Identification of Rice (Oryza sativa L.) Varieties ...

Indian JIndian Agric Biochem 22 (1), 18-25, 18 J Agric Biochem 22 (1),2009 2009

Identification of Rice (Oryza sativa L.) Varieties through Biochemical Markers DIWAKAR SINGH”, REETU MEHTA and JG TALATI

Fourteen rice varieties were compared for their biochemical markers viz., activity of peroxidase, esterase, acid phosphatase, polyphenol oxidase and ∝–amylase; isozyme analysis of peroxidase, esterase and acid phosphatase at different growth stages and SDS-PAGE profiles from Tris-HCl buffer soluble seed and leaf proteins. Peroxidase, polyphenol oxidase and ∝–amylase enzyme activity showed significant differences and could be applied for varietal identification, while acid phosphatase and esterase activity could not provide useful information regarding varietal identification. Peroxidase (5 and 10 DAG) and esterase (5, 10 and 20 DAG) isozymes were found useful for identification of varieties as well as parents and hybrid and served as marker isozymes for rice varieties. Similarly, SDS-PAGE of seed storage proteins and leaf proteins (5 DAG) showed variability and could be effectively used for identification of rice varieties. Key words: Rice, biochemical markers, peroxidase, esterase, acid phosphatase, ∝–amylase, polyphenol oxidase, electrophoresis, SDS-PAGE, isozymes Downloaded From IP - 202.41.108.114 on dated 29-Aug-2009

There are approximately 10,000 rice varieties throughout

been developed for varietal identification. These techniques

the world. Identification of the variety has been the major

have been found useful to characterize/identify a variety more

objective in seed analysis and is a major area of interest

accurately and to check the purity of hybrids. In view of the

for plant breeders, plant pathologists, seed technologists

above, present study was planned with the objective to find

and plant biochemists. Varietal identification is an important

out the marker enzyme activities and isozyme

criterion for registration of the newly developed variety by a

characteristics of particular genotype/variety and to find out

plant breeder to ensure the ultimate consumer for the

the proper stage of seedling and biochemical marker, where

availability of true to the type especially when the product

variations occur in male, female and hybrid genotypes.

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National Research Center for Groundnut, Junagadh-362001, Gujarat 1 Department of Biochemistry, BA College of Agriculture, Anand Agricultural University, Anand - 388 110, Gujarat

will be used for mechanized processing and to ensure the plant breeder for availability of pure parent in hybrid

Materials and Methods

development programme. Varietal purity testing is an

Rice seeds of different varieties viz., P-203, Gurjari, Jaya,

integral part of seed quality evaluation and control.

GR-6, GR-7, GR-11, GR-101, Krishna Kamod, IET-16310,

Increased number of new varieties/hybrids/transgenics and

NWGR-98124, NWGR-99004, genotypes viz., IR 58025 A,

the anticipated impact of biotechnology on varietal

IR 58025 B and hybrid GRH-1 were procured from Main

development will exacerbate the ability of seed analysts to

Rice Research Station, Navagam, Anand Agricultural

differentiate varieties by traditional approaches (1).

University. Seeds of all the cultivars were germinated under

Morphological descriptors that are widely used are time

controlled environment and leaves from 5, 10, 20 and 30

consuming and expensive. Further, many of the

days after germination (DAG) and 30 DOS were collected

morphological descriptors used are multigenic, quantitative

for enzyme and protein extraction. In order to get the extract

or continuous characters, the expression of which are

of peroxidase, esterase and polyphenol oxidase the rice

altered by environmental factors and the analysis of which

seedlings (1g) were ground with glass powder in cold with

requires statistical tests (1). To overcome the limitations of

0.1 M phosphate buffer (pH 7.2) containing 1 mM PVPP and

morphological descriptors few rapid and less expensive

10% glycerol. The homogenate was centrifuged at 10,000

laboratory techniques, such as, modified phenol test (2),

g for 20 minutes at 40C and the supernatant containing

protein electrophoresis (3, 4) and isozyme analysis (5) have

50 µg of protein was used for enzyme assay and

*Author for correspondence : Email : [email protected]

