4. Methods of genetic purity testing
KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE,
ARABHAVI

5. Genetic purity ?
Genotypic purity is simply defined as
true to type plants / seeds conforming to the
characteristics of the variety as described by
the breeders.
Principle
Genetic purity or genuiness of the
cultivar is tested by means of heritable
characters (morphological, physiological or
chemical) of seeds, seedlings or plants.

6. Minimum standards for genetic purity for different class of
seeds.
SL. No.
1
2
3
4
CLASS OF SEEDS
Breeder Seeds
Foundation seeds
Certified seeds
Hybrids
PURITY %
100%
99%
98%
95%
Factors affecting genetic purity.
1.
2.
3.
4.
5.
Natural crossing
Mechanical admixtures
Random drift
Mutation
Selective influence of pest and diseases.
(Basra, 2002)

7. Criteria for GOT to decide the genuineness of variety
Maximum
permissible off types
(%)
Minimum genetic
purity (%)
Number of plants
required/sample for
observation
0.10
99.9
4,000
0.20
99.8
2,000
0.30
99.7
1,350
0.50
99.5
800
1.00 and above
99.0 and below
400
(Basra, 2002)

8. The are two main approaches for genetic or
varietal purity testing:1. The use of computerized systems to capture and
process morphological information
(Machine vision).
2. The use of biochemical methods to analyze
various components of seeds
(Chemotaxonomy).
(Basra, 2002)

9. Methods to assess genetic purity
1. Morphological / Conventional grow out test
2. Chemical test
3. Electrophoresis method
 Biochemical markers (Proteins and Isozymes)
 Molecular markers (DNA)
(Basra, 2002)

10. Morphological test
In laboratory
• Examination features of seeds such as length, width,
thickness, shape, weight, colour, seed coat colour etc.
and comparing them with those of authentic sample.
• Which are examined with naked eye / with magnified
hand lens / with the help of scanning electron
microscope
(Basra, 2002)

11. In field or green house condition
• Grow out test
• The seed sample is sown in the controlled
condition with the authentic sample
• Genetic purity is determined on the basis of
observation made on the plant morphological
characters with reference to authentic sample.
• Genetic purity is always expressed in
percentage
(Basra, 2002)

12. The size of the submitted sample will be as follows: for GOT
• 1000 g
–
for maize, cotton, groundnut, soybean
and species of other genera with
seeds of similar size.
• 500 g
–
for sorghum wheat, paddy and species of
other genera with seeds of similar size.
• 250 g
–
Beta sp and species of other genera with seeds
of similar size.
• 100 g
–
for bajra, jute and species of all other genera.
• 250 tubers/ planting – seed potato, sweet potato and other
vegetatively propagating crops.
Stakes/roots/corns.
(Basra, 2002)

13. Procedure of GOT
Raising of desired population by following recommended
cultural practices e.g., field preparation, size of the plot, etc.
Provide equal opportunity to each and every plant for full
expression of genetically controlled characters
Sow the various samples of the same variety / cultivar in
succession and standard sample at suitable intervals
Adjust of seed rate depending on germination % of individual samples and
subsequent thinning is not recommended.
This test is preferably conducted in area to which the variety is recommended
A minimum of 200 plants from control samples should be raised along with the test
crop.
The analyst employed for conducting „grow out test‟ should possess the basic
qualification as identified under Seed Rules, 1968.

14. Traditional approach to purity testing
Morphological traits
ON SEED
IN LAB OR GREEN HOUSE
ON SEEDLING
IN FIELD

15. PLANT HABIT
FLOWERING HABIT
FRUITING HABIT

16. CHEMOTAXONOMY
Chemo taxonomists have recognized two groups of
compounds that are generally use full in classification of plant
species
1. Episemantic or secondary metabolites
(pigments or fatty acid etc.)
2. Semantides or sense carrying molecules
(Proteins, Nucleic Acids)
Methods of testing based on1. Analysis of secondary compounds
2. Protein analysis
3. Nucleic acid analysis

