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BGRI	
  Technical	
  Workshop	
  	
  
New	
  Delhi,	
  India,	
  19-­‐22	
  August	
  2013	
  
H.	
  Zegeye	
  
Outline
Ø Introduction	
  
	
  
Ø Materials	
  and	
  Methods	
  
Ø Results	
  
	
  
Ø Summary	
  	
  and	
  conclusio...
Major wheat growing areas
Minor wheat growing areas
Introduction	
  	
  
Ø Area	
  under	
  wheat	
  has	
  shown	
  
ste...
Factors	
  affecting	
  wheat	
  productivity	
  in	
  Ethiopia	
  
	
  
v Biotic	
  stress	
  	
  
Ø  Wheat	
  rusts	
 ...
Variety	
  	
  
Year	
  of	
  
release	
  
Epidemic	
  
Year	
  
Virulence	
  against	
  	
  
Estimated	
  	
  
loss	
  (%...
Wheat	
  Area	
  Affected	
  by	
  Yellow	
  Rust	
  Epidemics	
  
During	
  2010	
  in	
  Ethiopia	
  
Region	
  
Zones	
...
ü Heavy	
  Yellow	
  rust	
  
infestation	
  at	
  tillering	
  stage	
  
on	
  	
  Kubsa:	
  Farmers	
  bield	
  
ü Wit...
 
Ø Continuous	
  release	
  of	
  bread	
  wheat	
  cultivars	
  with	
  
similar	
  parentages	
  	
  
	
  
	
  
	
  
	...
 ....So	
  what?	
  
	
  
Ø It	
  is	
  imperative	
  to	
  search	
  for	
  new	
  sources	
  of	
  resistance	
  
in	
 ...
Ø Association	
  Mapping(AM)	
  is	
  one	
  of	
  the	
  techniques	
  being	
  
used	
   to	
   study	
   the	
   assoc...
 	
  	
  	
  Objectives	
  
	
  
v To	
  characterize	
  stripe	
  rust	
  resistance	
  in	
  a	
  collection	
  
of	
  ...
 	
  Materials	
  and	
  Methods	
  
	
  
v A	
  total	
  of	
  187	
  plant	
  materials	
  were	
  used	
  for	
  this	...
 
	
  	
  
Ø SHWs	
  were	
  screened	
  against	
  stripe	
  rust	
  under	
  bield	
  
conditions	
  in	
  Ethiopia	
  ...
Disease	
  scoring	
  
	
  
Ø  Consecutive	
  scores	
  were	
  taken	
  in	
  ten	
  days	
  interval	
  until	
  the	
 ...
Genotyping	
  
	
  
Ø DNA	
  extraction(Ogbonnaya	
  et	
  
al.,	
  2001)	
  
Ø 9000	
  SNP	
  Illumina’s	
  
inbinium	
...
Population	
  Structure	
  (Q)	
  
	
  
v Estimated	
  using	
  42	
  unlinked	
  SNP	
  markers	
  considering	
  at	
  ...
Linkage	
  Disequilibrium	
  (LD)	
  
	
  
Ø Software:	
  TASSEL	
  v3.0	
  
Ø 4040	
  	
  unlinked	
  SNPs	
  
Ø r2	
 ...
Association	
  Analysis	
  
	
  
Ø  TASSEL	
  V.3	
  
Ø  Mixed	
  Linear	
  model	
  (MLM)	
  
Ø  The	
  results	
  of	...
Results	
  
Reaction	
  of	
  some	
  SHWs	
  across	
  location	
  and	
  	
  
at	
  seedling	
  stage	
  to	
  stripe	
  rust	
  
Ge...
…con’t	
  
	
  
The	
  frequency	
  of	
  SHWs	
  under	
  different	
  severity	
  classes	
  
tested	
  at	
  Meraro,	
 ...
Population	
  Structure	
  (Q)	
  
	
  
