Methods of genetic purity testing

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

5. Genetic purity ?
Genotypicpurityissimplydefinedas
truetotypeplants/seedsconformingtothe
characteristicsofthevarietyasdescribedby
thebreeders.
Principle
Geneticpurityorgenuinessofthe
cultivaristestedbymeansofheritable
characters(morphological,physiologicalor
chemical)ofseeds,seedlingsorplants.

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

7. 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
Criteria for GOT to decide the genuineness of variety
(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
•Examinationfeaturesofseedssuchaslength,width,
thickness,shape,weight,colour,seedcoatcolouretc.
andcomparingthemwiththoseofauthenticsample.
•Whichareexaminedwithnakedeye/withmagnified
handlens/withthehelpofscanningelectron
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. •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 spand 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.
The size of the submitted sample will be as follows: for GOT
(Basra, 2002)

13. Raisingofdesiredpopulationbyfollowingrecommended
culturalpracticese.g.,fieldpreparation,sizeoftheplot,etc.
Provideequalopportunitytoeachandeveryplantforfull
expressionofgeneticallycontrolledcharacters
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.
Aminimumof200plantsfromcontrolsamplesshouldberaisedalongwiththetest
crop.
Thistestispreferablyconductedinareatowhichthevarietyisrecommended
Theanalystemployedforconducting„growouttest‟shouldpossessthebasic
qualificationasidentifiedunderSeedRules,1968.
Procedure of GOT

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

15. PLANT HABIT FLOWERING HABIT
FRUITING HABIT

16. Methods of testing based on-
1.Analysis of secondary compounds
2.Protein analysis
3.Nucleic acid analysis
CHEMOTAXONOMY
Chemotaxonomistshaverecognizedtwogroupsof
compoundsthataregenerallyusefullinclassificationofplant
species
1.Episemanticorsecondarymetabolites
(pigmentsorfattyacidetc.)
2.Semantidesorsensecarryingmolecules
(Proteins,NucleicAcids)

17. These test ranges from simple colour tests to complex
chromatographic separations of phenols, anthocyanin, flavonoids
and other compounds.
Different tests includes
1.Phenol test
2.Peroxidase test
3.Potassium hydroxide –bleach test
4.Fluorescence test
5.Hydrochloric HCI test
6.Ferrous sulphate test
7.NaOH test
8.Anthocyanin test
9.Seedling pigmentation
Analysis of secondary compounds
(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.Pureseedsof23ricegenotypes
2.Fivechemicaltestsviz.Phenol,modifiedphenol,FerrousSulphate,Potassium
hydroxideandsodiumhydroxide
3.50seedsofeachgenotypewereobserved

19. VARIETY Phenol test Modified Phenol testFeSO4 testKOH testNaOH Test
Very
strong
strongmoderat
e
no
colour
Very
strong
Strongmodera
te
no
colour
DGStBStBSpDWRNo
colou
r
DYLYNO
colou
r
1Chaitanya (-) (-) BSp (-) (-)
2Maruteru (-) (-) BSt (-) (-)
3Vijetha ++ (-) BSt (-) LY
4Tholakari ++ ++ BSp (-) LY
5vajaram (-) (-) BSt (-) LY
6Swarna ++ ++++ DGSt (-) LY
7Deepthi (-) (-) BSp (-) LY
8Krishanveni +++ ++++ DGSt (-)DY
9MTU 1004 ++ +++ BSt (-) LY
10Anjali ++ ++ BSp (-) (-)
11vikas +++ ++++ DGSt (-) LY
12rajendra ++ ++ BSt (-)DY
13ASD-7 +++ ++++ BSp + DY
14PR-113 ++ (-)++++ BSt (-) (-)
15QPE-2 ++ +++ DGSt (-) LY
16Rathuheenathi (-) +++ DGSt + LY
17mudgo ++ ++ BSt + DY
18Tadukan +++ +++ BSt (-) LY
19Varalu ++++ ++++ DGSt (-)DY
20CO-31 ++ ++ BSp (-)DY
21Pooja ++ +++ BSp (-) (-)
22Chenegi ++++ ++++ BSp + LY
23Supreme +++ ++++ DGSt (-) (-)
(Vijaylakshmiand Vijay, 2009)
Table 1: Response of different rice varieties for different chemical test

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

21. Becauseproteinsarethedirectgeneproductthe
analysisofseed,seedlingproteinsandenzymesismost
successfulandwidelyused.Hencemuchattentionhasbeen
focusedonseedstorageproteins.
There are two primary methods
Various types of gel electrophoresis
High pressure liquid chromatography
Protein analysis

