Semi-erect, 3 = Droopy

Leaf appearance (LA)


1 = Narrow, 2 = Intermediate, 3 = Broad

Lodging incidence (Lg)


1 = Heavy-lodging, 2 = Slight-lodging, 3 = Absent

Panicle exertion (PEx)


1 = Well exerted, 2 = Moderately well exerted, 3 = Just exerted, 4 = Partly exerted, 5 = Enclosed

Panicle type (PT)


1 = Compact, 2 = Intermediate, 3 = Open

Awning (An)


1 = Awned, 2 = Awnletted, 3 = Awnless

Awn color (AC)


1 = No awn, 2 = White, 3 = Light-brown, 4 = Brown, 5 = Dark-brown, 6 = Red

Seed coat (bran) color (SCC)


1 = White, 2 = Light brown, 3 = Speckled brown, 4 = Dark-brown, 5 = Red, 6 = Blackish-brown, 7 = Purple


2.2: Biochemical basis of genetic diversity

2.2.1: SDS-PAGE

Molecular evaluation involves the use of molecular techniques for assessing genetic diversity of plant germplasm and identification of molecular markers for crop improvements. Healthy and mature seed of 35 commercial varieties and primitive cultivars including two control varieties (Appendix 2) was used for molecular analysis of total seed protein. SDS-PAGE technique was used to identify molecular diversity of rice commercial varieties available. Different molecular level characteristics were studied.

Diversity of total seed protein of all 40 varieties and primitive cultivars were checked in laboratory phase. Electrophoresis was carried out in the discontinuous Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) system of Leammli (1970) using 15% (w/v) separating gel and 4.5% (w/v) stacking gel (Walter et al., 2003). Total Seed Protein Analysis: SDS-PAGE Electrophoresis

In Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) separations of polypeptides, migration is determined by molecular weight. Sodium Dodecylsulphate (SDS) is an anionic detergent that denatures proteins by wrapping the hydrophobic tail around the polypeptide backbone. For almost all proteins, SDS binds at a ratio of approximately 1.4g SDS per gram of protein, thus conferring a net negative charge to the polypeptide in proportion to its length. The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds the protein molecules, minimizing differences in molecular form by eliminating the tertiary and secondary structures. The proteins can be totally unfolded when a reducing agent is employed. The SDS denatured and reduced polypeptides are flexible rods with uniform negative charge per unit length. Thus, because molecular weight is essentially a linear function of peptide chain length, in sieving gels the proteins separate by molecular weight.

In a discontinuous system, a non restrictive large-pore gel called a stacking gel is layered on top of a separating (resolving) gel. The two gel layers are each made with a different buffer, and the tank buffers differ from the gel buffers. In this system the protein mobility, a quantitative measure of the migration rate of a charged species in an electric field, is intermediate between the mobility of the buffer ion of the same charge (usually negative) in the stacking gel (leading ion) and the mobility of buffer ion in the upper tank (trailing ion). When electrophoresis is started, the ions and the proteins begin migrating into the stacking gel. The proteins concentrate in a very thin zone, called the stack, between the leading ion and trailing ion. The proteins continue to migrate in the stack until they reach the separating gel. At that point, due to a pH or an ion change, proteins become the trailing ion and "unstuck" as they separate on the gel. Denaturing gel electrophoresis can resolve complex protein mixtures into hundreds of bands on a gel. Preparation of Buffers

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Following buffers were utilized for protein extraction and SDS-PAGE electrophoresis.

Protein Extraction Buffer

(0.05 M Tris-HCl pH 8.0, 0.2% SDS, 5M Urea, 1% ß-mercaptoethanol)

Tris 0.6057g

Sodium Dodecylsulphate (SDS)* 0.2g

Urea* 30.3g

Distilled water about 70ml

HCl (conc.) Adjust to pH 8.0

2-Mercaptoethanol 1ml

Total volume of 100ml

A little bit Bromophenol blue (BPB) was added. Buffer solution was stored in a refrigerator.