17. Analysis of secondary compounds
These test ranges from simple colour tests to complex
chromatographic separations of phenols, anthocyanin, flavonoids
and other compounds.
Different tests includes
1.
2.
3.
4.
5.
6.
7.
8.
9.
Phenol test
Peroxidase test
Potassium hydroxide – bleach test
Fluorescence test
Hydrochloric HCI test
Ferrous sulphate test
NaOH test
Anthocyanin test
Seedling pigmentation
(Basra, 2002)

18. Objective of the study- For the development of quick and reliable tests for
varietal identification particularly for those working in seed certification and
quality maintenance
Materials and method.
1.Pure seeds of 23 rice genotypes
2.Five chemical tests viz. Phenol, modified phenol, Ferrous Sulphate, Potassium
hydroxide and sodium hydroxide
3.50 seeds of each genotype were observed

19. Table 1: Response of different rice varieties for different chemical test
VARIETY
Phenol test
Modified Phenol test
FeSO4 test
Very strong moderat
no
Very Strong modera no DGSt BSt
strong
e
colour strong
te
colour
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Chaitanya
Maruteru
Vijetha
Tholakari
vajaram
Swarna
Deepthi
Krishan veni
MTU 1004
Anjali
vikas
rajendra
ASD-7
PR-113
QPE-2
Rathuheenathi
mudgo
Tadukan
Varalu
++++
CO-31
Pooja
Chenegi
++++
Supreme
(-)
(-)
(-)
(-)
(-)
(-)
++
++
++
++++
DGSt
(-)
+++
(-)
++++
++
++
DGSt
+++
BSt
++
+++
++++
DGSt
++
++
+++
(-)
BSt
++++
++++
BSt
+++
+++
(-)
++
DGSt
DGSt
++
+++
BSt
BSt
+++
++++
++
++
+++
BSt
++
(-)
++
++
BSt
BSt
DGSt
++
+++
++++
++++
DGSt
KOH test NaOH Test
BSp DWR No
colou
r
BSp
(-)
(-)
(-)
BSp
(-)
(-)
(-)
BSp
(-)
(-)
(-)
BSp
(-)
(-)
(-)
BSp
+
(-)
(-)
+
+
(-)
(-)
BSp
(-)
BSp
(-)
BSp
+
(-)
DY LY NO
colou
r
(-)
(-)
LY
LY
LY
LY
LY
DY
LY
(-)
LY
DY
DY
(-)
LY
LY
DY
LY
DY
DY
(-)
LY
(-)
(Vijaylakshmi and Vijay, 2009)

20. Figure 1- Schematic results of all five chemical tests
(Vijaylakshmi and Vijay, 2009)

21. Protein analysis
Because proteins are the direct gene product the
analysis of seed, seedling proteins and enzymes is most
successful and widely used. Hence much attention has been
focused on seed storage proteins.
There are two primary methods
Various types of gel electrophoresis
High pressure liquid chromatography

22. What is Electrophoresis ……..?
Migration of a charged particle through a medium
(agarose, polyacrylamide, starch) under the influence of an
electrical field. it is usually carried out in aqueous solution
•A mixture of molecules of various sizes will migrate at different
velocities and will be separated.
The varieties are verified on the basis of
banding pattern.
1. By measuring Rm of bands
2. Total number of bands
3. Presence or absence of specific band
4. Intensity of band

23. Gel Electrophoresis-based on type of separation
 Native: separation by size and charge (charge/mass)
 Denaturing: separation by size
 Others (IEF, 2-Dimenstional electrophoresis)
Gel Electrophoresis
Native continuous system--gel and tank buffers are the same, single phase
gel; examples are PAGE, agarose, and starch gels.
Discontinuous System--gel and tank buffers are different, two phase gel
(stacking gel); example is PAGE.
Gel Electrophoresis based on denaturation
SDS (sodium dodecyl sulphate) used to denature proteins (discontinuous
system). urea or form amide used to denature DNA or RNA.
Other types are
Isoelectric focusing: protein-separation based on isoelectric points in a pH
gradient.
2-D electrophoresis: combination of IEF and SDS-PAGE.