-­‐9500	
  
-­‐9000	
  
-­‐8500	
  
-­‐8000	
  
-­‐7500	
  
-­‐7000	
  
-­‐6500	
 ...
LD	
  decay	
  
	
  
R2	
  
Genetic	
  distance	
  (cM)	
  
R2=0.22	
  
Detected	
  QTLs	
  associated	
  with	
  stripe	
  rust	
  
resistance	
  at	
  adult	
  plant	
  stage	
  	
  
Marker	
 ...
Marker	
   Chromosome	
   Position	
  (cM)	
   P-­‐value	
   R2	
  
wsnp_Ku_rep_c69876_69364477	
   2AS	
   93.2	
   9.97E...
Map	
  position	
  of	
  signibicant	
  markers	
  
	
  
wsnp_Ex_c17884_266479520.0
wsnp_Ex_c10657_173764486.5
wsnp_Ku_c57...
wsnp_Ra_c9738_1617400215.9
wsnp_Ex_c44447_5049667637.1
wsnp_Ex_c742_145803340.9
wsnp_Ku_c40218_4848441043.3
wsnp_Ex_rep_c1...
SNP	
  markers	
  signibicantly	
  associated	
  with	
  resistance	
  to	
  
stripe	
  rust	
  at	
  seedling	
  stage	
 ...
Summary	
  and	
  Conclusion	
  
	
  
Genome-wide Association Mapping of Adult Plant Resistance to Stripe Rust in Synthetic Hexaploid Wheat
Genome-wide Association Mapping of Adult Plant Resistance to Stripe Rust in Synthetic Hexaploid Wheat
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Genome-wide Association Mapping of Adult Plant Resistance to Stripe Rust in Synthetic Hexaploid Wheat

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Transcript of "Genome-wide Association Mapping of Adult Plant Resistance to Stripe Rust in Synthetic Hexaploid Wheat"