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 Rmof 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:separationbysizeandcharge(charge/mass)
Denaturing:separationbysize
Others(IEF,2-Dimenstionalelectrophoresis)
GelElectrophoresis
Nativecontinuoussystem--gelandtankbuffersarethesame,singlephase
gel;examplesarePAGE,agarose,andstarchgels.
DiscontinuousSystem--gelandtankbuffersaredifferent,twophasegel
(stackinggel);exampleisPAGE.
GelElectrophoresisbasedondenaturation
SDS(sodiumdodecylsulphate)usedtodenatureproteins(discontinuous
system).ureaorformamideusedtodenatureDNAorRNA.
Othertypesare
Isoelectricfocusing:protein-separationbasedonisoelectricpointsinapH
gradient.
2-Delectrophoresis:combinationofIEFandSDS-PAGE.

24. Materialsandmethod
1.Itconsistsof8varietiesviz.,CSV15,SPV669,PVK400,PVK801,BTX623,
IS18551,R16andE36-1.
2.4hybridsalongwithparentsandmaintainerlineviz.,CSH14(ms14AXAKR
150),CSH9(ms296axCS3541),CSH18(IMS9AxIndore12)andSPH840
(ms70AxCS3541).
Proteinextraction-using1.5mlsodiumphosphatebuffer(0.1M,pH-7),centrifuged
at10kfor45mins@4
0
CproteinestimationbyLowrymethod
usingalkalinecopperandfolinreagent.

25. Low range protein markers High range protein markers
Sr.
No.
Protein molecular
weight marker
Molecular
weight (Da)
Sr. No.
Protein molecular
weight marker
Molecular
weight (Da)
1Phosphorylaseb 97000 1 Myosin 220000
2serum albumin 66000 2 α-2-macroglobulin 170000
3ovalbumin 45000 3 β-galactosidase 116000
4Carbonic anhydrase30000 4 Transferin 76000
5Trypsin inhibitor20100 5
glutamate
dehydrogenase
53000
6α-lactalbumin 14400
Table 2 : Low range and high range protein molecular markers and their molecular
weight
(Kavimandan and khan, 2012)
Da-Dalton

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. Fig 3: Zymogram of the PAGE patterns of the seed
globulins in cotton hybrids and their patterns
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
DS
-
28
CPD
-
423
B
-
82
-
1
-
1
CPD
-
428
SB (YF)
-
425
DCH
-
32
DHH
-
11
CPD
-
420
DHB
-
10
5
DS
-
28
CPD
-
423
B
-
82
-
1
-
1
CPD
-
428
SB (YF)
-
425
DCH
-
32
DHH
-
11
CPD
-
420
DHB
-
10
5
DS
-
28
CPD
-
423
B
-
82
-
1
-
1
CPD
-
428
SB (YF)
-
425
DCH
-
32
DHH
-
11
CPD
-
420
DHB
-
10
5
Rm value
Rm value
Rm value
1.0
-0.0
-0.0
1.0
-0.0
1.0
( Manjunath Reddy et al. 2008 )
Rm= Relative migration

29. MaterialsandMethods
PlantMaterial:FreshmatureseedsofselectedspeciesofBauhinia.
ProteinExtraction:bymethodgivenbyJensenandLixue.
overnightpresoakedseedsinproteinsolubilizationsolution(62mMTris–HCl,
pH6.8,10%glycerol,2%SDS,β-mercaptoethanolandtracesofbromophenolblue)then
centrifugedat14000rpmfor30seconds.Thesupernatentwascollectedplacedintoa
boilingwaterbathfor4minutes.
SDS-PAGEwasdonebymethodsuggestedbyLaemmli.
Itwasperformedonaverticalslabgel.Bromophenolbluewasaddedtothe
supernatantastrackingdyetowatchthemovementofproteininthegel.Seedproteinwas
analyzedthroughslabtypeSDS-PAGEusing10%Separatinggeland4%Stackinggel.

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

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

32. (Sinha et al. 2012)
Fig7:DiagramaticofSDSproteinprofileofseedof[A]B.variegata;[M]Marker;
[B]B.accuminata;[C]B.purpurae;[D]B.racemosa;[E]B.tomentosa
Fig6:SDSproteinprofileofseedof[A]
B.variegata;[M]Marker;[B]B.
accuminata;[C]B.purpurae;[D]
B.racemosa;[E]B.tomentosa

33. Materials:-
1.Mature seeds of F
1Hybrids of Tomato and their respective parental lines were used
a.F1-hybrid ( 6944 x 2413){parental line carries pollen sterility ms10
35
}
b.F
1 –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 TrisEDTA –boric acid
buffer with pH-8.3
•Staining for isozyme-Glumatedehydrogenase (GDH)

