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Chapter III

MATERIALS AND METHODS

The present investigation entitled “Genetic analysis of morphological, biochemical

and molecular traits of some novelty rices of Himachal Pradesh” was carried out at the

experimental farm of Rice and Wheat Research Centre, (RWRC) of Himachal Pradesh Krishi

Vishvavidyalaya situated at Malan (Kangra) during kharif 2005 and 2006. The details of the

materials used and methods employed in the present investigation are described in this chapter

under the following heads:

3.1 General description of the experimental site

3.2 Materials

3.3 Methods

3.3.1 Field evaluation of the experimental material

3.3.2 Observations recorded

3.3.2.1 Morpho-physiological traits

3.3.2.2 Morphological markers

3.3.2.3 Cooking, eating quality and nutritional traits

3.3.3 Statistical analysis

3.3.4 Molecular characterization

3.1 Experimental site

The experimental site at RWRC, Malan is situated at an elevation of 950 m above

mean sea level with 32o07' N latitude and 76

o23' E longitude commanding sub-humid mid-hill

conditions. The annual rainfall of the area is 1800 + 512 mm. Nearly 80 per cent of the total

precipitation is received during the crop season. The soil is silty clay loam with pH ranging

between 5.8 to 6.0.

3.2 Materials

The material comprised 17 land races of red pericarp rices, 19 land races of purple

leaved rice and 11 land races of quality rices collected from different parts of Chamba, Kangra,

Kullu, Shimla and Mandi Districts of Himachal Pradesh, and maintained at Rice and Wheat

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Research Centre, Malan. Besides, there were three checks, RP-2421, HPR-2143 and China-988.

The details of the genetic stocks are given in Table 3.1.

3.3 Methods

3.3.1 Field evaluation of the experimental material

The experiment was laid out in a Randomized Block Design with three replications.

The net plot size was 2.55 m x 0.8 m (4 rows of 17 plants each) with plant to plant and row to row

spacing of 15 and 20 cm, respectively. All recommended cultural practices were followed to raise

the crop.

3.3.2 Observations recorded

Five plants per replication were taken randomly and tagged to record data on the

following traits, except for days to flowering, which were recorded on plot basis.

3.3.2.1 Morpho-physiological traits

(i) Days to 50 per cent flowering

The number of days was recorded from the date of sowing to 50 per cent flowering.

(ii) Plant height (cm)

Measured in centimeters at maturity from the ground level to the tip of the main

panicle, including awns.

Table 3.1 List of material used in the study

Sr. No. Designation Source (District)

Red pericarp rices

1. Jhinjan Chamba

2. Kijun Chamba

3. Tiyun Chamba

4. Sukara Chamba

5. IC3131171 Chamba

6. IC3131180 Chamba

7. Sukara Dhan Chamba

8. Desi Dhan Kangra

9. Ram Jawain Kangra

10. Achhoo Baldhar Kangra

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11. Achhoo Kangra

12. Jattoo Kullu

13. Deval Kullu

14. Matali Kullu

15. Bhrighu Dhan Kullu

16. Chohatoo Shimla

17. IC3131159 Mandi

Purple leaved rices

18. RLC-3 Kangra

19. Lal Nakanda 41 Kangra

20. Totu Dhan Kangra

21. Kaloo Dhan Kangra

22. Lalzhini Kangra

23. R-575 Kangra

24. Purple Baldhar Kangra

25. Tapta Baldhar Kangra

Sr. No. Designation Source (District)

26. Kaladhan-1 Kangra

27. Kaladhan-2 Kangra

28. Krishan Dhan Kangra

29. China Purple Kangra

30. HPLC-130 Kangra

31. HPR-1194 Kangra

32. HPR-2089 Kangra

33. HPR-2178 Kangra

34. Palampur Purple Kangra

35. Nagrota Purple Kangra

36. IC3131183 Chamba

Quality Rices

37. IC3131155 Mandi

38. IC3131165 Kullu

39. IC3131166 Kullu

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40. LC99-1B Kangra

41. LC99-4B Kangra

42. LC99-5B Kangra

43. Local variety Kangra

44. Rajpur Basmati Kangra

45. Ram Jawain-100 Kangra

46. Kalizhini Kangra

47. Chitizhini

Kangra

Checks

48. RP-2421 Kangra

49. HPR-2143 Kangra

50. China-988 China

(iii) Tillers per plant (no.)

