Methods of genetic purity testing

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Methods of genetic purity testing

  1. 1. Methods of genetic purity testing KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI
  2. 2. 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.
  3. 3. 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)
  4. 4. 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)
  5. 5. 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)
  6. 6. 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)
  7. 7. 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)
  8. 8. 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)
  9. 9. 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)
  10. 10. 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.
  11. 11. Traditional approach to purity testing Morphological traits ON SEED IN LAB OR GREEN HOUSE ON SEEDLING IN FIELD
  12. 12. PLANT HABIT FLOWERING HABIT FRUITING HABIT
  13. 13. 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
  14. 14. 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)
  15. 15. 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
  16. 16. 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)
  17. 17. Figure 1- Schematic results of all five chemical tests (Vijaylakshmi and Vijay, 2009)
  18. 18. 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
  19. 19. 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
  20. 20. 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.
  21. 21. 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.
  22. 22. 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)
  23. 23. Fig 2 : Schematic diagram of the SDS-PAGE profiles of the seed albumins in the sorghum varieties, hybrids, parents & control. (Kavimandan and khan, 2012)
  24. 24. 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
  25. 25. 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 )
  26. 26. 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.
  27. 27. 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)
  28. 28. 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)
  29. 29. 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)
  30. 30. 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)
  31. 31. 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)
  32. 32. 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)
  33. 33. 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 )
  34. 34. Figure 12: PHI (a), PGM (b) and PGD (c) isozyme patters of sunflower hybrids ( Nikolic et al. 2008 )
  35. 35. 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 )
  36. 36. 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)
  37. 37. 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)
  38. 38. 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)
  39. 39. 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
  40. 40. 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)
  41. 41. 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
  42. 42. (Leist, 2005)
  43. 43. Genetic marker are any genetically determined trait (morphological, biochemical, molecular) that can distinguish among genotypes (Leist, 2005)
  44. 44. 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)
  45. 45. 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.
  46. 46. 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)
  47. 47. 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)
  48. 48. 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
  49. 49. 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)
  50. 50. 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)
  51. 51. Table 6: Genetic purity of 210 hybrid ‘Zaoxia 16’ individuals determined by identified molecular markers and field GOTs. (Liu et al. 2007).
  52. 52. 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).
  53. 53. 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).
  54. 54. 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).
  55. 55. 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)
  56. 56. 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.
  57. 57. 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)
  58. 58. 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)
  59. 59. 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
  60. 60. 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
  61. 61. Thank you

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