24. Materials and method
1. It consists of 8 varieties viz., CSV 15, SPV 669, PVK 400, PVK 801, BTX623,
IS 18551, R16 and E36-1.
2. 4 hybrids along with parents and maintainer line viz., CSH 14(ms14A X AKR
150), CSH 9 (ms 296a x CS 3541), CSH18 (IMS 9A x Indore12) and SPH 840
(ms70A x CS 3541).
Protein extraction- using 1.5 ml sodium phosphate buffer (0.1M, pH-7), centrifuged
at 10k for 45mins @ 40C protein estimation by Lowry method
using alkaline copper and folin reagent.

25. Table 2 : Low range and high range protein molecular markers and their molecular
weight
Low range protein markers
Sr.
No.
Protein molecular
weight marker
High range protein markers
Molecular
weight (Da)
Sr. No.
Protein molecular
weight marker
Molecular
weight (Da)
1
Phosphorylase b
97000
1
Myosin
220000
2
serum albumin
66000
2
α-2-macroglobulin
170000
3
ovalbumin
45000
3
β- galactosidase
116000
4
Carbonic anhydrase
30000
4
Transferin
76000
5
Trypsin inhibitor
20100
5
glutamate
dehydrogenase
53000
6
α- lactalbumin
14400
Da- Dalton
(Kavimandan and khan, 2012)

26. Fig 2 : Schematic diagram of the SDS-PAGE profiles of the seed albumins in the sorghum
varieties, hybrids, parents & control.
(Kavimandan and khan, 2012)

27. Materials:1.Hybrids and their respective parents of cotton
a. DCH-32 ( DS-28 X SB (YF) 425)
b. DHB-105 (CPD-428 X B-82-1-1)
c. DHH-11 (CPD-423 X CPD-420)
2.Seed globulin protein, seed enzyme and leaf enzyme extracted from the above material.
3.ELECTROPHORESIS-Acc to Davis (1964) using 7.7% running and 2.5% seperating P.A
gel carried out in Tris-glycine buffer (pH 8.3).
• Staining for protein 0.1% coomassie brilliant blue in methanol:aceticacid:water
•
(5:2:3).
for isozyme glumate oxaloacetate transminase
Destaining by – 7% acetic acid over night

28. Rm value
Rm value
1.0
CPD-420
DHH-11
CPD- 423
B-82-1-1
DHB-105
CPD- 428
SB (YF)-425
DCH-32
DS-28
1.0
Fig 3: Zymogram of the PAGE patterns of the seed
globulins in cotton hybrids and their patterns
-0.0
CPD-420
DHH-11
CPD- 423
B-82-1-1
DHB-105
CPD- 428
SB (YF)-425
DCH-32
DS-28
-0.0
Rm value
CPD-420
DHH-11
CPD- 423
B-82-1-1
DHB-105
CPD- 428
SB (YF)-425
DCH-32
DS-28
-0.0
1.0
Fig. 4: Zymogram of the PAGE patterns of leaf
esterase in cotton hybrids and their patterns
Fig. 5: Zymogram of the PAGE patterns of
alcohol dehydrogenase isozyme cotton hybrids
and their patterns
Rm= Relative migration
( Manjunath Reddy et al. 2008 )

29. Plant Material:
Materials and Methods
Fresh mature seeds of selected species of Bauhinia .
Protein Extraction: by method given by Jensen and Lixue .
overnight presoaked seeds in protein solubilization solution (62 m M Tris –HCl,
pH 6.8, 10% glycerol, 2% SDS, β- mercaptoethanol and traces of bromophenol blue ) then
centrifuged at 14000 rpm for 30 seconds. The supernatent was collected placed into a
boiling water bath for 4 minutes.
SDS-PAGE was done by method suggested by Laemmli.
It was performed on a vertical slab gel. Bromophenol blue was added to the
supernatant as tracking dye to watch the movement of protein in the gel. Seed protein was
analyzed through slab type SDS-PAGE using 10% Separating gel and 4% Stacking gel.