19

electrophoresis. Extracts of acid phosphatase were

also to differentiate indica x japonica, indica and japonica

obtained by homogenizing rice seedlings (1g) with ice cold

cultivars. In present investigation we observed wide

50 mM Citrate buffer, (pH 5.3) in a pre-chilled pestle and

variations in rice cultivars at different stages of growth for

mortar. The homogenate was filtered through four layers of

different enzymes viz., peroxidase, esterase, acid

cheese cloth and filtrate was centrifuged at 10,000 g for 10

phosphatase, polyphenol oxidase and ∝–amylase (Fig. 1

minutes at 40C. The supernatant was used for enzyme assay

to 5). Maximum variation for peroxidase activity among the

and electrophoresis. ∝-amylase extracts were obtained by

varieties/genotypes has been observed at 5 and 10 DAG

homogenizing rice seedlings (1g) with 1.5% NaCl and

(Fig. 1) and appeared to be variety specific. Little variations

homogenate was centrifuged at 5000 g for 15 minutes and

were observed for esterase among the varieties at all growth

the supernatant was used for enzyme assay. Total soluble

stages studied (Fig. 2), indicating that esterase enzyme

proteins were extracted from the defatted seed samples

activity does not give much variation for the rice varieties

(1g) and leaves (1g) using 50 mM Tris–HCl buffer (pH 6.8)

under study. The acid phosphatase enzyme activity was

containing 2% sodium dodecyl sulphate (SDS) and 1%

found to be maximum at 20 and 30 DAG but variations

β-mercaptoethanol. Protein estimation was done by Lowry’s

among the varieties/genotypes were very less (Fig. 3) to

method (6). SDS-PAGE was performed using 12%

distinguish all rice varieties/genotypes under study. Highest

acrylamide as separating slab gel and 4% acrylamide as

activitiy as well as maximum variations for polyphenol

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stacking gel according to the method described by Laemmli

oxidase were observed at 10 DAG (Fig. 4) and could be

(7) with some modifications. An aliquot (15 µl) was mixed

used for distinguishing the rice varieties. Maximum

with 2x sample buffer (0.25 M Tris-HCl, pH6.8, 0.2% SDS,



Identification of Rice Varieties through Biochemical Markers

10% glycerol, 10% β-mercaptoethanol and 0.002%

∝–amylase activity was observed at 30 DAG. Among all fourteen varieties, variety specific ∝–amylase activity was

bromophenol blue). All enzymes, viz., peroxidase (EC

recorded viz., IR 58025 A showed maximum ∝–amylase

1.11.1.7), esterase (EC 3.1.1.2), acid phosphatase (EC

activity at all growth stages (Fig. 5) and could be used as

3.1.3.2), polyphenol oxidase (EC 1.14.18.1) and ∝–amylase

marker enzyme to distinguish it from rest of the varieties.

(EC 3.2.1.1) were assayed by the methods described by Sadasivam and Manikam (8). Isozyme electrophoresis was conducted following the method described by Laemmli (7) on 7% resolving gel. The gels were stained with odianisidine dye for peroxidase (8), alpha-naphthyl acetate for esterase (9) and alpha-naphthyl phosphate for acid phosphatase (10). Protein electrophoresis was conducted at a constant current of 20mA for all lanes for about 1hr and thereafter, at 30mA until the tracking dye; bromophenol blue reached the bottom of the gel. After proper staining and destaining of the gel, the protein bands were assigned

Sivasubramanian and Ramakrishnan (13) also reported the usefulness of peroxidase and polyphenol oxidase activity for rice varietal identification and classified 14 rice varieties into 10 different groups. Kang and Nam (14) studied 5 rice cultivars each of indica x japonica, indica and japonica rice for varietal differences on the basis of peroxidase activity and banding pattern. They found that peroxidase activity from leaf blades could differentiate indica and indica x japonica cultivars. Abrol and Uprety (15) also reported that polyphenol oxidase activity could be the basis for grouping of rice varieties.

with numbers depending upon their relative mobilities (Rm).