Tris; Tris (hydroxymethyl) aminomethane

*SDS and urea solubilize and denature proteins. Solutions for Electrophoresis

Solution A

(3.0 M Tris-HCl pH 9.0, 0.4% SDS)

Tris 36.3g

SDS 0.4g

Distilled water About 70 ml

HCl (conc.) Adjusted to pH 8.8

Total volume of 100ml

Stored in a refrigerator

Solution B

(0.493 M Tris-HCl pH 7.0, 0.4% SDS)

Tris 5.98 g

SDS 0.4 g

Distilled water About 80 ml

HCl (conc.) Adjusted to pH 7.0

Stored in a refrigerator

Solution C

(30% Acryl amide, Acrylamide/Bis = 30: 0.8)

Acryl amide* 30g

Bis-acrylamide (Bis)* 0.8g

Distilled water Total volume of 100 ml

Stored in refrigerator

*Acryl amide and Bis-acrylamide are highly toxic and carcinogenic. Gloves were used while preparing solution using these reagents.

10% APS

Ammonium Per sulfate (APS) 0.1g

Distilled water Total volume 1 ml

Can be stored in a refrigerator for several days but it was prepared fresh all the times for better performance.

Electrode Buffer Solution

(0.025 M Tris, 0.129 M Glycine, 0.125% SDS)

Tris 3.0g

Glycine 14.4g

SDS 1.25g

Distilled water Total volume of 1000 ml

Stored at room temperature

Staining Solution

Methanol 440 ml

Acetic Acid 60 ml

Distilled water 500 ml

Coomassie Brilliant Blue (CBB)* R250 2.25g

Total volume of 1litre

Solution was stirred for 30 minutes and then filtered, stored at room temperature. *CBB is a protein staining dye.

Destaining Solution

Methanol 200 ml

Acetic Acid 50 ml

Distilled water 750 ml

Total volume 1 litre

Stored at room temperature Preparation of Seed Samples

Single seed of each variety and primitive cultivars was taken, crushed and grinded in mortar and pestle. 10mg (0.01g) seed flour was weighed by an electronic balance and put into 1.5ml micro tube. After each sample weighing mortar and pestle were cleaned with great care so that there should not be even a single particle of last seed flour. To extract proteins from flour, 500µl of the protein extraction buffer was put into the micro tube and mixed well by the test tube mixer (vortex). This sample was preserved in a freezer (- 20°C). Preparation of Electrophoretic Gel

Glass plates used for electrophoresis were cleaned up from internal side with 80% Ethanol and Kim wipe. Gaskets were used for sealing to the glass plates with spacer; it was kept in mind that gaskets should not overlap with spacer of plates. Sets of glass plates were fixed with double clips and marked 2cm from the top. To make sure that there is no leakage; glass plate set ups were filled wit water and placed for some time (Fig 2.2).

Figure 2.2: Electrophoretic Gel Assembly

Following separation gel solution was prepared after setting up the apparatus;

Separation Gel with 1mm thickness (For two mini gels)

Separation gel 15 %

Solution A 5ml

Solution C 10 ml

10% APS 200µl

Distilled water 5 ml

TEMED 15µl

TEMED (N-N-N-N-Tetramethylethylenediamine) was added at the end and shaken well.

Separation gel was put into the space between a set of glass plates (up to 2cm from the top). Small amount of distilled water (120µl) was added on separation gel gently to prevent gel surface from air and promote fixation. The set up was left for 30 minutes so that gel was fixed. During the fixation time of separation gel, stacking gel was prepared.

Stacking Gel (For two mini gels)

Stacking gel 4.5%

Solution B 2.5ml

Solution C 1.5ml

10% APS 70µl

Distilled water 6.0ml

TEMED 17µl

TEMED was added at the end and shaken well.

When separation gel was fixed, distilled water was removed from its top and stacking gel solution poured on it. Combs were fixed into the stacking gel. Combs were put with special care and it was confirmed that there was no any air bubble at the bottom of the combs. The set up was left for 15 minutes so that the stacking solution became gel. Combs, clips and gaskets were removed from glass plates carefully and confirmed there was no any air bubble at this stage. Gel plates were freshly used for electrophoresis but is was also possible that these would be wrapped in aluminum foil and could be used even for one week. Electrophoresis

Electrophoresis procedure was carried out using slab type SDS-PAGE model: AE-6530M, ATTA Japan, with 15% polyacrylamide gel. The molecular weight of dissociated proteins was estimated by using molecular weight standard proteins "MW-SDS-70 Kit".