30. Table 3: The Rf value of the various bands that appeared on gel of Bauhinia species
Band
No.
Rf value
Mol.Wt.in
KDa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
0.08
0.14
0.2
0.24
0.26
0.28
0.3
0.34
0.38
0.42
0.44
0.47
0.48
0.51
0.54
0.56
0.58
0.64
0.75
0.81
0.82
0.9
261.143
254.77
178.34
148.622
147.74
137.19
128.04
126.6
113.36
102.564
97.9
91.65
89.74
54.854
51.8
49.95
41.56
37.67
31.85
21.767
21.5
7.337
Marker B.acuminata B.parpurca B.racemosa B.tomentosa B.variegata
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(Sinha et al. 2012)

31. TABLE 4: Percentage Similarity Index between Bauhinia Species
S. No.
1
2
3
4
5
6
7
8
9
10
Species x Species
B. acuminata x B. purpurea
B. acuminata xB. Racemosa
B. acuminata x B.tomentosa
B. acuminata x B. varigata
B. purpurae X B. racemosa
B. purpurae X B. tomentosa
B. purpurae X B. varigata
B.racemosa X B. tomentosa
B.racemosa X B. variegata
B. tomentosa X B. variegata
Percentage similarity
45.45%
33.33%
31.25%
33.33%
38.46%
26.66%
40.00%
43.75%
20.00%
35.71%
(Sinha et al. 2012)

32. Fig 6: SDS protein profile of seed of [A]
B. variegata; [M] Marker; [B] B.
accuminata; [C] B. purpurae; [D]
B. racemosa; [E] B. tomentosa
Fig 7: Diagramatic of SDS protein profile of seed of [A] B. variegata; [M] Marker;
[B] B. accuminata; [C] B. purpurae; [D] B. racemosa; [E] B. tomentosa
(Sinha et al. 2012)

33. Materials:1. Mature seeds of F1Hybrids of Tomato and their respective parental lines were used
a. F1-hybrid ( 6944 x 2413) {parental line carries pollen sterility ms 10 35 }
b. F1 –hybrid (2197 x 2263)
2. Total 840 seeds were used 120 parental and 180 hybrid seeds imbibed in water for 36hr
3. Iso enzyme extraction in 0.05MTris HCL pH-7.2
ELECTROPHORESIS-VBE with 7.5% polyacrylamide gel with Tris EDTA –boric acid
buffer with pH-8.3
•
Staining for isozyme - Glumate dehydrogenase (GDH)

34. Fig 8: Electrophoretic patterns and scheme of GDH in tomato seeds a-maternal parent
line 6944; b- paternal line 2413; C-F1 hybrid; B1-contamination.
Fig 9: Electrophoretic patterns and scheme of GDH in tomato seeds a-maternal parent
line 2197; b- paternal line 2263; C-F1 hybrid.
(Markova and stilova, 2003)

35. Materials:1. 16 sunflower Hybrids
2. Protein was extracted form seed by adding 400µl 0.03M Tris HCL pH-8 containing 0.01% 2mercaptoethanol for 4hrs. Centrifuged at 11k for 15mins.
3. Proteins were than dissociated by denaturing buffer(0.15M Tris pH6.8 containg 3%SDS, 5%
mercaptaetanol & 7% glycerol)
ELECTROPHORESIS-Acc to Laemmli using 12.5% P.A gel under denaturing SDS and reducing
mercaptaethanol
Staining for protein using0.24g coomassie brilliant blue in 90 ml of 1:1 (v/v) methanol :
water and 10 ml of glacial acetic acid
for isozyme stem tissues of 5 days old seedling homozinized in 50mM TrisHCL, pH- 6.8 in 1% mercaptaethanol
ISOZYME SYSTEMS -(PHI), (PGM) & (PGD)

