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Methods of genetic purity testing

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Page 1: Methods of genetic purity testing
Page 2: Methods of genetic purity testing

What about seeds th

en???

What about seeds th

en???

Page 3: Methods of genetic purity testing
Page 4: Methods of genetic purity testing

KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI

Methods of genetic purity testingMethods of genetic purity testing

Page 5: Methods of genetic purity testing

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.

Page 6: Methods of genetic purity testing

Minimum standards for genetic purity for different class of seeds.

SL. No. CLASS OF SEEDS PURITY %

1 Breeder Seeds 100%

2 Foundation seeds 99%

3 Certified seeds 98%

4 Hybrids 95%

(Basra, 2002)

Factors affecting genetic purity.

1. Natural crossing2. Mechanical admixtures3. Random drift4. Mutation5. Selective influence of pest and diseases.

Page 7: Methods of genetic purity testing

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)

Page 8: Methods of genetic purity testing

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)

Page 9: Methods of genetic purity testing

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)

Page 10: Methods of genetic purity testing

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)

Page 11: Methods of genetic purity testing

In field or green house conditionIn 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)

Page 12: Methods of genetic purity testing

• 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.

The size of the submitted sample will be as follows: for GOT

(Basra, 2002)

Page 13: Methods of genetic purity testing

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.

A minimum of 200 plants from control samples should be raised along with the test crop.

This test is preferably conducted in area to which the variety is recommended

The analyst employed for conducting ‘grow out test’ should possess the basic qualification as identified under Seed Rules, 1968.

Procedure of GOT

Page 14: Methods of genetic purity testing

Traditional approach to purity testing

Morphological traitsON SEED ON SEEDLING

IN FIELDIN LAB OR GREEN HOUSE

Page 15: Methods of genetic purity testing

PLANT HABIT FLOWERING HABIT

FRUITING HABIT

Page 16: Methods of genetic purity testing

Methods of testing based on-

1.Analysis of secondary compounds

2.Protein analysis

3.Nucleic acid analysis

CHEMOTAXONOMYChemo 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)

Page 17: Methods of genetic purity testing

These test ranges from simple colour tests to complex chromatographic separations of phenols, anthocyanin, flavonoids and other compounds.

Different tests includes1.Phenol test2.Peroxidase test3.Potassium hydroxide – bleach test4.Fluorescence test5.Hydrochloric HCI test6.Ferrous sulphate test7.NaOH test8.Anthocyanin test9.Seedling pigmentation

Analysis of secondary compounds

(Basra, 2002)

Page 18: Methods of genetic purity testing

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.•Pure seeds of 23 rice genotypes •Five chemical tests viz. Phenol, modified phenol, Ferrous Sulphate, Potassium hydroxide and sodium hydroxide •50 seeds of each genotype were observed

Page 19: Methods of genetic purity testing

  VARIETY Phenol test Modified Phenol test FeSO4 test KOH test NaOH Test    Very

strongstrong moderat

e no

colourVery

strongStrong modera

te no

colourDGSt BSt BSp DWR No

colour

DY LY NO colou

r1 Chaitanya       (-)       (-)     BSp   (-)     (-)2 Maruteru       (-)       (-)   BSt     (-)     (-)3 Vijetha     ++         (-)   BSt     (-)   LY  4 Tholakari     ++       ++       BSp   (-)   LY  5 vajaram       (-)       (-)   BSt     (-)   LY  6 Swarna     ++   ++++       DGSt       (-)   LY  7 Deepthi       (-)       (-)     BSp   (-)   LY  8 Krishan veni   +++     ++++       DGSt       (-) DY    9 MTU 1004     ++     +++       BSt     (-)   LY  

