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بسم الله الرحمن الرحیمبسم الله الرحمن الرحیم
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
1,3PST-31964
1,3,11PST-38
1987
1,3,12 PST-22
1981
1,3,11,12PST-421990
PST-591998 1,3,11,12,16
1,3,8,11,12,16,17,18,19,20 PST-80
2000
PST-972002
1,3,10,11,12,16,17,18,19,20
PST-98
2002
1,3,8,10,11,12,16,17,18,19,20
New2003 1,3,8,9,10,11,12,16,17,18,19,20
Stepwise Evolution of PST Races Stepwise Evolution of PST Races
12345678
R 47-51
Lemhi Chinese 166
Heines VIIMoroPaha
Druchamp
Produra9
10
1112131415
YamhillStephens
Lee Fielder Tyee Tres Hyak
16 Express 17 Yr818 Yr9 19 Clement
PST-782000 1,3,11,12,16,17,18,19,20
20 Compair
1,3,9,11,12,16,17,18,19,20 PST-99
2002
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
YrSD, YrA, Yr25,Yr24, Yr10, Yr9, Yr7, Yr6, Yr4 and Yr2
Torabi et al., 1997
Yr 3 5 4 10 sp nd cv
Torabi et al., 1999
YrSD, Yr24,Yr23, Yr22, Yr19,
Yr9, Yr8, Yr7, Yr2, YrA and Yr1
Nazari et al., 1998
Yr24,Yr23, Yr22, Yr19, Yr9, Yr8, Yr7,Yr6, Yr24,YrA , YrSU,YrND,YrCV
Torabi 2000, Afshari 2003 and 2006
Yr24, Yr9, Yr7, Yr6, Yr2 and YrA
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Special Greenhouse for Yellow Rust Research
Seed and Plant
Improvement Institute
Glass house
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Sample Collection
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9
Multiplication of the Fungus7-days-old seedlings of cultivar BulaniMaleic hydrazide acid (0.25 g·liter−1) per pot
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11
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After 10 days, latent lesions could be observed for each successful infection
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Collecting Urediniospores
Collecting Urediniospores
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Dried in a Desiccator at 4°C for 3 days, and stored in microtubes
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Microtubes stored in Liquid Nitrogen
Race Determination
according to
Johnson et al., 1972
McNeal et al., 1990
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
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Molecular Research
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DNA extraction
Digestion
Adapter Ligation
Preamplification PCR
Selective amplication
Polyacryl -Amid gel Electrophoresis
Data Analysis
AFLP Analysis
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DNA Extraction1- Grinding ( 15 mg Urediniospores + Mercapto-Ethanol)65C 1h
2-Phenol-Chloroform- Isoamylalchohol 14000rpm 3min 4C
3-Chloroform 14000 3min 4C4-Chloroform 14000 3min 4C
5- IsoPropanple -20C 30min17000rpm 4C 30min
Drying TE BufferRnase 37C 1h
Nanodrop Spectrophotometer
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Selective amplification PCR EcoRI+AC / AG/ GC/ GT MseI+ AA/ AC/ AG/ GT13 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.
DigestionGenomic DNA (100 ng) EcoRI and MseI Restriction Enzyme (5 U each; Biolabs) at 37°C in 40 μl of RL buffer 1 h
Adapter Ligation 5 pmol EcoRI adapter, 50 pmol MseI adapter, 1 pmol ATP 1 U ligase (Fermentas), incubated 3 h more at 37°C diluted Three times
Preamplification PCR
EcoRI+0(5-GACTGCGTACCAATC-3) MseI+0(5-GATGAGTCCTGAGTAA-3)
30 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min.
