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Genetic Mapping of Powdery

Mildew Resistance Genes in Wheat

Ainong Shi

Advisors: Steven Leath, and Paul Murphy

North Carolina State University

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Wheat

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Wheat powdery mildew (Blumeria graminis f. sp. tritici) is a disease of major importance.

The use of single gene resistance is the primary method of control of this disease. Identification of molecular markers and genetic mapping can provide a tool for marker assisted breeding.

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Identify molecular markers Genetic mapping of genes for powdery mildew resistance in wheat

Objective

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Genetic mapping procedure

Linkage Analysis

Genetic Map

Primer Screening

Phenotype Evaluation

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An exampleIdentification and mapping of Pm25

gene for wheat powdery mildew resistance

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Coker 68-15 x NC96BGTA5

F1 x Coker 68-15

F2 BC1F1

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Obtaining segregating populations for genetic analysis

(S) (R)

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An example of detached leaf technique for reactions to powdery mildew in a F2 population. The culture dish contains 35 primary leaf segments from 35 F2 plants of a Coker 68-15/NCBGTA5 F2 population and six leaf segments from susceptible Check Chancellor on the each side.

Detached leaf technique

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A scale of 0 to 9• Resistant: 0-3• Intermediate: 4-6• Susceptible: 7-9

Assessment of Reaction to Powdery Mildew

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123

4

5

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9

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0

5

10

15

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25

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45

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0 1 2 3 4 5 6 7 8 9

209a2

Reactions to isolate 209a2 of Bgt in Coker 68-15/NCBGTA5 F2

and Coker 68-15*2/NCBGTA5 BC1F1 populations

Frequency

Infection type

Res. Sus. Exp. 2 Gene -------------------------------------------------------------------------- F2 189 77 3:1 2.210 Pm25BC1F1 55 52 1:1 0.08

F2

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• Method: Bulked segregant analysis (BSA)

• Segregating population: Coker 68-15*2/NCBGTA5 BC1F1

• R bulked from 30 high resistant plants

• S bulked from 30 high susceptible plants

• A total of 156 ten-base random primers

Primer Screening

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Amplification pattern from RAPD marker OPX061050 for Pm25 in four wheat materials:

1. Coker 68-15 (None),

2. NC96BGTA5 (Pm25),

3. S bulked (None),

4. R bulked (Pm25),

An example for BSA

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Amplification pattern of DNA detecting OPX061050, OPAG04950, and OPAI14600 RAPD fragments in the Coker 68-15*2/NC96BGTA5 BC1F1 population. Lane 1 to 5 from susceptible plants and lane 6 to 11 from resistant plants with lane 1 and 7 indicating recombinants.

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_______________________________________________ Rec Dist Marker

Frac cM id Name _______________________________________________

(4) OPAI14600

(4.2%) 4.4 (2) OPX061050

(4.2 %) 4.4 (3) OPAG04950

(11.3 %) 12.8 (1) Pm25

A genetic map of the region carrying Pm25 constructed from the Coker 68-15*2/NC96BGTA5 BC1F1 population

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Another exampleMapping Pm3b gene for resistance to wheat powdery mildew

• Method: NILs• Linkage analysis: F2 seg.

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Amplification patterns of DNA from seven ‘Chancellor’ near-isogenic lines with random primers.

1. Chancellor (Cc, recurrent parent),

2. Axminister/8*Cc (Pm1),

3. Ulka/8*Cc (Pm2),

4. Asosan/8*Cc (Pm3a),

5. Chul/8*Cc (Pm3b),

6. Sonora/8*Cc (Pm3c),

7. Khapli/8*Cc (Pm4a).

1400-bp

1 2 3 4 5 6 7

OPAN07

A total of 332 random Operon primers were used to screen for RAPD markers in the seven ‘Chancellor’ near-isogenic lines.

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Chancellor//Chul/8*Cc FChancellor//Chul/8*Cc F22

0 1 2 3 4 5 6 7 8 90

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10

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0 1 2 3 4 5 6 7 8 9

E314

Isolate Res. Sus. Exp. 2

E314 61 22 3:1 0.100 -------------------------------------------------------

Infection type

Frequency

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Amplification pattern of DNA detecting OPAN071400 RAPD fragments from the F2 population of Chancellor//Chul/8*Cc. PS=susceptible parent, PR=resistant parent, S=Susceptible individual in the F2 and R=resistant individual in the F2. Lane M is a 1-kb molecular-weight marker.

1400bp

1400bp

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Co-segregation of reaction of Pm3b to E314 isolate of Bgt and RAPDmarker OPAN071400 in the Chancellor/(Chul/8*Cc) F2 population.

________________________________________________________ Locus Phenotype 2

A 2B 2

AB Recombination

A B RM* Rm SM Sm (3:1) (3:1) (9:3:3:1) fraction

Pm3b AN071400 61 0 1 21 0.100 0.004 81.776** 1.2________________________________________________________

* RM = resistant plant with the marker, Rm = resistant without the marker, SM = susceptible with the marker, and Sm = susceptible without the maker.

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Pm3bOPAN071400

1.2

Linkage distance

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SUMMARY

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Sixteen RAPD markers linked to Pm1, Pm3b, Pm12, Pm21, Pm25, pmTD1 and Pm3 locus.

Gene RAPD markerPm1 OPU17750

Pm3b OPAN071400

Pm12 OPAE121350, OPAE12495

OPAH05580, OPAI13490

Pm21 OPAN031700, OPAI01700, OPAL03750

Pm25 OPAI14600, OPX061050, OPAG04950

pmTD1 OPQ09750

Pm3 locus OPS031400, OPN09600, OPN091200

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_______________________________________________ Rec Dist Marker

Frac cM id Name _______________________________________________

(4) OPAI14600

(4.2%) 4.4 (2) OPX061050

(4.2 %) 4.4 (3) OPAG04950

(11.3 %) 12.8 (1) Pm25

A genetic map of the region carrying Pm25 constructed from the NK-68-15*2/NC96BGTA5 BC1F1 population

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Pm1OPU17750

2.2Pm3bOPAN071400

1.2

OPAI13490

OPAE12495

OPAH05580

Pm12

OPAE121350

1.6

3.3

1.6

3.3

3.2

0.01.0

Pm3

OPN09600, OPN091200

OPS031400

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ACKNOWLEDGEMENTS

North Carolina State University:Dr. Steven Leath, Dr. J. Paul Murphy,Dr. Martin L. Carson, Dr. Ben-Hui Liu, and Dr. Rebeca C. Rufty.

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Thank you!

Thank all of you!