36. Figure 10: Electrophoretogram of
seed storage proteins of
sunflower hybrid H1
Figure 11: Electrophoretogram of
seed storage proteins of
sunflower hybrids H1-H10
( Nikolic et al. 2008 )

37. Figure 12: PHI (a), PGM (b) and PGD
(c) isozyme patters of sunflower hybrids
( Nikolic et al. 2008 )

38. Table 5: Comparative data of genetic purity level in sunflower hybrids measured
on the basis if isozyme and seed storage protein analyses
sample
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
genetic purity (%)
EI
98
90
91
98
92
99
98
93
94
97
89
88
91
96
97
95
EI - electrophoresis of isoenzymes
ESSP- electrophoresis of seed storage proteins
ESSP
87
95
89
98
94
96
95
89
92
98
97
92
95
96
92
98
( Nikolic et al. 2008 )

39. Iso electric focusing
This technique relies not on the rates of mobility but on the protein’s net
charge. Isozymes move through a pH gradient under the influence of an electric
field.
As the enzymes move through acidic regions of the gel and enters into
areas of higher alkalinity the net charge on the protein changes, eventually, it
reaches a pH region where the net charge equals zero. At this point the protein
will not migrate any further and is said to be “focused”.
IEF can be used to differentiate proteins with very subtle changes in
amino acid composition.
Proteins migrating through a pH gradient
will continue to move until their net
charge becomes zero.
(Leist, 2005)

40. How does the pH gradient work in the gel matrix
Carrier ampholytes are in the gel matrix that are low molecular
weight and have closely related isoelectric points
When electricity is applied to the gel the ampholytes forms a pH
gradient in the gel.
When an amphoteric protein from a sample is no longer
charged the electrical current will not have an effect on it. Thus, the
term “FOCUSING”.
(Leist, 2005)

41. Steps in Iso Electric Focusing
Crushed seed
(protein
extraction)
Vortexing by
adding extraction
solution
Loading of
sample on gel
Gel running
Taking off the gel once
done
Single bands
Staining the gel
Destaining the gel
Blotting the
stained gel
Gel ready to read
(Leist, 2005)

42. Materials:-
1. 5 maize hybrids and their respective parents
2. Protein was extracted form seed by crushing & adding 320µl 0.02% (w/v)NaCl
for 1hr .Centrifuged at 10k for 10mins.
ELECTROPHORESIS-UTLIEF gels caste don polyester film (gel-fix, GE) Gel
is made up of 0.8 urea, 0.16g taurine, 5 ml acrylamide, and
0.22 ml of pH 5-7 ampholytes, 4µl
tetramethylethylenediamine and 30µl of 20% ammonium
peroxydisulphate
sample size is 25µl per well is added

43. Chang
72
Zheng
dan
958
Zheng
58
Dan
598
Danyu
39
C-8605
FMB represents female marker band,
Shen
503
Shenyu
43
Shen
502
Shen
139
shenyu
20
Shen
151
Shen
137
Shenyu
17
Shen
151
MMB represents male marker band
Fig 13: The UTLIEF profile of maize hybrids and their parents.
(Dou et al. 2010)

44. MMB1
Fig 14: The salt soluble protein UTLIEF profile of genetic purity testing Shenyu 17. MMB1 represents male
marker band,
is the mixed seed or self pollinated seed
MMB1
MMB2
FBM1
Fig 15: The salt soluble protein UTLIEF profile of genetic purity testing Shenyu 20. MMB1 and
MMB2 represents male marker band, FBM1 represent female marker band, is the mixed
seed or self pollinated seed

46. (Leist, 2005)

47. Genetic marker are any genetically determined trait
(morphological, biochemical, molecular) that can distinguish
among genotypes
(Leist, 2005)

48. Polymerase Chain Reaction technique
 It requires very little DNA (single seed or leaf)
 It‟s a fast, simple and accurate method
 It is highly sensitive and specific method
(Leist, 2005)

49. Material and methods
Six petunia, five cyclamen hybrids and their parents
RAPD analysis twenty germinated petunia seedlings and 10 cyclamen seeds for DNA
isolation.
Modification- 10µl of proteinase and 10µl 1.0mM CaCl2were added and incubated 1.5hr
at 37ºc to digest protein
Genetic purity studies is done on 10 seeds of each hybrid and inbred parents of F197222
cyclamen cultivar.