The total number of tillers at maturity.

(iv) Panicle length (cm)

The length of the main tiller measured from base of main rachis to the tip of the top

most grain of panicle, including awns, if any.

(v) Spikelets per panicle (no.)

Total number of spikelets on the main panicle at maturity.

(vi) Grains per panicle (no.)

The number of spikelets bearing seeds on the main panicle.

(vii) 1000-grain weight (g)

A random sample of 1000 well filled grains per replication from the bulk produce of

each genotype were counted and weighed in grams (g).

(vii) Yield per plant (g)

Panicle harvested from each plant were hand threshed, grains cleaned, dried and

weighed.

3.3.2.2 Morphological markers

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The germplasm was characterized using different morphological descriptors viz.

pigmentation of basal leaf sheath, leaf blade and margins, collar, ligule, auricle, stigma, apiculus,

awn, hull and pericarp. In addition size of ligule, auricle and awns was also recorded. The

descriptors for classification are given below as per the details in the leaflet “National Guidelines

for the conduct of Test for distinctness, Uniformity and Stability” (Shobha Rani et al., 2006).

Sr. No. Characteristics States Code

1. Basal leaf sheath Green 1 Light Purple 2 Purple

3

2. Leaf blade and margins Green 1 Purple margin 2 Purple blotch 3 Light purple 4 Dark purple

5

3. Collar White 1 Purple 2 White with purple margins

3

4. Ligule White 1 Light purple 2 Dark purple

3

5. Auricle Absent 0 White 1 Purple

2

6. Stigma White 1 Purple

2

7. Apiculus White 1 Purple

2

8. Awns Absent 0 Green 1 Purple

2

9. Hull Golden 0 Yellow 1 Raddish Brown 2 Black

3

10. Pericarp White 0 Light brown 1 Dark brown 2 Light red 3 Dark red

4

11. Size of ligule 0.5-0.9 cm (small) 0 1.0-2.4 cm (medium) 1 2.5-3.8 cm (large)

2

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12. Size of auricle Absent 0 Small 1 Medium 2 Large

3

13. Size of awns Absent 0 Small 1 Medium 2 Large 3 Tip awn 4

3.3.2.3 Cooking, eating quality and nutritional traits

Data on quality and nutritional trait was recorded on two replications for each

genotype.

(i) Grain length (mm)

Length of ten dehusked grains from the bulk produce of each genotype, recorded in

mm using dial micrometer.

(ii) Grain width (mm)

Width of the same ten dehusked grains, recorded in mm.

(iii) Grains L/B ratio

L/B ratio was calculated by dividing the grain length by its width.

(iv) Grain length after cooking (mm)

Ten milled grains were put in a test tube to which 25 ml of distilled water was added.

The test tubes were immersed in water bath at 98oC for 10 minutes. The tubes were then

immersed in cold water until cooled to room temperature. Cooked grains were transferred to

properly labelled Petridishes. Length of the cooked grains was measured in mm.

(v) Elongation ratio

Elongation ratio was computed by dividing the length of cooked grains by its length

before cooking.

(vi) Amylose content (%)

Amylose content of polished rice was estimated following Juliano (1971) method

using acetate buffer and amylose-amylopectin (70% starch standard).

(a) Preparation of working standards for curve

100 mg of potato amylose (amylose standard) and polished rice of Palampur Purple

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a waxy line as (amylopectin standard) were weighed and kept in 100 ml volumetric flasks. To this

1 ml of 95 per cent ethanol and 9 ml of 1N NaOH was added. Swirled carefully, allowed to stand

overnight and final volume was made 100 ml with distilled water.

Then amylose and amylopectin standards as per table below were pipetted out and

made to 100 ml with 0.09 N NaOH in 100 ml volumetric flasks labelled 0, 10, 20 and 30 per cent

amylose.