Electrode buffer solution was put into the bottom pool of the apparatus. Gel plates were placed in the apparatus, here again air bubble formation was avoided. Electrode buffer solution was also put into the top pool of the apparatus; wells formed by combs were washed by syringe. Seed samples were centrifuged at 15,000 rpm for 10 minutes, 15 µl of supernatant was put into wells with the help of micropipette. Protein molecular weight marker was put in first well of each glass plate. The numbering of seed samples and wells were noted to avoid repetition. The apparatus was connected with + (red) and - (black) electrodes of power supply. The voltage of apparatus was kept constant at 100V and apparatus was left until a blue line of BPB came at the bottom of the gel plates. Detection of Proteins

(Staining and De-staining of Separation gel)

When blue line reached at the bottom of the gel plates, electric supply was disconnected. Gel plates were taken out from the apparatus and separated by spatula. Stacking gel was removed with the help of same spatula. Separation gel was put in the box which contained staining solution. Box was put on the shaker for two hours. Staining solution was exchanged by destaining solution and the box was shaked gently almost overnight until the background of the gel disappeared to absorb excess CBB, a piece of Kim wipe was put in the destaining solution to check absorbance. Drying of separation Gel

Wet filter paper was placed on the plate of gel dryer. Separation gel was carefully placed on the paper and covered with a wrap. It was dried in a drier for about 1.5 hours at 60°C. When gel sheet was completely dried it was taken out while the pump was still running. All gels were dried with the same manner. Data Analysis

Depending upon the presence or absence of polypeptide bands, similarity index was calculated for all possible pairs of protein types. To avoid taxonomic weighing, the intensity of bands was not taken into consideration rather only the presence of bands was taken as indicative. The score was '1' for presence and '0' for the absence of bands. Presence and absence of bands were entered in a binary data matrix. Based on result of electrophoresis band spectra, Nei & Li's similarity matrix was calculated for all possible pairs of protein type's electrophoregrams by the following formula (Sneath and Sokal, 1973).

S=W/ (A+B-W)

Where 'W' is the number of bands of common mobility, 'A' the number of bands in protein type A and B is the number of bands in protein type B. The similarity matrix thus generated was converted to a dissimilarity matrix (Dissimilarity = 1- Similarity) and used to construct dendrogram by the un-weighed pair group method with arithmetic means (Sneath and Sokal, 1973). All computations were carried out using the NTSYS-pc, Version 2.1 package (Rohlf 2000, Applied Biostatistics Inc., Exeter Software, NY, and USA).

2.2.2: Molecular basis of genetic diversity Plant Materials

Initially around 75 selected accessions (Appendix 2) and 35 commercial varieties of rice were used as starting material for molecular characterization. DNA Extraction from Dry Seed Samples

Total genomic DNA was also extracted from dried seeds of each cultivar according to the method described by Kang et al. (1998) with minor modifications which appears to be more useful in saving cost of extraction, labor and time being used while extracting DNA from seedling samples:

Remove seed coat and place 3-5 seeds containing the storage tissue in a micro centrifuge tube (1.5ml).

Add 400µl of extraction buffer (200mM Tris-HCl (pH 8.0), 25mM EDTA, 200mM NaCl, 0.5% SDS) containing Proteinase K (50µg).

Incubate at 37oC for 1 hour. Grind seeds in the buffer with a glass rod.

Add 400µl of 2%CTAB solution (100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4M NaCl, 2% CTAB (w/v), 1% PVP "polyvinylpyrrolidone 40,000).

Gently extract using chloroform: isoamyl alcohol (24:1) with 5% phenol.

Centrifuge at 12,000rpm for 10 min at 4oC and transfer supernatant into new tubes.

Add â…” volume of Isopropanol and incubate tubes at room temperature for 10 minutes to precipitate DNA.

Centrifuge tubes at 12,000 rpm for 5 minutes and remove supernatant.

Wash DNA pellet with 70% Ethanol (500µl). Centrifuge at 12,000 rpm for 5 minutes at room temperature and pour off 70% Ethanol.

Air dry DNA pellet for 5-10 minutes and re-suspend in 100µl of TE buffer.

Remove RNA by adding 1µl of RNase (10mg/ml).

After isolation of DNA from dried seed samples, DNA concentration and purity of each variety and primitive cultivar genotype was determined spectrophotometrically at a wavelength of 260 and 280nm using NanoDrop ND-1000 Spectrophotometer. The ratio between absorbance at 260 and 280nm (260/280) was used to estimate DNA purity. DNA of each cultivar was diluted to a working concentration of 20ng/µl for PCR analysis.


