1. 1 Genetic Mapping of Powdery Mildew Resistance Genes in Wheat Ainong Shi Advisors: Steven Leath, and Paul Murphy North Carolina State University

2. 2 Wheat

3. 3 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.

4. 4 l Identify molecular markers l Genetic mapping of genes for powdery mildew resistance in wheat Objective

5. 5 Genetic mapping procedure Linkage Analysis Genetic Map Primer Screening Phenotype Evaluation

6. 6 An example Identification and mapping of Pm25 gene for wheat powdery mildew resistance

7. 7 Coker 68-15 x NC96BGTA5 F 1 x Coker 68-15 F 2 BC 1 F 1 X Obtaining segregating populations for genetic analysis (S) (R)

8. 8 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

9. 9 A scale of 0 to 9 Resistant: 0-3 Intermediate: 4-6 Susceptible: 7-9 Assessment of Reaction to Powdery Mildew 0 1 2 3 4 5 6 7 8 9

10. 10 Reactions to isolate 209a2 of Bgt in Coker 68-15/NCBGTA5 F 2 and Coker 68-15*2/NCBGTA5 BC 1 F 1 populations Frequency Infection type Res.Sus. Exp.  2 Gene -------------------------------------------------------------------------- F2 189773:1 2.210 Pm25 BC1F155521:10.08 F2

11. 11 Method: Bulked segregant analysis (BSA) Segregating population: Coker 68-15*2/NCBGTA5 BC 1 F 1 R bulked from 30 high resistant plants S bulked from 30 high susceptible plants A total of 156 ten-base random primers Primer Screening

12. 12 Amplification pattern from RAPD marker OPX06 1050 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

13. 13 Amplification pattern of DNA detecting OPX06 1050, OPAG04 950, and OPAI14 600 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.

14. 14 _______________________________________________ Rec Dist Marker Frac cM id Name _______________________________________________ (4)OPAI14 600 (4.2%) 4.4 (2)OPX06 1050 (4.2 %) 4.4 (3)OPAG04 950 (11.3 %) 12.8 (1) Pm25 A genetic map of the region carrying Pm25 constructed from the Coker 68-15*2/NC96BGTA5 BC 1 F 1 population

15. 15 Another example Mapping Pm3b gene for resistance to wheat powdery mildew Method: NILs Linkage analysis: F 2 seg.

16. 16 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.

17. 17 Chancellor//Chul/8*Cc F 2 IsolateRes.Sus.Exp.  2 E 3 1461223:10.100 ------------------------------------------------------- Infection type Frequency

18. 18 Amplification pattern of DNA detecting OPAN07 1400 RAPD fragments from the F 2 population of Chancellor//Chul/8*Cc. P S =susceptible parent, P R =resistant parent, S=Susceptible individual in the F 2 and R=resistant individual in the F 2. Lane M is a 1-kb molecular-weight marker. 1400bp

19. 19 Co-segregation of reaction of Pm3b to E 3 14 isolate of Bgt and RAPD marker OPAN07 1400 in the Chancellor/(Chul/8*Cc) F 2 population. ________________________________________________________ Locus Phenotype  2 A  2 B  2 AB Recombination A B RM* Rm SM Sm (3:1)(3:1) (9:3:3:1) fraction Pm3bAN07 1400 61 0 1 210.1000.00481.776** 1.2 ________________________________________________________ * RM = resistant plant with the marker, Rm = resistant without the marker, SM = susceptible with the marker, and Sm = susceptible without the maker.

20. 20 Pm3b OPAN07 1400 1.2 Linkage distance

21. 21 SUMMARY

22. 22 Sixteen RAPD markers linked to Pm1, Pm3b, Pm12, Pm21, Pm25, pmTD1 and Pm3 locus. GeneRAPD marker Pm1OPU17 750 Pm3bOPAN07 1400 Pm12OPAE12 1350, OPAE12 495 OPAH05 580, OPAI13 490 Pm21 OPAN03 1700, OPAI01 700, OPAL03 750 Pm25OPAI14 600, OPX06 1050, OPAG04 950 pmTD1OPQ09 750 Pm3 locus OPS03 1400, OPN09 600, OPN09 1200

23. 23 _______________________________________________ Rec Dist Marker Frac cM id Name _______________________________________________ (4)OPAI14 600 (4.2%) 4.4 (2)OPX06 1050 (4.2 %) 4.4 (3)OPAG04 950 (11.3 %) 12.8 (1) Pm25 A genetic map of the region carrying Pm25 constructed from the NK-68-15*2/NC96BGTA5 BC 1 F 1 population

24. 24 Pm1 OPU17 750 2.2 Pm3b OPAN07 1400 1.2 OPAI13 490 OPAE12 495 OPAH05 580 Pm12 OPAE12 1350 1.6 3.3 1.6 3.3 3.2 0.0 1.0 Pm3 OPN09 600, OPN09 1200 OPS03 1400

25. 25 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.

26. 26 Thank you! Thank all of you!