  1. 1. BGRI  Technical  Workshop     New  Delhi,  India,  19-­‐22  August  2013   H.  Zegeye  
  2. 2. Outline Ø Introduction     Ø Materials  and  Methods   Ø Results     Ø Summary    and  conclusion   Ø Acknowledgements  
  3. 3. Major wheat growing areas Minor wheat growing areas Introduction     Ø Area  under  wheat  has  shown   steady  increase  to  about  2  M     ha  at  present   Ø National  average  yield     increased  by  3x  compared  to   1960s  (0.6    -­‐    2  t/ha),  below   the  world’s  average  yield  (3   t/ha)   v   Ethiopia  is  the  2nd  largest  wheat  producer  in  SSA    
  4. 4. Factors  affecting  wheat  productivity  in  Ethiopia     v Biotic  stress     Ø  Wheat  rusts  (yellow,  stem   and  leaf)  and  septoria   Ø  Weeds  (grass)   v Abiotc  stress   Ø  Water  logging     Ø  Low  soil  fertility   Ø  Drought-­‐low  moisture   in  lowlands   v Technical  constraints   Ø  Shortage  of  appropriate           varieties  for  d/f  AEZs   v   Socio-­‐economic  factors   Ø   Inputs     Yellow     Stem Leaf
  5. 5. Variety     Year  of   release   Epidemic   Year   Virulence  against     Estimated     loss  (%)    Laketch   1974   Sr?    ??    Dashen   1984   1988   Yr9   58    Enkoy   1974   1994   Sr36   67-­‐100    Kubsa      Galama   1995   2010   Yr27   Up  to  100   Major  Rust  Epidemics  and  Estimated  Yield   Losses  in  Ethiopia   Dead  Wheat  2010   Farmer’s  Field  around  Meraro,Ethio  Looking  at  the  Spot  
  6. 6. Wheat  Area  Affected  by  Yellow  Rust  Epidemics   During  2010  in  Ethiopia   Region   Zones   under   epidemics   Districts   under   epidemics   Wheat  area   affected  (ha)   Area   sprayed  by   fungicide   Percent  area   sprayed   Oromia   13   118   292,866   123,357   42.1   Amhara   8   77   161,348   26,579   16.5   SNNP   15   94   137,376   32,089   23.4   Total   36   289   591,590   182,026   30.8   Source:  MoA  report  2010  (unpublished)  
  7. 7. ü Heavy  Yellow  rust   infestation  at  tillering  stage   on    Kubsa:  Farmers  bield   ü With  no  timely  fungicide,   losses  estimated  up  to   100%   v International  Scientists:     Ø   Cornell  University   Ø   CIMMYT   Ø  ICARDA   Ø   India    visited  Farmers’  field  and  witness   the  Yellow  rust  epidemic  in  2010    
  8. 8.   Ø Continuous  release  of  bread  wheat  cultivars  with   similar  parentages             Ø Production  of  few  cultivars  for  long  time  in  the   same  area   ü Lack  of  continuous  cultivar  replacement  preferred   by  farmers  and  rust  resistance  wheat    varieties   diversification       The  recurrent  outbreak  of  wheat  rusts  could  be    
  9. 9.  ....So  what?     Ø It  is  imperative  to  search  for  new  sources  of  resistance   in   ü   Cultivated  wheat   ü   Wild  relatives   ü   Synthetics Ø Synthetic  hexaploid  wheat  provide  resistance  to  several   abiotic  and  biotic  stresses  (van  Ginkel  and  Ogbonnaya,  2007)   Ø The   use   of   molecular   marker   tools   that   aids   to     genotype   germplasm   to   identify   markers   with   desirable   traits   that   could   be   incorporated   in   breeding   programs   via   marker   assisted  selection  (MAS)  become  vital  and  contemporary  