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Formammid Temprature UREA
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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
AFLP Finger printing Gel
IGS (intergenic spacers) SEQUENCE RESEARCHIGS (intergenic spacers) SEQUENCE RESEARCH
L318 5-GCTACGATCCACTGAGGTTC-3
5SK 5-CTTCGCAGATCGGACGGGAT-3
PCR Reaction
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PCR Product
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Band Cutting
Biospin Gel Extraction Kit
DNA Extraction from the Gel
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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 15o C
Transformation of Ligated PCR:pGRM-T vectorTransformation of Ligated PCR:pGRM-T vector
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1- High efficiency Competent Cells( JM1o9)
2- LB/ Ampicillin/IPTG/X-gal plate
6 h 37oC
Incubate 4oC
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White colones are
selected as Positive
transformed colones
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+
_
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ABI PRISM BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA)
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Virulence to resistanse Genes Yr1 ، Yr2 ، Yr4 ،
Yr6 ، Yr7 ، Yr8 ، Yr9 ، Yr10 ، Yr19 ، Yr22 ، Yr23 ، Yr24 ،
Yr SD ، Yr ND ، Yr SU ، Yr CV و Yr SP
But Yr3 and Yr5
35 Phisiologic Races were Detected178E0A+ Khuzestan and Iranshahr64E 241 A+ Mazandaran Fars Khuzestan Hamadan
Virulence for Yr2, Yr24, Yr7 and YrA was detected in all around Iran
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Safi3 , Ahvz, Ilam and Ghazvin 4.6%
First time Detected 1999 Khorasan
Yr1
Persian Gulf
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Heines Kolben Yr2 77% Heines PEKO Yr2 and Yr6 37% Kalyansona Yr2
2000 reported
Yr2
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Yr 4 1997 for the first time34%
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Yr 6
Torabi et al., 1997Nazari et al., 1998 High F.Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
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Yr7
Yr 7
In Lee and Riechesberg 42
Torabi et al., 1997Nazari et al., 1998 Common.Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
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Yr 8
Nazari et al., 1998 Common.Torabi et al., 1999 Low F.Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
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Yr9
1993 Falat EpidemyTorabi et al., 1997Nazari et al., 1998 Common.Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
Yr10Torabi et al., 1997Mardukhi and Torabi., 1998 Shiraz and Moghan Torabi et al., 1999Torabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
Yr19Compare line ( Yr8)
Torabi et al., 1997Nazari et al., 1998
Yr22
Lee Yr7Yr22 and Yr23( Chen and Line 1995)Torabi et al., 1998Nazari et al., 1998
Yr23
Lee Yr7Yr22 and Yr23( Chen and Line 1995)Torabi et al., 1998Nazari et al., 1998
Yr 24
Meering+24 Isogenic lineTorabi et al., 1997Torabi et al., 1999Nazari et al., 1998 CommonTorabi et al., 2000 High F.Afshari et al., 2003, 2005 High F.
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A
Golestan and
Mazandadaran
B
Mazandaran
C
Khuzestan
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D Fars
E Fars
F Fars
G Khorassan
H Ardebil
I Center West and North west
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D Fars
E Fars
F Fars
G Khorassan
H Ardebil
I Center West and North west
B
D
E
H
A
C
F
G
I
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High Genetic Variation AND Relationship between Geographical regions and AFLP groups
Why:1- Different Environmental Condition Different cultivars Different History of EvolutionIn 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.
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Genetic Distance and Identity of Populations
54% Similarity
metapopulation
Golestan
Mazandaran
Khorassan
Khuzestan
West & nwest
Fars
Center
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D Fars
E Fars
F Fars
G Khorassan
H Ardebil
I Center West and North west
AB
C
F D
E
G
H
I
M & GW& NWKhoKhuF
Mediteranian Wind
Flow
Sudanian Wind Flow
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Conclusion:However the interplay of selection, Genetic drift, Migration and Mutation has a major effect on the genetic diversity of this pathogen population The relative roles of this factors may change markedly beteween different Pathogen-host association, Between stages in the epidemiological cycle and between association in agricultural and natural ecosystems.
It is inevitable that the layer of selection is imposed on individual pathogen demes by a range of biotic and abiotic factors.
Host related factors: Major resistance genes , Variation in presence, Quantity and relative balance of phenolic compounds , Variation in morphology and .. Are Important However Their effect on population diversity is not predictable.
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DamavandIRAN
Thanks
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