Working standard (% amylose)

Volume amylose (ml) Volume amylopectin (ml)

0.09 N NaOH (ml)

0 0 70 30

10 10 60 30

20 20 50 30

30 30 40 30

(b) Preparation of check and test samples

100 mg of finely powdered samples of each genotype and standard checks were

taken. To each, one ml of 95 per cent ethanol and 9 ml of 1N NaOH was added and allowed to

stand for 24 hours. Final volume was made to 100 ml with distilled water. Five ml of each sample

(working standards, checks and test samples) were pipetted out and transferred to 100 ml

volumetric flask. For blank, 50 ml of 0.09 N NaOH was taken. In each sample, one ml of 1N

acetic acid and 50 ml of distilled water was added and mixed. Thereafter, two ml of iodine was

added to each sample and volume was made to 100 ml with distilled water. The contents were

mixed thoroughly, allowed to stand for 15-20 minutes and the absorbance was recorded in

spectrophotometer at 620 nm. Amylose per cent was determined from the regression equation

derived from amylose-amylopectin standard curve.

(vii) Gelatinization temperature

Gelatinization temperature was estimated by the alkali digestion method of Little et al.

(1958). Six whole milled grains of each genotype were spaced evenly in transparent Petriplates

containing 10 ml of 1.7 per cent KOH solution. The Petriplates were covered and left undisturbed

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for 23 hours in an incubator maintained at 30oC. The spreading of each kernel was rated visually

on a seven point numeric scale as below:

Spreading scale

Rating Description

1 Kernel not affected

2 Kernel swollen

3 Kernel swollen, collar incomplete and narrow

4 Kernel swollen, collar incomplete and wide

5 Kernel split or segmented, collar complete and wide

6 Kernel dispersed, merging with collar

7 Kernel dispersed and intermingled

Classification

Rating Gel temperature

1.0 – 2.0 Very high (> 75oC)

2.1 – 4.0 High (high/intermediate)

4.1 – 6.0 Intermediate (70-75oC)

> 6.1 Low (< 70oC)

(viii) Protein content (%)

Protein content in mature seed was determined by Micro Kjeldhals method (A.O.A.C.,

1970).

Reagents

o Sulphuric acid (specific gravity 1.84)

o Mercuric oxide

o Potassium sulphate

o Sodium hydroxide – sodium thiosulphate solution : Dissolved 600 of NaOH and

50 g Na2S2O3.5H2O in distilled water and made to one litre with distilled water

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o Indicator solution : Methyl red 0.2 g/100 ml ethanol, methylene blue 0.2 g/100 ml

ethanol. For mixed indicator, added two parts of methyl red solution to one part of

methylene blue solution.

o 4 per cent boric acid : Diluted 20 g of boric acid and 10 ml of mixed indicator to

500 ml with distilled water.

o N sulphuric acid solution.

The grains after dehusking were ground to fine powder. 100 mg of the sample was

weighed and transferred to 30 ml digestion flask. 1.0 + 0.1 g potassium sulphate, 80 + 10 mg

mercuric oxide and 2 ml concentrated sulphuric acid was added to digestion flask. Boiling chips

were also added to it and the sample was digested till the solution became colourless. The digest

was then cooled and diluted with a small quantity of distilled ammonia free water and transferred

to the distillation apparatus. The kjeldhal flask was rinsed with successive small quantities of

water. After that 100 ml of conical flask containing 5 ml of boric acid solution containing a few

drops of mixed indicator was placed with the tip of the condenser dipping below the surface of the

solution. 10 ml of sodium hydroxide sodium thiosulphate solution was added to test solution in the

apparatus. Distillation was done and ammonia was collected on boric acid. The tip of the

condenser was rinsed and the solution was titrated against the standard acid till first appearance

of violet colour. Reagent blank was also run with an equal volume of distilled water.

Calculations

100X(g)sampleofWeightx(ml)takenAliquot

acidofNormalityxdigestofVolumex0.014x(ml)]titreblank(ml)titre[SampleN%

% Protein = 0% N x 5.95

(ix) Sugar content (% glucose)

Sugar content in brown rice seed was estimated by Dubois et al. (1956).

