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

BGRI  Technical  Workshop    New  Delhi,  India,  19-­‐22  August  2013  

H.  Zegeye  

Outline

Ø Introduction    

Ø Materials  and  Methods  

Ø Results    

Ø Summary    and  conclusion  

Ø Acknowledgements  

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    

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

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  

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Source:  MoA  report  2010  (unpublished)  

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  

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    

 ....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  

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  

ü Much  higher  mapping  resolution  

…con’t    

       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  

   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  

     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    

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)    

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  

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  

Linkage  Disequilibrium  (LD)    

Ø Software:  TASSEL  v3.0  

Ø 4040    unlinked  SNPs  

Ø r2    used  to  estimate  LD    

Ø r2    plotted  against  genetic  distance  

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  

Results  

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  

…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

Freq

uenc

y of

dis

ease

sev

erity

(%

)

Host Reaction0

10

20

30

40

50

60

R MR MS S

Host Reaction

Freq

uenc

y of

dis

ease

sev

erity

(%

)

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

LD  decay    

R2  

Genetic  distance  (cM)  

R2=0.22  

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%  

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  

wsnp_Ex_c25505_34772008   6DL   50.8   2.14E-­‐03   5.0  

…con’t    

Map  position  of  signibicant  markers    

wsnp_Ex_c17884_26647952 0.0 wsnp_Ex_c10657_17376448 6.5 wsnp_Ku_c5756_10191339 24.7

wsnp_Ex_c10595_17291999 52.3

wsnp_Ku_c5210_9290700 100.5

wsnp_JD_rep_c49006_33254974 150.1

wsnp_RFL_Contig3850_4199825 175.6 wsnp_Ex_c52086_55808824 181.2

1A wsnp_Ra_c19335_28504388 21.9 wsnp_RFL_Contig2449_2013497 34.8 wsnp_Ex_c11177_18096010 wsnp_Ex_c14_27570 35.3 wsnp_Ku_c62848_63784645 35.5 wsnp_Ku_c30982_40765341 37.2 wsnp_BE442716B_Ta_2_2 38.2 wsnp_BE591501B_Ta_2_1 38.8 wsnp_Ex_c10233_16784994 39.5 wsnp_Ku_c66585_65967792 39.7 wsnp_CAP7_c940_480745 46.9 wsnp_JD_c5659_6814240 59.7 wsnp_Ku_c6158_10825959 66.5 wsnp_Ex_rep_c69766_68723140 69.6

1B

wsnp_Ex_rep_c68599_67447880 90.2 wsnp_Ku_rep_c69876_69364477 93.2 wsnp_bg606625A_Ta_2_1 103.4

wsnp_Ex_c2887_5330787 127.4

wsnp_Ex_c5984_10493714 150.3 wsnp_Ex_c1604_3060855 160.7 wsnp_Ku_c54793_58953037 165.0

2A

wsnp_Ra_rep_c71290_69343893 150.5 wsnp_Ex_rep_c66522_64795143 158.2 wsnp_CAP7_c3519_1643682 165.4 wsnp_Ku_c4319_7847988 168.0

2D

wsnp_Ra_c9738_16174002 15.9

wsnp_Ex_c44447_50496676 37.1 wsnp_Ex_c742_1458033 40.9 wsnp_Ku_c40218_48484410 43.3 wsnp_Ex_rep_c106152_90334299 46.2 wsnp_JG_c2509_1153697 56.7 wsnp_BG263769A_Td_2_1 62.5 wsnp_RFL_Contig429_4978628 81.4 wsnp_Ku_c4886_8753646 97.8 wsnp_RFL_Contig4734_5671036 104.8 wsnp_BF292596A_Ta_1_3 119.1 wsnp_Ex_c20250_29303152 135.5 wsnp_BE604885A_Ta_2_1 151.5 wsnp_Ex_c55096_57733894 163.0 wsnp_Ex_rep_c104141_88935451 163.6 wsnp_Ku_c35632_44814164 171.5

3A

wsnp_Ex_c2288_4293430 83.5 wsnp_Ra_rep_c87547_79842909 87.1 wsnp_Ex_c41074_47987860 93.5 wsnp_JD_c38619_27992279 95.4 wsnp_Ku_c8059_13763683 99.5

4A

wsnp_Ex_rep_c107564_91144523 0.5 wsnp_CAP11_c356_280910 8.0 wsnp_Ku_c25831_35797676 10.0 wsnp_RFL_Contig2828_2623246 12.1 wsnp_Ku_c9140_15390166 15.3 wsnp_BE444858D_Ta_1_1 18.9

4D

wsnp_RFL_Contig44_5147697 103.3 wsnp_Ku_c15816_24541162 107.5 wsnp_Ku_rep_c103857_90489662 110.1 wsnp_Ku_c35386_44598937 112.5 wsnp_Ku_c14139_22353229 114.5 wsnp_Ra_c21347_30731133 118.3

5A

wsnp_Ex_c6942_11966469 0.0 wsnp_BE471213D_Ta_2_1 2.4 wsnp_Ex_c4518_8119503 6.1 wsnp_Ex_c62371_62036044 7.1 wsnp_JD_c7795_8867843 8.5

6D

…con’t    

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%  

Summary  and  Conclusion    

���  Usefulness  of  AM  for    marker  trait  association  is  demonstrated  on  SHWs  

���  Identibied  list  of  SNP  markers  closely  linked  to  Yr  resistance    

���  SHW  genotypes  possessing  potentially  novel   resistance  alleles   at   different   QTL   should   be   further   investigated  and  could  be  used  as  parental  lines  in  a  marker-­‐assisted  backcrossing  scheme  

���  About  26  signibicant  associations  obtained  (needs  validation)  

Acknowledgment    

ICARDA K. Nazari W. Tadesse O. Abdalla A. Jighly    

EIAR-KARC Bedada Girma – DRRW Solomon G. – KARC Director Firdisa Eticha – KARC-Wheat      

Advisors Francis. C. O Ayele B. Hussien M    

EIAR

Study leave Funding All 2013 BGRI Organizers

Cristobal  Uauy-­‐Jonns  Center