Glaszmann (16) reported that Isozymes of plant tissues

Dendogram was prepared using phylip 3.6 version (11).

are useful biochemical index reflecting changes in

The data were subjected to statistical analysis of variance

metabolic activity during growth development and

for completely randomized design as described by

differentiation. The isozyme electrophoresis has been

Snedecor and Cochran (12).

applied extensively for the identification of plant cultivars.

Results and Discussion

The differences in the banding pattern provide accurate and reliable results and hence it proved to be a useful and

Activity of Enzyme, viz., peroxidase, esterase, acid

rapid technique for varietal identification. All fourteen varieties

phosphatase, polyphenol oxidase, ∝–amylase, etc. has

were tested at four different growth stages viz., 5, 10, 20

been found useful to identify and classify rice cultivars and

and 30 DAG.

Indian J Agric Biochem 22 (1), 2009

Fig. 1: Peroxidase activity at different stages of rice seedling


Fig. 2: Esterase activity at different stages of rice seedling Downloaded From IP - 202.41.108.114 on dated 29-Aug-2009


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Fig. 3: Acid Phosphatase activity at different stages of rice seedling

Fig. 4: Polyphenol oxidase activity at different stages of rice seedling

Fig. 5: α-Amylase activity at different stages of rice seedling


Peroxidase banding pattern of leaves at 5 DAG were found

parents and hybrid at 5 and 10 DAG. At 30 DAG three bands

to be effective for comparison. At 5 DAG stage four bands

were observed with Rm values 0.04, 0.22 and 0.50 and

were observed in leaves having Rm value of 0.10, 0.13,

these three bands were common for all fourteen varieties

0.18 and 0.42. Banding pattern for 5 DAG leaves (Fig. 9)

(Fig. 12). Thus, with acid phosphatase no polymorphism

showed that band no. 1 (Rm value = 0.10) was present only

could be detected for the varieties, hence, not found useful

in three varieties i.e. GR-101, GRH-1 and NWGR-99004.

for identification of rice varieties studied.

Band no. 2 (Rm value = 0.13) was absent in the varieties P203, Gurjari, GR-11, Krishna Kamod, IR 58025 A, IR 58025 B and NWGR-98124. Parent IR 58025 A and IR 58025 B showed one similar band with Rm value 0.18, which was also present in the hybrid with high intensity. Band no. 4 with Rm value 0.42 was present in the parent IR 58025 A as well as hybrid GRH-1. Thus, the bands, which were present in the parent were also present in the hybrid. Through differences in peroxidase banding patterns, most of the rice varieties could be identified, as clear differences were Downloaded From IP - 202.41.108.114 on dated 29-Aug-2009

visible at 5 DAG (Fig. 9) and also found to be useful for identification of parents and hybrid.

Chu (17) applied the technique of electrophoretic analysis of peroxidase for identification of rice varieties and found to be useful for varietal identification. Kang and Nam (14) studied 5 rice cultivars each of indica x japonica, indica and japonica rice for varietal differences on the basis of peroxidase activity and banding pattern. They found that differences in banding pattern could be used for identification of rice cultivars. Santhy et al. (18) differentiated IR 58025 A line from its B line on the basis of greater intensity of a peroxidase band. Yen (19) studied the identification of the genuineness and purity of F1 hybrids of rice and their parental lines using electrophoretic analysis of esterase

The results of 5 and 10 DAG were comparable and have

and they concluded that the genuineness and purity of both

been discussed. Four different esterase bands were

F1 hybrids and their parental lines may be determined by

observed at 5 DAG (Fig. 10). Band No. 1 (Rm = 0.05) and

the presence or absence of 8 or 9 bands of esterase activity.