34. Fig 9: Electrophoretic patterns and scheme of GDH in tomato seeds a-maternal parent
line 2197; b-paternal line 2263; C-F
1 hybrid.
Fig 8: Electrophoretic patterns and scheme of GDH in tomato seeds a-maternal parent
line 6944; b-paternal line 2413; C-F
1 hybrid; B
1-
contamination.
(Markova and stilova, 2003)

35. Materials:-
1.16 sunflower Hybrids
2.Protein was extracted form seed by adding 400µl 0.03M TrisHCL pH-8 containing 0.01% 2-
mercaptoethanol for 4hrs. Centrifuged at 11k for 15mins.
3.Proteins were than dissociated by denaturing buffer(0.15M TrispH6.8 containg3%SDS, 5%
mercaptaetanol& 7% glycerol)
ELECTROPHORESI S-Accto Laemmliusing 12.5% P.A gel under denaturing SDS and reducing
mercaptaethanol
Staining for protein using0.24g coomassiebrilliant blue in 90 ml of 1:1 (v/v) methanol :
water and 10 ml of glacial acetic acid
for isozymestem tissues of 5 days old seedling homozinizedin 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. sample
number
genetic purity (%)
EI ESSP
1 98 87
2 90 95
3 91 89
4 98 98
5 92 94
6 99 96
7 98 95
8 93 89
9 94 92
10 97 98
11 89 97
12 88 92
13 91 95
14 96 96
15 97 92
16 95 98
EI -electrophoresis of isoenzymes
ESSP-electrophoresis of seed storage proteins
Table 5: Comparative data of genetic purity level in sunflower hybrids measured
on the basis if isozyme and seed storage protein analyses
( Nikolic et al. 2008 )

39. Iso electric focusing
Thistechniquereliesnotontheratesofmobilitybutontheprotein’snet
charge.IsozymesmovethroughapHgradientundertheinfluenceofanelectric
field.
Astheenzymesmovethroughacidicregionsofthegelandentersinto
areasofhigheralkalinitythenetchargeontheproteinchanges,eventually,it
reachesapHregionwherethenetchargeequalszero.Atthispointtheprotein
willnotmigrateanyfurtherandissaidtobe“focused”.
IEFcanbeusedtodifferentiateproteinswithverysubtlechangesin
aminoacidcomposition.
ProteinsmigratingthroughapHgradient
willcontinuetomoveuntiltheirnet
chargebecomeszero.
(Leist, 2005)

40. 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”.
How does the pH gradient work in the gel matrix
(Leist, 2005)

41. Vortexingby
adding extraction
solution
Loading of
sample on gel
Single bands
Steps in Iso Electric Focusing
Crushed seed
(protein
extraction)
Staining the gel Destainingthe gel
Gel running
Blotting the
stained gel
Gel ready to read
Taking off the gel once
done
(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.
ELECTROPHORESI S-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
tetramethylethylenediamineand 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
Shen
137
Shen
151
shenyu
20
Shen
139
Shenyu
17
Shenyu
43
Shen
503
Shen
502
Shen
151
C-8605
Fig 13: The UTLIEF profile of maize hybrids and their parents.
(Dou et al.2010)
FMBrepresents female marker band, MMBrepresents male marker band

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
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
MMB1
MMB2
FBM1

46. (Leist, 2005)

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

48. It requires very little DNA (single seed or leaf)
It‟s a fast, simple and accurate method
It is highly sensitive and specific method
Polymerase Chain Reaction technique
(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 CaCl
2were 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 F
197222
cyclamen cultivar.

50. M 1 2 3 4 5 6 7 8 9 10 11 C
M 1 2 3 4 5 6 7 8 9 10 C
Kb
1.5
6
Kb
1.5
6
Fig16:RAPDmarkersfor20bulkedseedlingand
10seedsamplesforpetuniaandcylamen
cultivars,M=molecularweightDNAmarker
lanes1-6petuniacultivars(fantasypink,
primetimeblue,ultracrimsonstar,ultra
blue,primetimeredvein,fantasysalmon)
andLanes7-11cylamencultivars(F
180360,
F
197222,F
197294,F
197358,72229721)and
C=controllane
Fig 17: RAPD markers for individual
cylamen seeds of hybrid F
197222 cultivar,
M= mol. wt. DNA marker, lanes 1-10=
individual hybrid seeds, C= control lane.
(Zhang et al. 1997)