50. Kb
M 1 2 3 4 5 6 7 8 9 10 11 C
1.5
6
Fig 16: RAPD markers for 20 bulked seedling and
10 seed samples for petunia and cylamen
cultivars, M= molecular weight DNA marker
lanes 1-6 petunia cultivars (fantasy pink,
prime time blue, ultra crimson star, ultra
blue, prime time red vein, fantasy salmon)
and Lanes 7-11 cylamen cultivars ( F180360,
F197222, F197294, F197358,72229721) and
C= control lane
Kb
Fig 17: RAPD markers for individual
cylamen seeds of hybrid F197222 cultivar,
M= mol. wt. DNA marker, lanes 1-10=
individual hybrid seeds, C= control lane.
M 1 2 3 4 5 6
7 8 9 10 C
1.5
6
(Zhang et al. 1997)

51. Fig 18: RAPD markers for individual cylamen seeds of the female and male inbred
parents of F197222. M= Mol. Wt. DNA marker, Female and male lanes 1-10=
invidual inbred seeds, C= control lane.
(Zhang et al. 1997)

52. CURRENT SCIENCE, VOL. 93.NO. 4. 25 AUGUST
2007
NAMRATA SINGH, MAJOR SINGH, SANJEET KUMAR, RAJESH KUMAR, H. C. PRASANNA, MATHURA
RAI.
Indian Institute Of Vegetable Research
Material and methods
Two hybrids & Parents- NTH-1 (DVRT X Flora Dade) and NTH-7 (DVRT-2 X 97/754) from IIVR
RAPD Reaction mixture-Each amplification mixture of 25 µl contained 2.5 mM MgCl , 10 mM of
each dNTP, 0.5 µl of each primer, 2.5 units of Taq polymerase, and 50 ng of template DNA.
The thermal profile for RAPD-PCR
•
•
•
•
•
Initial Denaturation at 94 C for 1 min.
Complete denaturation-35 cycles of 94 C for 5sec.
Annealing- 35 C for 25secs
Primer extention- 70 C for 30secs
Finally extention- 70 C for 3 min.
PCR products were then subjected to 1.2% agarose gel electrophoresis

53. Fig 19: RAPD marker (0pb161193) present in individual male plant ( Flora Dade, 1-4)
Hybrids (NTH-1, lanes 5-10), and absent in female plant ( DVRT-1, lanes 10-20). M.
(SINGH et al. 2007)

54. Materials and methodsF 1 hybrid -Zaoxia 16 along with the parents was used
Plant part used young leaves of 20 days old seedling
Out of 157 RAPD primers- Three primers (NAURP2006), (NAURP2020) and (NAU2032).
Out of 54 ISSR primers- Two Primers (NAUISR1058) and (NAUISR1060)
Out of 84 SRAP primers- one primer combination (NAUSR04/NAURS05)
Out of 44 SSR primers- Two primers (NAUSSR1011) and (NAUSSR1031)

55. Table 6: Genetic purity of 210 hybrid ‘Zaoxia 16’ individuals determined by identified
molecular markers and field GOTs.
(Liu et al. 2007).

56. Fig. 20. RAPD analysis of ‘Zaoxia 16’ individuals and parents. F hybrids were screened with the
identified primers (a) NAURP2006, (b) NAURP2020, and (c) NAURP2031: lane 1, female parent;
lane 2, male parent; lanes 3–16, individuals 62–75; lane M, DL 2000 DNA ladder (Takara Bio,
Japan). Arrows indicate male parent- and female parent-specific markers.
(Liu et al. 2007).