Reagents

o 80% ethanol

o Lead acetate

o Sodium acetate

o 5% phenol

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o 95.5% sulphuric acid

Five hundred mg of brown rice was macerated in 50 ml of ethanol (80%) and

transferred to a conical flask. The contents of the flask were then boiled upto half of the volume

(25 ml). The contents were filtered and filtrate was made to 98 ml with distilled water. 1 ml of

saturated lead acetate solution was added to it. To remove the lead ions a pinch of sodium

oxalate was added and volume was made to 100 ml with distilled water. Aliquot 0.5 ml was taken

in the test tube and 1 ml of 5% phenol (freshly prepared) and 5 ml of 95.5% of concentrated

sulphuric acid was added from the top, not from the side of test tube in ice cold solution. The

intensity of pink colour was read at 490 nm. The amount of sugars present in the extract was then

calculated using a standard curve from glucose (0.1 mg ml-1

).

(x) Iron and Zinc

Iron and Zinc contents in polished rice grains were estimated using atomic absorption

spectrophotometer (Varian Model) after wet digestion of the sample (Piper, 1950).

Five gram dried sample of polished rice of each genotype were taken in 100 ml

digestion tubes. 25 ml of nitric acid and perchloric acid in the ratio of 9:4 was poured in each

digestion tube. The digestion tubes were shaken carefully. The contents in digestion tubes were

digested by heating on hot plate till clear and colourless liquid was left. A blank was treated

similarly with each set of samples. Then this content was transferred to 100 ml volumetric flask

after repeated washings with distilled water. The digests were filtered through Whatman No. 42

filter paper and final volume was made 100 ml with distilled water.

Mineral content = Concentration of sample (ppm) x dilution factor

Standard curve

Ferrous ammonium sulphate and zinc sulphate were used for preparation of 1000

ppm concentration of both the minerals. These stock solutions were diluted for Fe (0.75, 2.25 and

4.50 ppm) for Zn (3, 9 and 18 ppm) for various concentrations with distilled water and standard

curves were prepared using atomic absorption spectrophotometer. Fe and Zn contents were

analyzed only during 2006.

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3.3.3 Statistical analysis

Average value for each genotype in each replication were used for statistical

analysis.

3.3.3.1 Analysis of variance

The data were statistically analyzed as per the following model given by Panse and

Sukhatme (1984).

Yij = + gi + rj + eij

where,

Yij = phenotypic observation of ith genotype in the j

th replication

= general mean

gi = effect of ith genotype

rj = effect of jth replication

eij = random error associated with ith genotype in the j

th replication

Analysis of variance

Source of variation

df Mean square Expected MS ' F' value

Replications (r-1) Mr 2e+g

2r Mr/Me

Genotypes (g-1) Mg 2e+r

2g Mg/Me

Error (r-1) (g-1) Me 2e -

where,

r = number of replications

g = number of genotypes

2r = variance due to replications = (Mr–Me)/g

2g = variance due to genotypes = (Mg–Me)/r

2e = error variance

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2p = phenotypic variance =

2g +

2e

The standard error of mean SE (m) (+) and critical difference (CD) for comparing the

means of any two lines were computed as follows:

SE (m) = ± r

Me

SE (d) = ±r

Me2

Critical difference = SE (d) x „t‟ value at 5% error degrees of freedom.

3.3.3.2 Estimation of parameters of variability

The genotypic, phenotypic and environmental coefficients of variation were estimated

following Burton and De Vane (1953):

2g

Genotypic coefficient of variation (GCV%) = ––––– X 100

X

2p

Phenotypic coefficient of variation (PCV%) = ––––– X 100

X

2e

Environmental coefficient of variation (ECV%)= ––––– X 100

X where,

2g = genotypic standard deviation

2p = phenotypic standard deviation

2e = environmental standard deviation

X = population mean

3.3.3.3 Heritability (%)

Heritability in broad sense (h2bs) was calculated as per the following formula given by

Burton and De Vane (1953) and Johnson et al. (1955a).

2g

Heritability (h2

bs) = ––––––––– X 100

2p

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3.3.3.4 Genetic advance (%)

The expected genetic advance (GA) resulting from the selection of 5 per cent

superior individuals was calculated as per Burton and De Vane (1953) and Johnson et al.