  10. 10. Ø Association  Mapping(AM)  is  one  of  the  techniques  being   used   to   study   the   association   between   genotype   and   phenotype   that   could   help   to   identify   marker-­‐   trait   association   Ø Advantages  of  AM   ü No  new  crosses   ü Several   traits   can   be   studied   in   the   same   population   using  same    genotype   …con’t    
  11. 11.        Objectives     v To  characterize  stripe  rust  resistance  in  a  collection   of  synthetic  hexaploid  wheat     v To   identify   genomic   regions   with   QTLs   linked   to   stripe  rust  resistance  under  bield  conditions  
  12. 12.    Materials  and  Methods     v A  total  of  187  plant  materials  were  used  for  this  study.     Ø 181  SHWs:  ICARDA   Ø 6  bread  wheat:  Kulumsa  Research  Center,  Ethiopia     ü The  check  cultivars  show  variable  reaction  to  stripe  rust   under  Oield  conditions  in  hot  spot  areas  in  Arsi  and  Bale,   Ethiopia  
  13. 13.       Ø SHWs  were  screened  against  stripe  rust  under  bield   conditions  in  Ethiopia  at  Meraro  and  Arsi  Robe  from   2010-­‐2011   Ø simple  design  used  with  2  rows  of  1m  length   Ø SHWs  were  Characterized  at  seedling  stage  in  greenhouse     Phenotyping    
  14. 14. Disease  scoring     Ø  Consecutive  scores  were  taken  in  ten  days  interval  until  the   disease  progress  ceased   Ø  Modibied  Cobb’s  scale  was  adopted  for  Severity  while  bield   responses  (R,  MR,  MS,  S)-­‐  Roelfs  et  al.,  1992   Ø  The  terminal  scores  were  converted  to  coefbicient  of  infection     Ø  0  to  4  scale  was  adopted  for  scoring  seedling  reaction   (McIntosh  et  al.,  1995)    
  15. 15. Genotyping     Ø DNA  extraction(Ogbonnaya  et   al.,  2001)   Ø 9000  SNP  Illumina’s   inbinium  assay   Ø Genome  Studio®  for  ‘Allele   calling’   Ø Monomorphic  &  polymorphic   SNPs  markers  with  low  allelic   frequency  of  <  5%  were   culled  out  from  the  analysis   Ø 4040  polymorphic  SNPs  used 17  days  old  seedlings  collected  for   DNA  extraction  
  16. 16. Population  Structure  (Q)     v Estimated  using  42  unlinked  SNP  markers  considering  at  least   50  cM  apart  between  markers  within  the  same  chromosome   v Software  ‘STRUCTURE  v.2.3.4’  (Pritchard  et  al.,  2000)   v K=1…15  sub-­‐populations  assumed  
  17. 17. Linkage  Disequilibrium  (LD)     Ø Software:  TASSEL  v3.0   Ø 4040    unlinked  SNPs   Ø r2    used  to  estimate  LD     Ø r2    plotted  against  genetic  distance  
  18. 18. Association  Analysis     Ø  TASSEL  V.3   Ø  Mixed  Linear  model  (MLM)   Ø  The  results  of  the  MLM  analysis  that  accounted  for   population  structure  (Q  and  K  matrices)  was  adopted   ü  P<0.01  
  19. 19. Results  
