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

2 Wheat

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

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

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

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

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 A scale of 0 to 9 Resistant: 0-3 Intermediate: 4-6 Susceptible: 7-9 Assessment of Reaction to Powdery Mildew

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 F : Pm25 BC1F155521:10.08 F2

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 Amplification pattern from RAPD marker OPX for Pm25 in four wheat materials: 1. Coker (None), 2. NC96BGTA5 (Pm25), 3. S bulked (None), 4. R bulked (Pm25), An example for BSA

13 Amplification pattern of DNA detecting OPX , OPAG04 950, and OPAI 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 _______________________________________________ Rec Dist Marker Frac cM id Name _______________________________________________ (4)OPAI (4.2%) 4.4 (2)OPX (4.2 %) 4.4 (3)OPAG (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 Another example Mapping Pm3b gene for resistance to wheat powdery mildew Method: NILs Linkage analysis: F 2 seg.

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) bp OPAN07 A total of 332 random Operon primers were used to screen for RAPD markers in the seven ‘Chancellor’ near-isogenic lines.

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

18 Amplification pattern of DNA detecting OPAN 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 Co-segregation of reaction of Pm3b to E 3 14 isolate of Bgt and RAPD marker OPAN 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 Pm3bAN ** 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 Pm3b OPAN Linkage distance

21 SUMMARY

22 Sixteen RAPD markers linked to Pm1, Pm3b, Pm12, Pm21, Pm25, pmTD1 and Pm3 locus. GeneRAPD marker Pm1OPU Pm3bOPAN Pm12OPAE , OPAE OPAH05 580, OPAI Pm21 OPAN , OPAI01 700, OPAL Pm25OPAI14 600, OPX , OPAG pmTD1OPQ Pm3 locus OPS , OPN09 600, OPN

23 _______________________________________________ Rec Dist Marker Frac cM id Name _______________________________________________ (4)OPAI (4.2%) 4.4 (2)OPX (4.2 %) 4.4 (3)OPAG (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 Pm1 OPU Pm3b OPAN OPAI OPAE OPAH Pm12 OPAE Pm3 OPN09 600, OPN OPS

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 Thank you! Thank all of you!