10 Anjali     ++       ++       BSp   (-)     (-)11 vikas   +++     ++++       DGSt       (-)   LY  12 rajendra     ++       ++     BSt     (-) DY    13 ASD-7   +++     ++++           BSp +   DY    14 PR-113     ++ (-) ++++         BSt     (-)     (-)15 QPE-2     ++     +++     DGSt       (-)   LY  16 Rathuheenathi       (-)   +++     DGSt     +     LY  17 mudgo     ++       ++     BSt   +   DY    18 Tadukan   +++       +++       BSt     (-)   LY  19 Varalu ++++       ++++       DGSt       (-) DY    20 CO-31     ++       ++       BSp   (-) DY    21 Pooja     ++     +++         BSp   (-)     (-)22 Chenegi ++++       ++++           BSp +     LY  23 Supreme   +++     ++++       DGSt       (-)     (-)

(Vijaylakshmi and Vijay, 2009)

Table 1: Response of different rice varieties for different chemical test

Page 20: Methods of genetic purity testing

(Vijaylakshmi and Vijay, 2009)

Figure 1- Schematic results of all five chemical tests

Page 21: Methods of genetic purity testing

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

Protein analysis

Page 22: Methods of genetic purity testing

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

Page 23: Methods of genetic purity testing

Gel Electrophoresis-based on type of separationNative: separation by size and charge (charge/mass)Denaturing: separation by sizeOthers (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 denaturationSDS (sodium dodecyl sulphate) used to denature proteins (discontinuous system). urea or form amide used to denature DNA or RNA.

Other types areIsoelectric focusing: protein-separation based on isoelectric points in a pH gradient.2-D electrophoresis: combination of IEF and SDS-PAGE.

Page 24: Methods of genetic purity testing

Materials and method1.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.

Page 25: Methods of genetic purity testing

 Low range protein markers High range protein markers

Sr. No.

Protein molecular weight marker

Molecular weight (Da)

Sr. No.Protein molecular

weight markerMolecular

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 5glutamate dehydrogenase

53000

6 α- lactalbumin 14400   

Table 2 : Low range and high range protein molecular markers and their molecular weight

(Kavimandan and khan, 2012)Da- Dalton

Page 26: Methods of genetic purity testing

Fig 2 : Schematic diagram of the SDS-PAGE profiles of the seed albumins in the sorghum varieties, hybrids, parents & control.

(Kavimandan and khan, 2012)

Page 27: Methods of genetic purity testing

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

Page 28: Methods of genetic purity testing

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

CP

D-

42

3

B-8

2-1

-1

CP

D-

42

8

SB

(Y

F)-

425

DC

H-3

2

DH

H-1

1

CP

D-4

20

DH

B-1

05

DS

-28

CP

D-

42

3

B-8

2-1

-1

CP

D-

42

8

SB

(Y

F)-

425

DC

H-3

2

DH

H-1

1

CP

D-4

20

DH

B-1

05

DS

-28

CP

D-

42

3

B-8

2-1

-1

CP

D-

42

8

SB

(Y

F)-

425

DC

H-3

2

DH

H-1

1

CP

D-4

20

DH

B-1

05

Rm

val

ue

Rm

val

ue

Rm

val

ue

1.0

-0.0-0.0

1.0

-0.0

1.0

( Manjunath Reddy et al. 2008 )

Rm= Relative migration

Page 29: Methods of genetic purity testing

Materials and Methods Plant Material: 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.

Page 30: Methods of genetic purity testing

(Sinha et al. 2012)

Band No.

Rf valueMol.Wt.in

KDaMarker

B.acuminata

B.parpurca B.racemosa B.tomentosa B.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

Page 31: Methods of genetic purity testing

(Sinha et al. 2012)

S. No. Species x Species Percentage similarity1 B. acuminata x B. purpurea 45.45%2 B. acuminata xB. Racemosa 33.33%3 B. acuminata x 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. purpurae X B. varigata 40.00%8 B.racemosa X B. tomentosa 43.75%9 B.racemosa X B. variegata 20.00%

10 B. tomentosa X B. variegata 35.71%

TABLE 4: Percentage Similarity Index between Bauhinia Species

Page 32: Methods of genetic purity testing

(Sinha et al. 2012)

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

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

Page 33: Methods of genetic purity testing

Materials:-

1.Mature seeds of F1Hybrids of Tomato and their respective parental lines were useda. 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 36hr3.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)

Page 34: Methods of genetic purity testing

Fig 9: Electrophoretic patterns and scheme of GDH in tomato seeds a-maternal parent line 2197; b- paternal line 2263; C-F1 hybrid.