(1955a).

GA = K x p x h2bs

where,

K = 2.06 (selection differential at 5% selection intensity)

h2bs = heritability (broad sense)

pσ

gσ2

2

p = phenotypic standard deviation

Expected GA Genetic advance as percentage of mean = ––––––––––––– X 100 Grand Mean For categorizing the magnitude of different parameters, the following limits were

used:

PCV and GCV > 30% - High

15% - 30% - Moderate

< 15% - Low

Heritability (h2bs) > 80% - High

50% - 80% - Moderate

< 50% - Low

Genetic advance (GA) > 50% - High

25 % - 50% - Moderate

< 25% - Low

3.3.3.5 Estimation of correlation co-efficients at the phenotypic, genotypic and

environmental levels For computing phenotypic, genotypic and environmental correlation coefficients,

analysis of covariance were carried out in all possible combinations of the characters.

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Analysis of variance

Source of variation

df MSP Expected MS P ' F' value

Replications (r-1) Mr12 2e12+g

2r12 Mr12/Me12

Genotypes (g-1) Mg12 2e12+r

2g12 Mg12/Me12

Error (r-1) (g-1) Me12 2e12 -

where,

r = number of replications

g = number of genotypes

2g12= genotypic covariance of characters X1 and X2

2e12= error covariance of characters X1 and X2

The genotypic, phenotypic and error covariances were calculated as follow:

Mg12 – Me12

Genotypic covariance (g12) = ––––––––––– r

Phenotypic covariance (p12) = g12 + e12 The phenotypic, genotypic and environmental coefficients of correlation were

calculated as per Al-Jibouri et al. (1958).

p12 Phenotypic correlation = r12 (P) = ––––––––––––––––

2p (X1) X

2p (X2)

g12 Genotypic correlation = r12 (G) = ––––––––––––––––

2g (X1) X

2g (X2)

e12 Environmental correlation = r12 (E) = ––––––––––––––––

2e (X1) X

2e (X2)

where,

p12 = phenotypic covariance between characters X1 and X2

g12 = genotypic covariance between characters X1 and X2

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e12 = environmental covariance between characters X1 and X2

2p(X1) and

2p(X2) = phenotypic variance of traits X1 and X2,

respectively

2g(X1) and

2g(X2) = genotypic variance of traits X1 and X2,

respectively

2e(X1) and

2e(X2) = environmental variance of traits X1 and

X2, respectively The significance of phenotypic coefficients of correlation were tested against „r‟

values as given by (Fisher and Yates, 1963) at n-2 degree of freedom, where (n) is the number of

genotypes.

3.3.3.6 Path analysis

Path-coefficient is a standardized partial regression coefficient, which permits the

partitioning of the correlation coefficients into direct and indirect effects. The path-coefficient

analysis of important morphological traits as well as quality traits with yield was done following

Dewey and Lu (1959) as under:

Py1 + Py2.r12 + Py3.r13 + ……………………………….. + Pyn.r1n = ry1

Py1.r12 + Py2 + Py3.r23 + ……………………………….. + Pyn.r2n = ry2

Py1.r13 + Py2.r23 + Py3 + ……………………………….. + Pyn.r3n = ry3 : : : : Py1.r1n + Py2.r2n + Py3.r3n + ……………………………….. + Pyn = ryn

where,

Py1, Py2, Py3 ………….. Pyn are the direct path effects of 1, 2, 3, ………….., n

variables on the dependent variable „y‟.

r12, r13, ………….. r (n-1) n are the coefficients of correlation between various

independent variables and ry1, ry2, ry3, …………… ryn are the correlation coefficients

of independent variables with dependent variable „y‟.

The variation in the dependent variables was assumed to be due to variable (s) not

included in the present investigation. The degree of determination of such variables was

calculated as follows:

Residual effect (P X R) = 1 – R2

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where,

R2 =

n

1i piy riy

where, R

2 is the square multiple correlation coefficient and is the amount of variation in yield

that can be accounted for any yield component characters included in the present investigation.