A modified RAPD method based on Williams et al (1990) was used with a model 9700 thermal cycler (Applied Biosystems, USA). To establish RAPD protocols for rice, PCR analysis was performed by changing and checking the concentrations of total genomic DNA from 5~50ng/20µl reaction volume, MgCl2 from 1.5~3.0mM, dNTPs mixture from 100~400µM each, random primer from 0.1~1.0µM and Taq DNA polymerase from 0.2~1.25 units. After standardization of PCR, 20µl reaction mixture containing 1x PCR buffer [10mM Tris HCl (pH 8.3), 50mM KCl], 1.5mM MgCl2, 200µM each deoxynucleotide triphosphate (dNTP), 0.4µM of 10-mer primer (Operon Technologies Inc., Alameda, CA), 1 unit AmpliTaq Gold DNA polymerase and approximately 20ng of template DNA was found optimum for the amplification of rice genomic DNA (Table 2.2). Taq DNA polymerase and reaction buffer were purchased from Applied Biosystems, Japan. DNA amplification was performed in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, USA). The thermal cycler was programmed to 1 cycle of 5 minutes at 94oC for initial strand separation. This was followed by 45 cycles of 1 minute at 94oC for denaturation, 1 minute at 36oC for annealing and 2 minutes at 72oC for primer extension. Finally, 1 cycle of 7 minutes at 72oC was used for final extension, followed by soaking at 40oC (Table 2.3). The reproducibility of the amplification products was checked twice for each experiment. Primer Selection

Initially, three cultivars one each from aromatic, non-aromatic and japonica type was used to optimize the RAPD protocols and select the suitable primers which exhibit polymorphisms between the three cultivars. Altogether, forty arbitrary decamer oligonucleotides, belonging to kit OPA and OPB from Operon Technologies Inc. (Alameda, California, USA), were tested as single primers to identify the most promising ones for detecting polymorphism. After an initial screen, thirty-two primers were ultimately chosen for further use on the basis of their ability to detect the polymorphism and produce the reliable and easily scorable banding patterns in rice cultivars. Among them, 7 primers could not amplify the DNA from some of the cultivars used. Therefore, finally the data of twenty-five primers were used and compiled to examine the genetic diversity and relationship among 40 commercial varieties and primitive cultivars of Pakistani rice. Electrophoresis of Amplified Products

After amplification, 3µl of gel loading dye buffer (0.02% Bromophenol blue, 0.02% xylene cyanol FF, 50% glycerol and 1% SDS) were added directly to the reaction tubes and spun for few seconds in a micro centrifuge after mixing with the entire reaction mixtures. Aliquots of 15µl of amplification products plus loading dye were then loaded in 1.5% agarose gels for electrophoresis in 1 x TBE (10mM Tris-Borate, 1mM EDTA) buffer and run at 100V for 40 minutes to separate the amplified products. 1kb plus was used as a molecular size weight marker. After electrophoresis, the gels were photographed under UV light using black and white film # 667 (Polaroid, Cambridge, Mass., USA). Data Analysis

Photographs from ethidium bromide stained agarose gels were used to score the data for RAPD analysis. Each DNA fragment amplified by a given primer was treated as a unit character and the RAPD fragments were scored as present (1) or absent (0) for each of the primer-cultivar combinations. Bands were scored from the top of the gel (band number 1) to the bottom. The left lane of the gel was considered as lane-1. Since DNA samples consisted of a bulk sample of DNA extracted from 5~10 seeds, a low intensity for any particular fragment may be explained by the lesser representation of that specific sequence in the bulk sample of DNA. Therefore, the intensity of the bands was not taken into account and the fragments with the identical mobility were considered to be the identical fragments. Only major bands were scored and faint bands were not considered. The molecular size of the amplification products was calculated from a standard curve based on the known size of DNA fragments of a 1kb plus molecular size weight marker. The presence and absence of the bands was scored in a binary data matrix. Pair-wise comparisons of the cultivars based on the presence or absence of unique and shared amplification products were used to generate similarity coefficients. Estimates of genetic similarity (F) were calculated between all pairs of the cultivars by the Dice algorithm. The Dice algorithm is identical to that of Nei and Li (1979) as follows:

Similarity (F) = 2Nab/ (Na + Nb)

Where Na = the number of scored fragments of individual 'a',

Nb = the number of scored fragments detected in individual 'b' and

Nab = the number of shared fragments between individuals 'a' and 'b'.