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.

(Markova and stilova, 2003)

Page 35: Methods of genetic purity testing

Materials:-1.16 sunflower Hybrids 2.Protein was extracted form seed by adding 400µl 0.03M Tris HCL pH-8 containing 0.01% 2-mercaptoethanol 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 acidfor isozyme stem tissues of 5 days old seedling homozinized in 50mM TrisHCL, pH- 6.8 in 1% mercaptaethanol ISOZYME SYSTEMS -(PHI), (PGM) & (PGD)

Page 36: Methods of genetic purity testing

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 )

Page 37: Methods of genetic purity testing

Figure 12: PHI (a), PGM (b) and PGD (c) isozyme patters of sunflower hybrids

( Nikolic et al. 2008 )

Page 38: Methods of genetic purity testing

sample number

genetic purity (%) 

  EI ESSP1 98 872 90 953 91 894 98 985 92 946 99 967 98 958 93 899 94 92

10 97 9811 89 9712 88 9213 91 9514 96 9615 97 9216 95 98

EI - electrophoresis of isoenzymesESSP- 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 )

Page 39: Methods of genetic purity testing

Iso electric focusingThis 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)

Page 40: Methods of genetic purity testing

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)

Page 41: Methods of genetic purity testing

Vortexing by adding extraction

solution

Loading of sample on gel

Single bands

Steps in Iso Electric Focusing

Crushed seed (protein

extraction)

Staining the gel Destaining the gel

Gel running

Blotting the stained gel

Gel ready to read

Taking off the gel once done

(Leist, 2005)

Page 42: Methods of genetic purity testing

Materials:-1.5 maize hybrids and their respective parents2.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

Page 43: Methods of genetic purity testing

Chang72

Zhengdan958

Zheng58

Dan598

Danyu39

Shen 137

Shen151

shenyu20

Shen139

Shenyu17

Shenyu43

Shen503

Shen502

Shen 151

C-8605

Fig 13: The UTLIEF profile of maize hybrids and their parents.(Dou et al. 2010)

FMB represents female marker band, MMB represents male marker band

Page 44: Methods of genetic purity testing

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

MMB2FBM1

Page 45: Methods of genetic purity testing
Page 46: Methods of genetic purity testing

(Leist, 2005)

Page 47: Methods of genetic purity testing

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

(Leist, 2005)

Page 48: Methods of genetic purity testing

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)

Page 49: Methods of genetic purity testing

Material and methodsSix 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.

Page 50: Methods of genetic purity testing

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

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

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.

(Zhang et al. 1997)

Page 51: Methods of genetic purity testing

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)

Page 52: Methods of genetic purity testing

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

Page 53: Methods of genetic purity testing

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)

Page 54: Methods of genetic purity testing

Materials and methods-

F 1 hybrid -Zaoxia 16 along with the parents was usedPlant 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)

Page 55: Methods of genetic purity testing

Table 6: Genetic purity of 210 hybrid ‘Zaoxia 16’ individuals determined by identified molecular markers and field GOTs.

(Liu et al. 2007).

Page 56: Methods of genetic purity testing

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).

Page 57: Methods of genetic purity testing

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).

Page 58: Methods of genetic purity testing

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).

Page 59: Methods of genetic purity testing

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)

Page 60: Methods of genetic purity testing

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.

Page 61: Methods of genetic purity testing

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)

650 bp

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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)

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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

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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

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Thank you