3.3.3.7 Estimation of genetic diversity

A measure of group distance based on multiple characters was given by

Mahalanobis, 1936 (Rao, 1952b).

With x1, x2, x3 ……………. xp as the multiple measurements available on each

individual and d1, d2, d3 …….. dp as x1-1

– x1-2

, x2-1

– x2-2

………xp-1

– xp-2

, respectively, being the

difference in the means of two populations, Mahalabosis D2 statistics is defined as follows:

pD2 = b1d1 + b2d2 + ……………. bpdp

Here, the b1 values are to be estimated such that the ratio of variance between the

populations to the variance within the populations is maximized. In terms of variances and

covariances, the D2 value is obtained as follows:

pD2 = W

ij (xi

-1 – xi

-2) (xj

-1 – xj

-2)

where,

Wij is the inverse of estimated variance covariance matric.

3.3.3.7.1 Test of significance

Using (V) statistics which, in turn, utilizes Wilk‟s criteria, simultaneous test of

difference mean values of a number of correlated variables/characters at „pq‟ d.f. (where p =

number of variables/ characters and q = number of germplasm-1) done as suggested by Rao

(1952b).

3.3.3.7.2 Grouping of genotypes into various clusters

Using D2 values, different genotypes were grouped into various clusters following

Toucher‟s method as suggested by Rao (1952b).

3.3.3.7.3 Grouping of genotypes into various clusters

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Average intra-cluster D2 = Di

2/n

where,

Di2 = sum of all distances between all possible combinations (n) of the genotypes

included in the cluster.

Average inter-cluster distance D2 = Dij

2/ni…..nj

where,

Dij2 = sum of all distances between all possible combinations (ni.nj) of the

genotypes between the clusters.

ni = number of genotypes in ith cluster

nj = number of genotypes in jth cluster

3.3.3.7.4 Cluster mean

Character means of rice genotypes falling under different clusters in individuals as

well as combined over environments were also calculated.

3.3.3.7.5 Contribution of individual towards divergence

In all combinations each character was ranked on the basis of di = Yij – Yi

k values.

Rank 1 was given to all the highest mean difference and rank „p‟ to the lowest mean difference,

where „p‟ is the total number of characters. The contribution of individual character to the

divergence has been worked out in terms of „n‟ number of times it appeared first.

3.3.4 Molecular characters

RAPD and ISSR markers were used to generate DNA fingerprints of 50 genotypes of

red pericarp rices, purple leaved rices and quality rices including checks were used.

3.3.4.1 Isolation of genomic DNA

Genomic DNAs of the 50 accessions were isolated following the CTAB method of

Murray and Thompson (1980). In brief, about 1 g fresh juvenile leaves were collected from each

genotype, cut into small pieces with sterilized scissor and ground to fine powder in liquid nitrogen

(-196oC) in a oven baked pestle and mortar. Approximately 500 mg of ground tissue was

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transferred to 2 ml eppendroff tube and 700 l of pre-warmed (at 65oC water bath) extraction

buffer [100 mM Tris-HCl, 140 mM NaCl, 20 mM EDTA, 2% CTAB, 1% PVP and 0.5% -

mercaptoethanol, pH 8.0] was added. The tubes were vortexed to suspend the tissue in the

buffer. After incubation at 60oC for 1 hr in shaking water bath, equal volume (700 l) of chloroform

: isoamyl-alcohol (24:1) was added to each tube. The contents were mixed thoroughly and tubes

centrifuged at 10,000 rpm for 10 min. The aqueous phase was transferred to new tubes and 500

l pre-chilled propanol-2 (isopropanol) was added and left for 1 hr at -20oC to precipitate the

DNA. Tubes were then spun at 10,000 rpm for 10 min and the supernatant discarded. The DNA

pellet was washed with 200 l of 70% pre-chilled ethanol and centrifuged for 1 min at 10,000 rpm.

All the ethanol was drained from tubes and given washing again with 70% ethanol followed by 1

min centrifugation at 10,000 rpm. Finally the pellets were dried and dissolved in 100 l of TE

buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). After quantification, DNA was stored at -20oC till

used. A working DNA solution was made by diluting DNA stock.