The resulting similarity coefficients were used to evaluate the relationships among commercial varieties and primitive cultivars with a cluster analysis using an un-weighted pair-group method with arithmetic averages (UPGMA) and then plotted in the form of a dendrogram. All computations were carried out using the computer program NTSYS, version 2.1 (Applied Biostatistics Inc., USA).

EXPERIMENT -IV Microsatellite or Simple Sequence Repeat (SSR) Analysis

Thirty five primer pairs covering all twelve chromosomes were selected for the genetic diversity analysis on the basis of published rice microsatellite framework map. Three primers (RM5, RM210 and RM229) exhibited monomorphic fragments and were therefore excluded from further analysis. The original source, repeat motifs, primer sequences and chromosomal positions for these markers can be found in the rice genome database ( Microsatellite primer pairs were obtained from Hokkaido Science System (Sapporo, Hokkaido, Japan).

SSR analysis was performed following the protocol of Ravi et al. (2003) with minor modifications. PCR amplification reactions were carried out in a total volume of 20µl containing; 10mM Tris HCl (pH 8.3); 50mM KCl; 1.5mM MgCl2; 200µM each of deoxynucleotide triphosphate (dNTP); 0.2µM of each forward and reverse primer; 1 unit Taq DNA polymerase (Fermentas Life Sciences); and 20ng of template DNA. The PCR amplifications were carried out using a MyGene Series Peltier Thermal Cycler (UniEquip GmbH, Munich, Germany). Thermal cycler was programmed to 1 cycle of 5 min at 94oC as an initial hot start and strand separation step. This was followed by 35 cycles of 1 min at 94oC for denaturation, 1 min for annealing temperature depending on the marker used (55oC - 65oC) and 2 min at 72oC for primer elongation. Finally, 1 cycle of 7 min at 72oC was used for final extension. Amplified products were stored at -20oC until further use. The reproducibility of the amplification products was checked twice for each primer.

Electrophoresis of amplified products: After amplification, a 15μl aliquot of the amplified SSR samples was combined with 3μl of a loading buffer (0.4% (w/v) bromo-phenol blue, 0.4% (w/v) xylene cyanole and 5 ml of glycerol) and was analyzed directly on 3% (w/v) Gene Choice High Resolution agarose (CLP, USA) gels in 1xTBE buffer (10mM Tris-Borate, 1mM EDTA) containing 0.5µg per ml of ethidium bromide. A 25bp DNA ladder (Biolabs, New England, UK) was used as a size marker to compare the molecular weights of amplified products. After electrophoresis, the gels were documented using an UVI Doc Gel Documentation System (UVITEC, Cambridge, UK).

Allele scoring and data analysis: Ethidium bromide staining of agarose gels generally showed several bands. The size of the most intensively amplified band for each microsatellite marker was determined based on its electrophoretic mobility relative to molecular weight markers (increments of 25bp). Amplified products from SSR analysis were scored qualitatively for presence and absence of each marker allele-genotype combination. Each SSR band amplified by a given primer was treated as a unit character. Data was entered into a binary matrix as discrete variables, 1 for presence and 0 for absence of the character. The most informative primers were selected based on the extent of polymorphism. The polymorphic information content (PIC) value of a marker was calculated according to Anderson et al. (1993). Mean allele numbers, PIC values, and genetic similarities were calculated on the basis of different rice landraces, chromosomes and microsatellite classes. Pair-wise comparisons of the genotypes based on the proportion of unique and shared amplification products (alleles) were used to measure the genetic similarity by Dice coefficients using PAST(Paleontological Statistical Software Package for Education and Data Analysis) program (Hammer et al., 2001). Genetic similarities (F) between all pair of the landraces were calculated according to Nei and Li (1979). A dendrogram was constructed using pair-group method to get genetic relationships among landraces. The reliability of the dendogram was tested by bootstrap analyses with 10,000 replications to assess branch support. Some workers consider that the confidence limits obtained in bootstrap must be over 95% in order to consider the grouping of taxa (a group of genetically similar organisms that are classified together as e.g. species, genus, or family) at a branch to be statistically significant (Felsenstein, 1985). Others use a lower limit (above 50% or at least 50%) as indicating statistical support for the topology at a node (Highton, 1993). In our study we used the lower limits to assess grouping of taxa to be statistically significant because we observed that as the number of test sample increases the confidence interval decreases.