band No. 4 (Rm = 0.50) were present in all fourteen varieties

Nakagahra et al. (20) studied isozyme polymorphism of

while, intensity of band No.1 was faint for three varieties (P-

esterase in Oryza sativa L. with leaves of 776 native rice

203, IR 58025 A and GRH-1) and intensity of band No. 4

varieties collected from known sites of Asian countries and

was faint for five varieties (GR-7, GR-11, GR-101, GRH-1

classified all varieties to study the genetic relatedness. They

and IR 58025 B) respectively. Band No. 2 was absent only

concluded that esterase could be used for identification

in P-203, GR-11 and IR 58025 B. IR58025 A and IR 58025 B

and classification of rice varieties. Glaszmann (16) studied

showed 3 bands each, while, the respective hybrid GRH-1

peroxidase and esterase isozymes for rice and found to be

showed all four bands. Band No. 2 was present in IR 58025

useful for variety identification. Munu and Hazarika (21)

A and absent in IR 58025 B but it was present in the hybrid

observed variations among twenty-six rice cultivars through

GRH-1. Thus, differences in banding pattern enabled to

isozyme analysis of peroxidase and esterase utilizing

distinguish the parents and hybrid as well as other varieties.

Polyacrylamide Gel Electrophoresis. Beena et al. (22) also

At 10 DAG four esterase bands were observed (Fig. 11).

observed distinct variation between the parents for the

Band No. 2 (Rm value = 0.11) was common for all fourteen

isozymes of esterase, peroxidase, acid phosphatase and

rice varieties. Eight varieties viz., GR-7, GR-11, GR-101, IET-

superoxide dismutase. Abdel et al. (23) verified cultivar

16310, IR 58025 B, GRH-1, NWGR-98124 and NWGR-

identity in rice using electrophoretic profiles of esterase

99004 possessed common banding pattern for all four

and peroxidase. Santhy et al. (18) also detected highest

bands with Rm value; 0.06, 0.11, 0.49 and 0.75. P-203,

polymorphism in the esterase and peroxidase isozyme

Gurjari, Jaya and GR-6 possessed only three bands with

patterns which differentiated all A and R lines. The greater

Rm values of 0.11, 0.49 and 0.75. IR 58025 A was observed

intensity of a peroxidase band differentiated IR 58025 A

only for two bands with Rm value 0.11 and 0.49. Thus, it

from its B line. Acid phosphatase isozyme was also found

enabled to distinguish most of the varieties form each other.

to be useful for rice variety identification when studied with

Thus, differences in banding pattern of esterase isozymes

21 different Oryza species (24) and for other rice varieties

enabled to distinguish most of the varieties along with

(25).



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http:\\www.IndianJournals.com 22 Indian J Agric Biochem 22 (1), 2009


Protein electrophoresis is one of the more exciting tests,

0.25

0.50

0.75

1.0

officials, particularly because of the increased need for

GR-6 GR-7 IR-58025 A GRH-1 NWGR-99004 NWGR-98124 P-203 KRISHNA KAMOD IR-58025 B JAYA GURJARI GR-11

varietal identification with the advent of the plant protection

GR-101 IET-16310

which may have much potential for varietal distinction, is the SDS-PAGE of proteins that can be used on seeds as well as on other plant parts. It has been used as an aid in varietal identification. Protein electrophoresis has generated much interest among certification and seed quality control

act. Seed storage protein and leaf protein (at 4 different growth stages) have been analyzed using 12% resolving gel through SDS-PAGE. Seed proteins resolved a total of 17 bands of which two bands (Rm = 0.42 and 0.67) were common to all varieties (Fig. 6). Total number of bands present in a variety ranged from 4 (IET-16310) to 14 (P203). Though the varieties differed for the number of polypeptides, polymorphisms were noted mostly in the Downloaded From IP - 202.41.108.114 on dated 29-Aug-2009

molecular weight region of 36 to 18 kD. Several bands were present or absent in genotypes and formed the basis for identification (Fig. 6). For example, band No. 3 (Rm = 0.15)



