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The 10th  Solanaceae    Conference:  Genome  versus  Phenome   October  13  -­‐  17,  2013    -­‐  Beijing,  China    

Novel candidate  genes  for  quan3ta3ve  resistance  to  late   blight  in  potato   Teresa Mosquera-Vásquez, María Fernanda Álvarez, José M. Jiménez-Gómez, Astrid Draffehn, Christiane Gebhardt


Types of genetic resistance identified Basal resistance  

Qualita.ve resistance  

Quan.ta.ve resistance  

Ajjamada C. Kushalappa and Raghavendra Gunnaiah, Trends in Plant Science XX (2013) 1-10


Objectives 1.  To select novel candidate genes for quantitative resistance to late blight from genes showing high level of polymorphism in differential transcriptome analysis. 2.  To validate the identified allelic variants for association with resistance to late blight in diploid and tetraploid germplasm.


Methodology Differen.al  transcriptome  analysis  and     data  genera.on  (RNA  seq)  

Novel candidate     genes  selec.on      

Quan.ta.ve evalua.on  of   resistance  to  late  blight  

PCR -­‐  amplicon  sequencing         Tetraploid     SNP  iden.fica.on   Gene.c  analysis  

Associa3on mapping  

Diploid  


Plant material Diploid genotypes (Colombian germplasm)

Colombian Core  Collec.on:  Solanum  phureja  

Tetraploid genotypes (German germplasm)


Evaluation for resistance to late blight: AUDPC and Maturity Corrected Resistance (MCR)

Late blight evaluation under field conditions


Methodology Differen.al  transcriptome  analysis  and     data  genera.on  (RNA  seq)   Astrid  Draffehn  

Novel candidate     genes  selec.on      

José Jiménez-­‐Gómez  

Quan.ta.ve evalua.on  of   resistance  to  late  blight  

PCR -­‐  amplicon  sequencing         Tetraploid     SNP  iden.fica.on   Gene.c  analysis  

Associa3on mapping   María  Fernanda  Álvarez  

Diploid  


Differential transcriptome analysis (RNA seq experiment) StAOS2      Allene  Oxide  Synthase  2  

Three .me  points:  

Eight  resistant  and  eight  suscep3ble    genotypes  were  selected  

BNA16 x BNA20 BNA16 x BNA20 BNA16 x BNA20 BNA40 X BNA22 BNA40 X BNA22 PapPhy20 x PapPhy14 PapPhy20 x PapPhy16 PapPhy20 x PapPhy14

StAOS2 SNP691 AAAA AAAG AAAG AAAG AAAG AAAA AAAA AAAA

StAOS2 SNP692 CCCC CCCG CCCG CCCG CCCG CCCC CCCC CCCC

BNA16 x BNA20 BNA16 x BNA20 BNA16 x BNA20 BNA16 x BNA20 PapPhy20 x PapPhy16 PapPhy20 x PapPhy16 PapPhy20 x PapPhy16 PapPhy20 x PapPhy16

GGGG AGGG AGGG AGGG AGGG AGGG AGGG AGGG

GGGG CGGG CGGG CGGG CGGG CGGG CGGG CGGG

Genotype

Family

BL114 BL196 BL201 BL426 BL499 SL155 SL312 SL194 BL013 BL024 BL141 BL238 SL317 SL316 SL314 SL433

rAUDPC mean 0.24909 0.36605 0.31999 0.24014 0.24384 0.33 0.23 0.465 0.30551 0.53493 0.5308 0.4886 0.554 0.53 0.615 0.62 0.595 0.55854

Pool R8 R8 R8 R8 R8 R8 R8 R8 S8 S8 S8 S8 S8 S8 S8 S8

•   Before  inocula.on   •     One  day  aRer  inocula.on     •     Two  days  aRer  inocula.on  

Pooled   Resistance  vs.  suscep.ble    

454 Sequencing     Normalized  cDNA  library    


Criteria for candidate genes selection for quantitative resistance to late blight

v   Genes that co-localize in the genetic map with QTL for resistance to late blight.

v  Genes that are involved in fundamental pathways and participate in different regulations that might influence the defense response. v  Genes showing at least two SNPs (haplotype) that differentiate (significantly) resistant from susceptible genotypes. v  Genes across the 12 potato chromosomes.  