  20. 20. Reaction  of  some  SHWs  across  location  and     at  seedling  stage  to  stripe  rust   Gentype   2010   2011   Seedling   (0  to  4)   Meraro   A.Robe   Meraro   A.Robe    Kubsa   100S   90S   100S   70S   2    68.111/RGB-­‐U//WARD  RESEL/3/STIL/4/AE.SQUARROSA  (164)   30MS   10S   5MR   TR   2C    DOY1/AE.SQUARROSA  (322)   20SMS   5S   0   5S   3    68.111/RGB-­‐U//WARD  RESEL/3/STIL/4/AE.SQUARROSA  (631)   10MS   0   0   0   3    68.111/RGB-­‐U//WARD  RESEL/3/STIL/4/AE.SQUARROSA  (684)   10MR   0   0   0   1    RABI//GS/CRA/3/AE.SQUARROSA  (190)   TR   0   TR   TS   3    SORA/AE.SQUARROSA  (191)   20MS   5S   TR   0   0    SORA/AE.SQUARROSA  (208)   5MS   5S   TR   0   3    DOY1/AE.SQUARROSA  (415)   10MR   0   TR   5S   :N    SCA/AE.SQUARROSA  (409)   5R   0   TR   0   3    STY-­‐US/CELTA//PALS/3/SRN-­‐5/4/AE.SQUARROSA  (502)   5SMS   TR   TR   5S   3    DOY1/AE.SQUARROSA  (516)   5MSMR    5MS   TR   TS   4    DOY1/AE.SQUARROSA  (1024)   5MR   TS   TR   TR   4  
  21. 21. …con’t     The  frequency  of  SHWs  under  different  severity  classes   tested  at  Meraro,  Ethiopia  in  2011   0 10 20 30 40 50 60 R MR MS S Frequencyofdiseaseseverity (%) Host Reaction 0 10 20 30 40 50 60 R MR MS S Host Reaction Frequencyofdiseaseseverity (%)
  22. 22. Population  Structure  (Q)     -­‐9500   -­‐9000   -­‐8500   -­‐8000   -­‐7500   -­‐7000   -­‐6500   -­‐6000   -­‐5500   -­‐5000   2   3   4   5   6   7   8   9   10   11   12   13   14   15              K 1                          2      3                        4        5                      6                        7                                          8                9                  10  11  12    13          14   Ln(P)   Using 42 unlinked markers
  23. 23. LD  decay     R2   Genetic  distance  (cM)   R2=0.22  
  24. 24. Detected  QTLs  associated  with  stripe  rust   resistance  at  adult  plant  stage     Marker   Chromosome   Position  (cM)   P-­‐value   R2   wsnp_Ex_c10657_17376448   1AL   6.5   8.79E-­‐03   3.5   wsnp_RFL_Contig3850_4199825   1AS   175.6   3.72E-­‐03   4.5   wsnp_RFL_Contig2449_2013497   1BS   34.8   3.96E-­‐03   4.2   wsnp_Ex_c11177_18096010   1BS   35.3   2.86E-­‐03   4.5   wsnp_Ex_c14_27570   1BS   35.3   1.68E-­‐03   5.1   wsnp_Ku_c62848_63784645   1BS   35.5   1.51E-­‐03   5.2   wsnp_Ku_c30982_40765341   1BS   37.2   5.04E-­‐03   5.4   wsnp_BE442716B_Ta_2_2   1DS   38.2   8.55E-­‐03   5.2   wsnp_BE591501B_Ta_2_1   1BS   38.8   4.03E-­‐03   4.6   wsnp_Ex_c10233_16784994   1BS   39.5   7.38E-­‐03   5.0   wsnp_Ku_c66585_65967792   1BS   39.7   9.18E-­‐03   4.8   wsnp_Ku_c6158_10825959   1BS   66.5   3.72E-­‐03   4.3        Total=26                                        R2=  3.4%  to  8.1%  
  25. 25. Marker   Chromosome   Position  (cM)   P-­‐value   R2   wsnp_Ku_rep_c69876_69364477   2AS   93.2   9.97E-­‐04   5.6   wsnp_Ex_c1604_3060855   2AS   160.7   5.67E-­‐03   3.9   wsnp_CAP7_c3519_1643682   2DS   165.4   1.74E-­‐04   7.3   wsnp_Ra_c9738_16174002   3AL   15.9   2.52E-­‐03   6.1   wsnp_Ex_c742_1458033   3AS   40.9   7.39E-­‐03   3.6   wsnp_Ex_rep_c106152_90334299   3AL   46.2   5.12E-­‐04   7.8   wsnp_JG_c2509_1153697   3AL   56.7   7.48E-­‐04   7.4   wsnp_RFL_Contig4734_5671036   3AL   104.8   3.90E-­‐03   5.8   wsnp_Ex_c55096_57733894   3AL   163.0   7.50E-­‐03   3.6   wsnp_Ex_rep_c104141_88935451   3AS   163.6   5.02E-­‐04   8.1   wsnp_Ex_c41074_47987860   4AL   93.5   3.96E-­‐03   4.2   wsnp_RFL_Contig2828_2623246   4DL   12.1   9.67E-­‐03   3.4   wsnp_Ku_c35386_44598937   5A   112.5   5.32E-­‐04   6.2   …con’t    