0.0

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Fig. 13: Dendogram of 14 rice varieties from Tris-HCl soluble seed proteins

IR-58025 B P-203 GURJARI IR-58025 A NWGR-98124 GRH-1 IET-16310 GR-101 NWGR-99004 GR-7 JAYA KRISHNA KAMOD GR-11 GR-6

Fig. 14: Dendogram of 14 rice varieties from Tris-HCl soluble leaf proteins at 5 DAG

was only present in the varieties GR-6 and GR-7. Similarly, band No. 5 (Rm = 0.20), band No. 12 (Rm = 0.61) and band No. 13 (Rm = 0.61) were only absent in the varieties GR-11, IET-16310 and IR 58025 B, respectively. The dendrogram was prepared (Fig. 13-15) using phylip 3.6 version (11) in order to evaluate the degree of closeness among different genotypes. The dendrogram showed similarity from 44% (P-203 and IET-16310) to 100% (NWGR98124 and NWGR-99004 and Gurjari and Jaya). Relatively higher SI value was also obtained between cultivar pair IR

GR-11 P-203 KRISHNA KAMOD GR-101 IET-16310 JAYA GURJARI IR-58025 A GRH-1 NWGR-98124 NWGR-99004 IR-58025 B GR-7 GR-6

Fig. 15: Dendogram of 14 rice varieties from Tris-HCl soluble leaf proteins at 20 DAG

58025 A and GRH-1 (96%). The high SI value indicated a closeness which may be either due to common parentage

conveniently used for variety identification. Peruanskii and

or accumulation of similar genes from different parents in

Savich (27) used alcohol soluble seed proteins for

the development of varieties, e.g. IR-58025 A and GRH-1

identification of rice varieties. Buquing et al. (28) analyzed

which showed 96% SI value, where GRH-1 is the hybrid

seed protein with PAGE for identification of hybrid rice

and IR 58025 A is parental line. Sengupta and

genotypes. Rice seedling (5 DAG) electrophoresis showed

Chattopadhyay (26) identified 12 rice varieties (Oryza sativa

total 18 bands out of which 4 bands were common to all

indica) on the basis of SDS-PAGE of Tris-Cl soluble seed

varieties (Fig. 7). Total number of bands present in a variety

proteins using 12% gel. Sarkar and Bose (4) identified rice

ranged from 8 (Jaya) to 17 (NWGR-99004). Mostly

varieties using single seed (salt soluble) proteins. They

polymorphisms were observed in the molecular weight

found that the banding patterns appear to be unique for

region 45 to 18 kD (Fig. 7). On the basis of presence or

each of the varieties investigated and same to be genetical

absence of particular bands the varieties were identified,

in nature, as they remain constant under different

for example, band No. 9 (Rm = 0.41) was present only in

environmental conditions, and therefore could be

three varieties viz., IET-16310, NWGR-98124 and NWGR-

Indian J Agric Biochem 22 (1), 2009

99004. Band No. 18 was absent only in variety GR-101.

PAGE technique on the basis of leaf protein patterns of rice

The similarity index (SI) was worked out for the evaluation

varieties using SDS-PAGE. Gonzalez et al (30) identified

of degree of closeness among different genotypes. SI

and characterized 19 rice accessions using leaf proteins

values were in the range of 47% (Jaya and GR-101) to 97%

with SDS-PAGE. On the basis of protein pattern and some

(IET-16310 and NWGR-99004). Relatively higher SI value

isozymes banding pattern they grouped the accessions

was also obtained between cultivar pairs P-203 and Gurjari

into 10 groups, with the main similarities in groups IV and

(96%) and P-203 and IR 58025 B (96%). The

V. They found differences in electrophoretic pattern of protein

electrophoresis of leaf protein at 10 DAG showed maximum

and concluded that it could be useful for identification of

similarity with the results of 5 DAG for both banding pattern

rice cultivars. Variety specific banding patterns have also

as well as SI value. Electrophoresis of leaf protein at 20

been found for salt soluble (4) and alcohol soluble (27)