57. Fig. 21. ISSR analysis of ‘Zaoxia 16’ individuals and parents. F1hybrids were identified with
primers (a) NAUISR1058 and (b) NAUISR1062: lane 1, female parent; lane 2, male parent;
lanes 3–16, individuals 62–75; lane M, DL 2000 DNA ladder (Takara Bio, Japan). Arrows
indicate male parent and female parent-specific markers.
(Liu et al. 2007).

58. Fig. 22. SSR analysis of „Zaoxia 16‟ individuals using primers (a) NAUSSR1011 and (b)
NAUSSR1031: lane 1, female parent; lane 2, male parent; lanes 3–16, individuals 62–75;
lane M, 50-bp DNA ladder. Arrows indicate male parent- and female parent-specific
markers.
(Liu et al. 2007).

59. Materials
MZEs of ‘Tenera’ hybrid, derived from the cross 366 (D) 72 (P), 180 DAP
Hybrid verification via RAPD analysis-carried out using 7 decamer random
oligonucleotide primers (OPB08, OPR11, OPT06, OPT19, OPAB01, OPAB09, and
OPAB14)
Hybrid verification via SSR analysis- carried out using 9 microsatellite loci
amplified in oil palm using 9 primers (EgCIR008, EgCIR0243, EgCIR0337,
EgCIR0409, EgCIR0446, EgCIR0465, EgCIR0781, EgCIR0905, and EgCIR1772)

60. RAPD Reaction mixture-Each amplification mixture of 25 µl contained 2.5
mM MgCl , 10× Taq buffer, 100 µM of each dNTP, 0.3 mM of each primer, 1.5
units of Taq polymerase,and 20 ng of template DNA.
The thermal profile for RAPD-PCR was started from 1 cycle of 95 °C for 1
min, 39 cycles of 95 °C for 1 min, 37 °C for 1 min, 72 °C for 2 min, followed by
1 cycle of 95 °C for 1 min, 37 °C for 1 min, and finally 72 °C for 10 min. PCR
products were then electrophoresed
SSR reaction mixture- each amplification mixture of 10 µl mixture containing
2.5 mM MgCl , 10× Taq buffer, 100 µM of each dNTP, 0.3 mM of each primer,
1.5 units of Taq polymerase and 20 ng of template
The thermal profile for SSR-PCR carried out using the following program:
denaturation at 95 °C for 1 min, 35 cycles of 94 °C for 30 s, 52 °C for 60 s, 72
°C for 120 s, and a final elongation step at 72 °C for 8 min.

61. 650 bp
Fig. 23 RAPD patterns in hybrids and parents of the cross 366 (D) 72 (P) obtained with
primers OPT06. In this and the next figure, the amplification products were compared
on the basis of molecular size. Lane M: standard DNA (100 bp plus DNA ladder).
Lanes P and D: fragments from parents. Lanes 1–15: fragments from hybrids.
(Thawaro and Te-chato, 2009)

62. Fig. 24: SSR patterns in hybrids and parents of the cross 366 (D) 72 (P) obtained with
primers EgCIR1772.
650 bp
Fig. 25:
RAPD pattern of somatic embryo line derived from MZE obtained with primers OPT06.
The amplification products were compared on the basis of molecular size. Lane M:
standard DNA (100 bp plus DNA ladder). Lane P and D: profile of DNA fragments from
parents. Lane 1–15: profile of DNA fragments from hybrids.
(Thawaro and Te-chato, 2009)

63. Advantages of genetic purity
1. It is helpful in plant variety protection, registration,
certification and patents
2. to detect the even the minute genetic differences
between cultivars visa-a-versa for existence of
novelty among essentially derived varieties
3. Assurance of genetic purity for ensuring better
agronomic performance and predicted expectations
4. Prevention of misappropriation and willful
admixture of seed/ cultivars at commercial or
farmers level

64. Let me conclude now…
Genetic purity analysis is THE IMPORTANT
FACTOR for quality seed
For farmer – No loss because of poor seeds +
Higher returns
For producer – Market grip
Technologies in hand – use for the benefit of
humankind

65. Thank you