  26. 26. Map  position  of  signibicant  markers     wsnp_Ex_c17884_266479520.0 wsnp_Ex_c10657_173764486.5 wsnp_Ku_c5756_1019133924.7 wsnp_Ex_c10595_1729199952.3 wsnp_Ku_c5210_9290700100.5 wsnp_JD_rep_c49006_33254974150.1 wsnp_RFL_Contig3850_4199825175.6 wsnp_Ex_c52086_55808824181.2 1A wsnp_Ra_c19335_2850438821.9 wsnp_RFL_Contig2449_201349734.8 wsnp_Ex_c11177_18096010wsnp_Ex_c14_2757035.3 wsnp_Ku_c62848_6378464535.5 wsnp_Ku_c30982_4076534137.2 wsnp_BE442716B_Ta_2_238.2 wsnp_BE591501B_Ta_2_138.8 wsnp_Ex_c10233_1678499439.5 wsnp_Ku_c66585_6596779239.7 wsnp_CAP7_c940_48074546.9 wsnp_JD_c5659_681424059.7 wsnp_Ku_c6158_1082595966.5 wsnp_Ex_rep_c69766_6872314069.6 1B wsnp_Ex_rep_c68599_6744788090.2 wsnp_Ku_rep_c69876_6936447793.2 wsnp_bg606625A_Ta_2_1103.4 wsnp_Ex_c2887_5330787127.4 wsnp_Ex_c5984_10493714150.3 wsnp_Ex_c1604_3060855160.7 wsnp_Ku_c54793_58953037165.0 2A wsnp_Ra_rep_c71290_69343893150.5 wsnp_Ex_rep_c66522_64795143158.2 wsnp_CAP7_c3519_1643682165.4 wsnp_Ku_c4319_7847988168.0 2D
  27. 27. wsnp_Ra_c9738_1617400215.9 wsnp_Ex_c44447_5049667637.1 wsnp_Ex_c742_145803340.9 wsnp_Ku_c40218_4848441043.3 wsnp_Ex_rep_c106152_9033429946.2 wsnp_JG_c2509_115369756.7 wsnp_BG263769A_Td_2_162.5 wsnp_RFL_Contig429_497862881.4 wsnp_Ku_c4886_875364697.8 wsnp_RFL_Contig4734_5671036104.8 wsnp_BF292596A_Ta_1_3119.1 wsnp_Ex_c20250_29303152135.5 wsnp_BE604885A_Ta_2_1151.5 wsnp_Ex_c55096_57733894163.0 wsnp_Ex_rep_c104141_88935451163.6 wsnp_Ku_c35632_44814164171.5 3A wsnp_Ex_c2288_429343083.5 wsnp_Ra_rep_c87547_7984290987.1 wsnp_Ex_c41074_4798786093.5 wsnp_JD_c38619_2799227995.4 wsnp_Ku_c8059_1376368399.5 4A wsnp_Ex_rep_c107564_911445230.5 wsnp_CAP11_c356_2809108.0 wsnp_Ku_c25831_3579767610.0 wsnp_RFL_Contig2828_262324612.1 wsnp_Ku_c9140_1539016615.3 wsnp_BE444858D_Ta_1_118.9 4D wsnp_RFL_Contig44_5147697103.3 wsnp_Ku_c15816_24541162107.5 wsnp_Ku_rep_c103857_90489662110.1 wsnp_Ku_c35386_44598937112.5 wsnp_Ku_c14139_22353229114.5 wsnp_Ra_c21347_30731133118.3 5A wsnp_Ex_c6942_119664690.0 wsnp_BE471213D_Ta_2_12.4 wsnp_Ex_c4518_81195036.1 wsnp_Ex_c62371_620360447.1 wsnp_JD_c7795_88678438.5 6D …con’t    
  28. 28. SNP  markers  signibicantly  associated  with  resistance  to   stripe  rust  at  seedling  stage     No   Marker   Chrom.   Pos(cM)   P-­‐value   R2   1   wsnp_Ku_c6158_10825959   1BS   66.5   6.52E-­‐03   4.1   2   wsnp_Ku_c30982_40765341   1BS   37.2   6.55E-­‐03   5.5   3   wsnp_Ex_c1318_2520916   1DS   85.0   6.67E-­‐03   5.5   4   wsnp_Ex_c25974_35235456   1DS   85.7   3.72E-­‐03   6.2   5   wsnp_Ex_c1318_2519998   1DS   86.5   6.81E-­‐03   5.4   6   wsnp_Ex_c6920_11929171   1DS   89.4   6.71E-­‐03   5.5   7   wsnp_BG275030D_Ta_2_2   2DL   41.9   9.01E-­‐03   5.1   8   wsnp_Ex_c25945_35206573   3DS   12.4   1.28E-­‐04   8.2   9   wsnp_Ex_c19724_28721580   5B   126.3   7.67E-­‐03   3.9   10   wsnp_Ex_c19928_28951983   6A   178.4   8.08E-­‐03   3.8   11   wsnp_Ex_c4480_8056013   6DS   6.4   5.81E-­‐03   4.1   12   wsnp_Ku_rep_c71225_70941765   6DS   6.4   5.81E-­‐03   4.1   13   wsnp_Ex_c4480_8055475   6DS   6.4   4.88E-­‐03   4.3   14   wsnp_Ex_c62371_62036044   6DS   7.1   8.87E-­‐03   5.2   R2  =  3.8  to  8.2%  
  29. 29. Summary  and  Conclusion    
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