3.3.4.2 Assaying of RAPD and ISSR markers

3.3.4.2.1 PCR amplification of DNA

PCR amplification was performed in 20 l volume consisting of 1.6 l of dNTP mix

(0.2 mM each of dATP, dGTP, dCTP and dTTP), 0.16 l Taq DNA polymerase, 2 l DNA

template, 1.6 l of 100 M primer, 2 l of 10x PCR buffer, 1.2 l of MgCl2 (25 mM) and 11.44 l of

sterilized distilled water. Reaction mixture was vortexed and centrifuged briefly. Amplification was

carried out in a thermal cycler, programmed for 5 min at 94oC for initial denaturation and 39

cycles consisting of 1 min at 94oC, 1 min at 37

oC and 2 min at 72

oC with final 7 min extension at

72oC using the fastest ramp times between the temperature transitions. For ISSR assay, 10 pmol

of each primer was used keeping other ingredients the same. For this, the initial denauration was

at 95oC for 4 min, followed by 45 cycles consisting of 30 sec at 94

oC, 45 sec at 52

oc and 2 min at

72oC with final 5 min extension at 72

oC.

3.3.4.2.2 Electrophoresis resolution of the amplified products

After amplification, 12 l of the amplified product from each sample was resolved on

agarose gel (1.4% for RAPD and 2% ISSR) in 1x Tris acetate-EDTA (TAE) buffer (242 g Tris,

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57.1 ml glacial acetic acid, 100 ml EDTA, pH 8.0). Ethidium bromide (0.5 g/ml) was added in the

buffer as inter calating agent. To estimate the size of amplified DNA fragments, 1 kb DNA ladder

was used as marker. The gel was run at 120V for 2 hr. After electrophoresis, the gel was viewed

and stored in the Gel Documentation System.

3.3.4.2.3 Primers

Fifteen random primers (Operon technologies, USA) and eleven ISSR primers were

used for DNA fingerprinting of 50 genotypes. The base sequence of the primers used are given in

the Table 3.2.

3.3.4.3 Analysis of DNA fingerprinting

The DNA profiles of genotypes were scored for the presence or absence of each

band of a particular molecular weight for different primers. A binary data matrix with „1‟ indicating

the presence of a particular molecular weight band and „0‟ indicating its absence was generated

separately for each primer. The binary data were used to generate a similarity matrix using

Jaccard‟s coefficient [Cjij = Cij/[ni + nj – Cij], where Cij is the number of positive matches between

two genotypes, while ni and nj are the total number of bands in genotype i and j, respectively] in

SIMQUAL programme of NTSYS-pc package (Rohlf, 1993). Cluster analysis of genotypes based

on similarity values was done by unweighted paired group arithmetic mean method (UPGMA) in

SAHN programme of NTSYS-pc package to construct a dendrogram.

Table 3.2 Base sequences of RAPD and ISSR primers used for DNA fingerprinting

RAPD primers 5'-------------------3' sequence

OPF-05 CCGAATTCCC

OPF-09 CCAAGCTTCC

OPF-16 GGAGTACTGG

OPJ-13 CCACACTACC

OPJ-20 AAGCGGCCTC

OPX-13 ACGGGAGCCA

OPX-20 CCCAGCTAGA

OPA-10 GTGATCGCAG

OPA-13 CAGCACCCAC

OPQ-05 CCGCGTCTTG

OPQ-06 GAGCGCCTTG

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OPQ-10 TGTGCCCGAA

OPD-02 GGACCCAACC

OPD-05 TGAGCGGACA

OPU-15 ACGGGCCAGT

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ISSR primers 5'--------------------3' sequence

UBC8 10 (GA)8 T

UBC8 14 (CT)8 A

UBC8 15 (CT)8 G

UBC8 40 (GA)8 YT

UBC8 41 (GA)8 YC

UBC8 43 (CT)8 RA

UBC8 45 (CT)8 RG

UBC8 50 (GT)8 YC

UBC8 58 (TG)8 RT

UBC8 59 (TG)8 RC

UBC8 73 (GACA)4

Y = Pyrimidines (C/T)

R = Purines (A/G)