Hojjat Rabbani, General Manager of North khorassan Agricultural and Natural Resources Research Center

1. بسم الله الرحمن الرحیم

2. Virulence and Molecular diversity in Puccinia striiformis f.sp . tritici from IRAN Hojjat Rabbani General Manager of North khorassan Agricultural and Natural Resources Research Center

3. 1,3 PST-3 1964 Stepwise Evolution of PST Races 1 2 3 4 5 6 7 8 R 47-51 Lemhi Chinese 166 Heines VII Moro Paha Druchamp Produra 9 10 11 12 13 14 15 Yamhill Stephens Lee Fielder Tyee Tres Hyak 16 Express 17 Yr8 18 Yr9 19 Clement 1,3, 11 PST-38 1987 1,3, 12 PST-22 1981 1,3, 11,12 PST-42 1990 PST-59 1998 1,3,11,12, 16 1,3, 8 ,11,12,16,17,18,19,20 PST-80 2000 PST-97 2002 1,3, 10 ,11,12,16,17,18,19,20 PST-98 2002 1,3, 8,10 ,11,12,16,17,18,19,20 New 2003 1,3, 8,9,10 ,11,12,16,17,18,19,20 PST-78 2000 1,3,11,12,16, 17,18,19,20 20 Compair 1,3, 9 ,11,12,16,17,18,19,20 PST-99 2002

4. Start of Race studing 20A & 25A 1960 Neiman et al .,1968 Bamdadian 1972 14/8 ، 14/8A ، 19, 2D 20A and 25A 96E16 on Falat and Qods cultivars Bamdadian 1984 Yr SD , Yr A, Yr 25, Yr 24, Yr 10, Yr 9, Yr 7, Yr 6, Yr 4 and Yr 2 Torabi et al., 1997 Reviwes Yr 3 5 4 10 sp nd cv Torabi et al., 1999 Yr SD, Yr 24, Yr 23, Yr 22, Yr 19, Yr 9, Yr 8, Yr 7, Yr 2, YrA and Yr 1 Nazari et al., 1998 Yr 24, Yr 23, Yr 22, Yr 19, Yr 9, Yr 8, Yr 7, Yr 6, Yr 24, Yr A , Yr SU, Yr ND, Yr CV Torabi 2000, Afshari 2003 and 2006 Yr 24, Yr 9, Yr 7, Yr 6, Yr 2 and YrA

5. Material & Methods Special Greenhouse for Yellow Rust Research Seed and Plant Improvement Institute Glass house

6. Sampling 2005-2006 Material & Methods

7. Sample Collection Material & Methods

8. Material & Methods

9. Material & Methods Multiplication of the Fungus 7-days-old seedlings of cultivar Bulani Maleic hydrazide acid (0.25 g·liter −1 ) per pot

10. Material & Methods

11. Material & Methods

12. Material & Methods After 10 days, latent lesions could be observed for each successful infection

13. Material & Methods Collecting Urediniospores

14. Material & Methods Dried in a Desiccator at 4°C for 3 days, and stored in microtubes

15. Material & Methods Microtubes stored in Liquid Nitrogen

16. Race Determination according to Johnson et al ., 1972 McNeal et al ., 1990 Material & Methods two-leaf-stage onto seedlings of each variety. Incubation: dark climatic room for 24 h at 8°C and 100% relative humidity greenhouse at 20°C for 10 days

17. Molecular Research

18. DNA extraction Digestion Adapter Ligation Preamplification PCR Selective amplication Polyacryl -Amid gel Electrophoresis Data Analysis AFLP Analysis Material & Methods

19. DNA Extraction 1- Grinding ( 15 mg Urediniospores + Mercapto-Ethanol) 65C 1h 2-Phenol-Chloroform- Isoamylalchohol 14000rpm 3min 4C 3-Chloroform 14000 3min 4C 4-Chloroform 14000 3min 4C 5- IsoPropanple -20C 30min 17000rpm 4C 30min Drying TE Buffer Rnase 37C 1h Material & Methods Nanodrop Spectrophotometer

20. Selective amplification PCR Eco RI+ AC / AG/ GC/ GT MseI + AA/ AC/ AG/ GT 13 cycles at 94°C for 30 s, 65°C for 30 s with a 0.7°C decrement per cycle, and 72°C for 1 min, and then 23 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min . Digestion Genomic DNA (100 ng) Eco RI and Mse I Restriction Enzyme (5 U each; Biolabs) at 37°C in 40 μl of RL buffer 1 h Adapter Ligation 5 pmol Eco RI adapter , 50 pmol Mse I adapter , 1 pmol ATP 1 U ligase (Fermentas), incubated 3 h more at 37°C diluted Three times Preamplification PCR Eco RI+0(5-GACTGCGTACCAATC-3) Mse I+0(5-GATGAGTCCTGAGTAA-3) 30 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min. Material & Methods

21. Formammid Temprature UREA Material & Methods

22. Silver staining 1- Fix ( 10% acetic acid) 2- Rinse ( Ultr pure water) 3- Impregnate (AgNo3 1) 4- Rinse 5- Develop 6- Stop 7- rinse and store Material & Methods AFLP Finger printing Gel