DAG from rice seedling resolved a total of 22 bands of

seed proteins of rice. Similar trends were also reported by

which 9 bands were common to all varieties (Fig. 8). Total

Sengupta and Chattopadhyay (26) for Tris-Cl soluble seed

number of bands present in a variety ranged from 13 (P-

storage proteins from 12 rice (Oryza sativa var. indica)

203) to 21 (IET-16310) (Fig. 8). On the basis of presence or

cultivars using SDS-PAGE on 12% gels. Chu (17) also

absence of several bands the genotypes were identified.

applied the technique of electrophoretic analysis of proteins

For example band No. 3 (Rm = 0.15) was only absent in the Downloaded From IP - 202.41.108.114 on dated 29-Aug-2009

varieties P-203 and GR-11, Band No. 11 (Rm = 0.42) was

for identification of rice varieties.

only absent in two varieties viz., IR 58025 B and NWGR-

Thus, peroxidase, polyphenol oxidase and ∝-amylase

99004, similarly, band No. 22 (Rm = 0.89) was absent only

enzyme activity showed significant differences and could



24

in the varieties P-203 and IR 58025 B. IET-16310 having

be applied for varietal identification at 5 and 10 DAG, 10

maximum 21 bands, where, only one band, band No. 19

DAG and 20 and 30 DAG, respectively, while, acid

with Rm value of 0.70 was absent. SI value was in the

phosphatase and esterase enzyme activity could not provide

range of 70% (IET-16310 and P-203) to 100% (Gurjari and

useful information regarding varietal identification and thus,

Jaya). Relatively higher SI value was also obtained between

was not found useful. Electrophoresis of peroxidase (5 and

cultivar pairs GR-6 and GR-7 (97%), IR 58025 A and GRH-

10 DAG) and esterase (5, 10 and 20 DAG) isozymes was

1 (97%) and IR 58025 A and IET-16310 (97%). Leaf proteins

useful for identification of varieties as well as identification

at 30 DAG were also resolved and the results, showed

of parents and hybrid and may serve as marker isozyme for

maximum similarity with the results of 20 DAG. Variations

rice varieties. Similarly, SDS-PAGE of seed storage protein

in band intensity observed within each electrophoregram

and leaf protein (5 DAG) showed variability and could be

which may be attributed to large variations in the amount of

effectively used for identification of rice varieties on the basis

various polypeptides present in extract. Most of the

of variations in banding pattern and intensity of bands. All

genotypes were identified on the basis of either presence

the tests conclusively proved that SDS-PAGE and isozyme

or absence of bands. Hybrid and parents were also

electrophoresis offer a potentially quick, simple, rapid and

identified on the basis of higher SI value, e.g. 96% SI value

reliable method for rice varieties identification and

and 97% SI value for the pair IR 58025 A and GRH-1 (hybrid)

recognition of lines. SDS-PAGE was more useful than

which was calculated from seed protein and leaf protein

isozyme electrophoresis because potential number of loci

(20 DAG), respectively. The above results indicated that

were more as compared to isozymes.

rice varieties, hybrid and parents could be identified from seed proteins and leaf proteins at 5 DAG. At 20 DAG less

Acknowledgement

polymorphism was detected, where SI value ranged from

The authors express their sincere gratitude to Dr. A.R.

70% to 100%, thus, at these stages leaf proteins do not

Pathak, Senior Research Scientist and Head, Main Rice

provide much information regarding varietal identification,

Research Station, Navagam, AAU, Anand for providing seed

while, could be useful for identification of parents and hybrid.

material. Thanks are also due to Dr. G.C. Jadeja, Prof and

Park and Stegmann (29) also identified rice varieties through

Head, Dept. of Plant Breeding and Genetics, AAU, Anand


and Dr. Atul Mehta, Asso. Prof., Dept. of Plant Breeding and

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Genetics, Navagam, AAU, for useful comments. Received April 23, 2005; accepted February 20, 2009

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