51. Fig18:RAPDmarkersforindividualcylamenseedsofthefemaleandmaleinbred
parentsofF
197222.M=Mol.Wt.DNAmarker,Femaleandmalelanes1-10=
invidualinbredseeds,C=controllane.
(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
RAPDReactionmixture-Eachamplificationmixtureof25µlcontained2.5mMMgCl,10mMof
eachdNTP,0.5µlofeachprimer,2.5unitsofTaqpolymerase,and50ngoftemplateDNA.
ThethermalprofileforRAPD-PCR
•InitialDenaturationat94Cfor1min.
•Completedenaturation-35cyclesof94Cfor5sec.
•Annealing-35Cfor25secs
•Primerextention-70Cfor30secs
•Finallyextention-70Cfor3min.
PCRproductswerethensubjectedto1.2%agarosegelelectrophoresis

53. Fig 19: RAPD marker (0pb16
1193) 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 methods-
F
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.RAPDanalysisof‘Zaoxia16’individualsandparents.Fhybridswerescreenedwiththe
identifiedprimers(a)NAURP2006,(b)NAURP2020,and(c)NAURP2031:lane1,femaleparent;
lane2,maleparent;lanes3–16,individuals62–75;laneM,DL2000DNAladder(TakaraBio,
Japan).Arrowsindicatemaleparent-andfemaleparent-specificmarkers.
(Liuetal.2007).

57. Fig.21.ISSRanalysisof‘Zaoxia16’individualsandparents.F
1hybridswereidentifiedwith
primers(a)NAUISR1058and(b)NAUISR1062:lane1,femaleparent;lane2,maleparent;
lanes3–16,individuals62–75;laneM,DL2000DNAladder(TakaraBio,Japan).Arrows
indicatemaleparentandfemaleparent-specificmarkers.
(Liuetal.2007).

58. Fig.22.SSRanalysisof„Zaoxia16‟individualsusingprimers(a)NAUSSR1011and(b)
NAUSSR1031:lane1,femaleparent;lane2,maleparent;lanes3–16,individuals62–75;
laneM,50-bpDNAladder.Arrowsindicatemaleparent-andfemaleparent-specific
markers.
(Liuetal.2007).

59. Materials
MZEsof‘Tenera’hybrid,derivedfromthecross366(D)72(P),180DAP
HybridverificationviaRAPDanalysis-carriedoutusing7decamerrandom
oligonucleotideprimers(OPB08,OPR11,OPT06,OPT19,OPAB01,OPAB09,and
OPAB14)
HybridverificationviaSSRanalysis-carriedoutusing9microsatelliteloci
amplifiedinoilpalmusing9primers(EgCIR008,EgCIR0243,EgCIR0337,
EgCIR0409,EgCIR0446,EgCIR0465,EgCIR0781,EgCIR0905,andEgCIR1772)

60. RAPDReactionmixture-Eachamplificationmixtureof25µlcontained2.5
mMMgCl,10×Taqbuffer,100µMofeachdNTP,0.3mMofeachprimer,1.5
unitsofTaqpolymerase,and20ngoftemplateDNA.
ThethermalprofileforRAPD-PCRwasstartedfrom1cycleof95°Cfor1
min,39cyclesof95°Cfor1min,37°Cfor1min,72°Cfor2min,followedby
1cycleof95°Cfor1min,37°Cfor1min,andfinally72°Cfor10min.PCR
productswerethenelectrophoresed
SSRreactionmixture-eachamplificationmixtureof10µlmixturecontaining
2.5mMMgCl,10×Taqbuffer,100µMofeachdNTP,0.3mMofeachprimer,
1.5unitsofTaqpolymeraseand20ngoftemplate
ThethermalprofileforSSR-PCRcarriedoutusingthefollowingprogram:
denaturationat95°Cfor1min,35cyclesof94°Cfor30s,52°Cfor60s,72
°Cfor120s,andafinalelongationstepat72°Cfor8min.

61. Fig.23RAPDpatternsinhybridsandparentsofthecross366(D)72(P)obtainedwith
primersOPT06.Inthisandthenextfigure,theamplificationproductswerecompared
onthebasisofmolecularsize.LaneM:standardDNA(100bpplusDNAladder).
LanesPandD:fragmentsfromparents.Lanes1–15:fragmentsfromhybrids.
(ThawaroandTe-chato,2009)
650 bp

62. Fig. 24: SSR patterns in hybrids and parents of the cross 366 (D) 72 (P) obtained with
primers EgCIR1772.
650 bp
Fig.25:RAPDpatternofsomaticembryolinederivedfromMZEobtainedwithprimersOPT06.
Theamplificationproductswerecomparedonthebasisofmolecularsize.LaneM:
standardDNA(100bpplusDNAladder).LanePandD:profileofDNAfragmentsfrom
parents.Lane1–15:profileofDNAfragmentsfromhybrids.
(ThawaroandTe-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