23. IGS (intergenic spacers) SEQUENCE RESEARCH L318 5-GCTACGATCCACTGAGGTTC-3 5SK 5-CTTCGCAGATCGGACGGGAT-3 Material & Methods PCR Reaction

24. Material & Methods PCR Product

25. Band Cutting Bio spin Gel Extraction Kit DNA Extraction from the Gel Material & Methods

26. DNA Cloning according to pGEM - T Vector System R 1- Ligation of the pGEM-T vector and PCR product Promega Cooporation T4 DNA Ligase T4 DNA Ligase 10 X buffer pGEM-T vector Insert DNA At least 3 h. at 15 o C Material & Methods

27. Transformation of Ligated PCR:pGRM-T vector 1- High efficiency Competent Cells( JM1o9) 2- LB/ Ampicillin/IPTG/X-gal plate 6 h 37 oC Incubate 4 oC Material & Methods

28. White colones are selected as Positive transformed colones Material & Methods

29. + _ Material & Methods

30. ABI PRISM  BigDye  Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) Material & Methods

31. Results & Discussion

32. V irulence to resistanse Genes Yr 1 ، Yr 2 ، Yr 4 ، Yr 6 ، Yr 7 ، Yr 8 ، Yr 9 ، Yr 10 ، Yr 19 ، Yr 22 ، Yr 23 ، Yr 24 ، Yr SD ، Yr ND ، Yr SU ، Yr CV و Yr SP But Yr 3 and Yr 5 35 Phisiologic Races were Detected 178E0A+ Khuzestan and Iranshahr 64E 241 A+ Mazandaran Fars Khuzestan Hamadan Virulence for Yr 2, Yr 24 , Yr 7 and Yr A was detected in all around Iran Results & Discussion

33. Safi3 , Ahvz, Ilam and Ghazvin 4.6% First time Detected 1999 Khorasan Yr 1 Results & Discussion Persian Gulf

34. Heines Kolben Yr2 77% Heines PEKO Yr2 and Yr6 37% Kalyansona Yr2 2000 reported Yr 2 Results & Discussion

35. Yr 4 1997 for the first time 34% Results & Discussion

36. Yr 6 Results & Discussion Torabi et al ., 1997 Nazari et al ., 1998 High F. Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F .

37. Yr7 Results & Discussion Yr 7 In Lee and Riechesberg 42 Torabi et al ., 1997 Nazari et al ., 1998 Common. Torabi et al ., 1999 Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F .

38. Yr 8 Results & Discussion Nazari et al ., 1998 Common. Torabi et al ., 1999 Low F. Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F .

39. Yr 9 Results & Discussion 1993 Falat Epidemy Torabi et al ., 1997 Nazari et al ., 1998 Common. Torabi et al ., 1999 Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F .

40. Yr 10 Torabi et al ., 1997 Mardukhi and Torabi., 1998 Shiraz and Moghan Torabi et al ., 1999 Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F . Results & Discussion

41. Yr19 Compare line ( Yr 8) Torabi et al ., 1997 Nazari et al ., 1998 Results & Discussion

42. Yr22 Lee Yr 7 Yr22 and Yr23 ( Chen and Line 1995) Torabi et al ., 1998 Nazari et al ., 1998 Results & Discussion

43. Yr23 Lee Yr 7 Yr22 and Yr23 ( Chen and Line 1995) Torabi et al ., 1998 Nazari et al ., 1998 Results & Discussion

44. Yr 24 Meering+24 Isogenic line Torabi et al ., 1997 Torabi et al ., 1999 Nazari et al., 1998 Common Torabi et al ., 2000 High F . Afshari et al ., 2003, 2005 High F . Results & Discussion

45. A Golestan and Mazandadaran B Mazandaran C Khuzestan Results & Discussion

46. D Fars E Fars F Fars G Khorassan H Ardebil I Center West and North west Results & Discussion

47. D Fars E Fars F Fars G Khorassan H Ardebil I Center West and North west Results & Discussion B D E H A C F G I

48. High Genetic Variation AND Relationship between Geographical regions and AFLP groups Why: 1- Different Environmental Condition Different cultivars Different History of Evolution In a uniform environment selection may favor particular genotypes This metapopulation view of evolution and diversity of pathogen population differed markedy from models that are based on evolution in a uniform invironment where mutation rate and frequency dependent selection are the most important pressure. 2- In Addition to Different Climatic condition Each Provinces Prefer special Cultivars Special History of Evolution Individuals respond asynchronously to differences in the local selective environment.

49. Genetic Distance and Identity of Populations 54% Similarity metapopulation Golestan Mazandaran Khorassan Khuzestan West & nwest Fars Center

50. D Fars E Fars F Fars G Khorassan H Ardebil I Center West and North west Results & Discussion A B C F D E G H I

51. M & G W& NW Kho Khu F Mediteranian Wind Flow Sudanian Wind Flow

53. Damavand IRAN Thanks