SNPs associated  to  quan.ta.ve  resistance  to     late  blight  in  diploid  potatoes  

TM18 TM14  

                                             1                                                      2                                        3                                  45      6      7                      8                        10                                  11                            12  

Chromosomes

Chr.

SNP Identification

-­‐log10(P)

Effect

3

TM14  SNP61

2.6

10.5%

3

TM14  SNP199

2.7

10.5%

3

TM14  SNP211

2.6

10.5%

6

TM18  SNP88

3.5

13%

6

TM18  SNP108

3.5

13%

1            2        3            4          6          7          8        10   chromosomes    


Quan.ta.ve resistance  and  allele  dosage   TMV  -­‐  induced  protein  I    

Chromosome 3,  SNP  211:    CATC(T/A)GAAG    

TM14  SNP  211   7000  

AUDPC

6000 5000   4000   3000   2000  

7                                              45                                            51  

1000 0   AA   AA                                          TA  TA                                            TT  TT  

Allele dosage  

p-­‐value 0.009  

Allele dosage  


Novel candidate gene validated for quantitative resistance to late blight in diploid potatoes 17.220.914 bp  

TMV-­‐ induced     protein  1  

SNP 211 TMV – induced protein I:

29.900.189 bp  

v  has a lypoxigenase domain. v  is found in a variety of membrane or lipid associated proteins. v  is a protein binding domain.

53.228.703 bp  

Chromosome  III   QTL  for  late  blight  resistance    

PoMaMo database     hbp://www.gabipd.org/projects/Pomamo/  


Quan.ta.ve resistance  and  allele  dosage   Thylakoid  lumenal  15  kDa  protein  1,  chloroplas3c   Chromosome  6,  SNP  88:    CCTT(T/C)CCT     TM  18  SNP  88  

TM18 SNP  88   AUDPC  

AUDPC

6000 5000   4000   3000   2000   1000  

24                                  53                                    13  

0 TT  

TC

10000        8000          6000         4000         2000  

CC

TT                                TC                                      CC  

Allele dosage   p-­‐value  <.001  

Allele dosage  


Novel candidate gene validated for quantitative resistance to late blight in diploid potatoes

Phu  TM18    CCTT(TC)CCTG              p.88  

Chromosome VI   QTL  for  late  blight  resistance    

50.394.411  bp   Thylakoid     lumenal  15  kDa    

52.355.675 bp    

58.913.746  bp   PoMaMo  database    

hbp://www.gabipd.org/projects/Pomamo/


Thylakoid lumenal 15 kDa protein 1, chloroplastic: v  is involved in light dependent reactions. v  has a tetratricopeptide repeat (TPR)-like. v  presents protein – protein interaction.


SNPs associated  to  quan.ta.ve  resistance  to     late  blight  in  tetraploid  potatoes       :    maturity  corrected  resistance  

Chr.

1 5 8 8

SNP Identification  

MF22    SNP  8   TM35    SNP  14     TM27    SNP  505   TM27    SNP  548  

-­‐log10(P)

Effect

2.7 3.1 5.3   10.3  

15% 11.5% 12.5% 14.5%

1            2        3            5          6          7          8        10   chromosomes    


Quan.ta.ve resistance  and  allele  dosage  

AUDPC

Cytochrome  P450  71D11   Chromosome  8,  SNP  505:  CTGA(A/G)GTAT  

160

Maturity

140

7

120

6

100

5

80

Series1

Series1

3

60 40

4

 1  5    150    1    48                                1    7                                                  98                                                      2        2  

2

20

   150                    17                          9                            2  

1

0

0

AAAA

AAAG

AAGG

AAAA

AGGG

P-­‐value 0.000346780  

0,05

Maturity Corrected  Resistance  (MCR)  

0 AAAA

AAAG

AAGG

AGGG

MCR

-­‐0,05 -­‐0,1

150                      17                                              9                                          2  

-­‐0,15 -­‐0,2

p-­‐value 5.52E-­‐06  

12.5% of  phenotypic  variance  

AAAG

AAGG

AGGG

P-­‐value 0.44971309  


Chromosome 8: cytochrome P450 71D11 SNP CTGA(A/G)GTAT


Novel candidate gene validated for quantitative resistance to late blight in tetraploid potatoes

Cytochrome P450 71D11: ü  has oxidoreductase activity, acting on paired donors. ü  incorporates or reduces molecular oxygen and is oxygen binding. ü  participates in regulation of defense response, response to bacterium, systemic acquired 51.488.760 bp   resistance with incorporation or reduction of molecular oxygen. Cytochrome  P450  71D11  

29.253.8 82  bp   Chromosome  VIII   QTL  for  late  blight  resistance    

PoMaMo database    

hbp://www.gabipd.org/projects/Pomamo/


Novel candidate genes validated for quantitative resistance to late blight in tetraploid potatoes

4.037.739 bp

AAA ATPase  domain

5.575.541 bp 31.442.042  bp     Defec3ve  in  induced    resistance  protein  1     76.955.498  bp  

Chromosome V  

Chromosome I   QTL  for  late  blight  resistance    

PoMaMo database     hbp://www.gabipd.org/projects/Pomamo/  


Summary Candidate genes  s elected Candidate  genes  analyzed Populations  s tudied   Number  of  individuals  analyzed   Total  of  SNPs  s cored

57 30 3 327 564

SNPs s cored  in  tuberosum  populations SNPs  s cored  in   S.  phureja

322 242

SNPs are  s howing  association  with  late  blight  resistance Candidate  genes  associated    to  late  blight  resistance Chromosomes  covered  with    11  s ignificant  SNPs

9 5 1, 3,  5,  6,  8


Genes associated  with  resistance  to  late  blight   In  diploid  and  tetraploid  germplasm   Chr. Gene  (ID)

Function described

Population SNP (ID) -­‐log10(P) Effect  % Primer  Name

1 PGSC0003DMG400011323 Defective in  induced  resistance  1  protein Tub  (4x) 3 PGSC0003DMG400016749 TMV  -­‐  induced  protein  I    

Phu (2X)

5 PGSC0003DMB000000609 AAA ATPase  domain Thylakoid  lumenal  15  kDa  protein  1,   6 PGSC0003DMG400034939 chloroplastic

Tub (4X)

8 PGSC0003DMG400020809 Cytochrome P450  71D11

Tub (4x)

Phu (2X)

8 61 199 211 14 88 108 505 548

2.7 2.6 2.7 2.6 3.1 3.5 3.5 5.3 10.3

15 10.5 10.5 10.5 11.5 13 13 12.5 14.5

MF22 TM14 TM35 TM18 TM27


Conclusions and perspectives v  Differential transcriptome analysis has shown to be useful to identify novel candidate genes for quantitative resistance to late blight.

v  The results regarding independence between traits: maturity and resistance to late blight open new opportunities for potato breeding. v  Our results show novel genes involved with basic process like light dependent reactions, growth and development, pest resistance, and senescence or responses to wounding, effects that are of potential relevance for potato resistance. v  The next step is to develop diagnostic molecular markers for resistance to late blight and to validate them in breeding programs for tetraploid and diploid germplasm.


Max Planck Institute for Plant Breeding Research

Acknowledges

Contract to  access  gene.c  resources  RGE0069  


Thanks for your attention

2013 10 sol conf genome vs phenome novel candidate genes for resistance to late blight in potato ter  
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