The worldwide Pst population structure and its temporal maintenance in Pakistan Sajid Ali The University of Agriculture, Peshawar, Pakistan & Aarhus University, Denmark #bgri2014

In the context of invasion Pst: Puccinia striiformis f.sp. tritici PstS1 & PstS2 2000 2003 Post-2000 Cases of recent invasions in Pst Australia 1979 South American Early 1900s South African population 1996 Warrior-Kranich type strains in Europe 2011-present North American Early 1900s Virulence to Yr9 1986 1998 (Milus et al., 2006, Hovmller et al., 2008; Wellings et al., 2004; Singh et al., 2004) Population subdivision for this long distance migrating pathogen?

Worldwide Pst population structure Worldwide representative set of 409 isolates, selected from INRA, France and AU, Denmark collection Genotyped with 20 SSR markers

FST values for geographically spaced populations NWEurope Mediterranean Region CentralAsia EastAfrica MiddleEast Nepal Pakistan China NW Europe - 0.420 0.380 0.500 0.380 0.370 0.410 0.390 Mediterranean Region 0.000 - 0.020 0.150 0.009 0.280 0.280 0.390 Central Asia 0.000 0.030 - 0.160 0.040 0.230 0.260 0.340 East Africa 0.000 0.000 0.000 - 0.140 0.380 0.280 0.540 Middle-East 0.000 0.020 0.000 0.000 - 0.260 0.250 0.360 Nepal 0.000 0.000 0.000 0.000 0.000 - 0.220 0.210 Pakistan 0.000 0.000 0.000 0.000 0.000 0.000 - 0.450 China 0.000 0.000 0.000 0.000 0.000 0.000 0.000 - Non-significant FST values (> 0.01) are shown in bold. (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision

Assignment with STRUCTURE at different K-values K2 K3 K4 K5 K7 K6 K8 China Nepal Pakistan Middle-East Central Asia Mediterranean region NW Europe East Africa (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision

Discriminant analysis of principal components (ADEGENET) Identification six distinct genetic groups of Pst (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision NW Europe China Nepal Pakistan Central Asia- Mediterranean region Eigen values contribution Number of clusters C Middle East- East Africa 200 3 2 4 6 8 10

France G1 G2 G3 G4 G5 G6 H Un Azerbaijan G1 G2 G3 G4 G5 G6 H Un France G1 G2 G3 G4 G5 G6 H Un Kyrgyzstan G1 G2 G3 G4 G5 G6 Eritrea G1 G2 G3 G4 G5 G6 Afghanistan G1 G2 G3 G4 G5 G6 Cyprus G1 G2 G3 G4 G5 G6 Iran Israil G1 G2 G3 G4 G5 G6 Lebanon G1 G2 G3 G4 G5 G6 Turkey G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un MoroccoG1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Tunisia G1 G2 G3 G4 G5 G6 Nepal G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Pakistan G1 G2 G3 G4 G5 G6 China G1 G2 G3 G4 G5 G6 -Geographically spaced genetic groups identified (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision and source of recent invasions Denmark G1 G2 G3 G4 G5 G6 H Un Sources of these recent invasions? Early 1900s 1979 1996 2003 2000 Early 1900s

FST Values NWEurope North America South America Mediterrane anRegion CentralAsia SouthAfrica EastAfrica MiddleEast Nepal Pakistan China NW Europe - 0.039 0.001 0.420 0.380 0.498 0.500 0.380 0.370 0.410 0.390 North America 0.100 - 0.046 0.409 0.368 0.485 0.490 0.378 0.364 0.400 0.398 South America 0.410 0.100 - 0.435 0.396 0.514 0.511 0.393 0.379 0.416 0.405 Mediterranean Region 0.000 0.000 0.000 - 0.020 0.109 0.150 0.009 0.280 0.280 0.390 Central Asia 0.000 0.000 0.000 0.030 - 0.044 0.160 0.040 0.230 0.260 0.340 South Africa 0.000 0.000 0.000 0.010 0.190 - 0.229 0.160 0.298 0.314 0.419 East Africa 0.000 0.000 0.000 0.000 0.000 0.006 - 0.140 0.380 0.280 0.540 Middle-East 0.000 0.000 0.000 0.020 0.000 0.000 0.000 - 0.260 0.250 0.360 Nepal 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 - 0.220 0.210 Pakistan 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 - 0.450 China 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 - FST for aggressive strains 0.420 0.408 0.434 0.000 0.010 0.099 0.150 0.010 0.270 0.270 0.390 P-value for aggressive strains 0.000 0.000 0.000 0.860 0.070 0.040 0.000 0.020 0.000 0.000 0.000 Non-significant FST values (> 0.01) are shown in bold. (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision

STRUCTURE G1 G2 G3 G4 G5 G6 America China Nepal Pakistan Middle-East Central Asia Mediterranean region NW Europe South Africa PstS1 and PstS2 PstS3 East Africa G1 G2 G3 G4 G5 G6 DAPC Recent invasions (Ali et al. 2014; PLOS Pathogens) Source of recent invasion

France G1 G2 G3 G4 G5 G6 H Un Azerbaijan G1 G2 G3 G4 G5 G6 H Un France G1 G2 G3 G4 G5 G6 H Un Kyrgyzstan G1 G2 G3 G4 G5 G6 Eritrea G1 G2 G3 G4 G5 G6 Afghanistan G1 G2 G3 G4 G5 G6 Cyprus G1 G2 G3 G4 G5 G6 Iran Israil G1 G2 G3 G4 G5 G6 Lebanon G1 G2 G3 G4 G5 G6 Turkey G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un MoroccoG1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Tunisia G1 G2 G3 G4 G5 G6 Nepal G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Pakistan G1 G2 G3 G4 G5 G6 China G1 G2 G3 G4 G5 G6 -Geographically spaced genetic groups identified -Sources of recent invasions identified (Ali et al. 2014; PLOS Pathogens) Worldwide population subdivision and source of recent invasions Diversity and recombinaIon in the worldwide populaIons?

Recombination in Pakistan, Nepal and China (Ali et al. 2014; PLOS Pathogens) Diversity and recombination in worldwide populations High genetic and genotypic diversity in Pakistan, Nepal and China

France G1 G2 G3 G4 G5 G6 H Un Azerbaijan G1 G2 G3 G4 G5 G6 H Un France G1 G2 G3 G4 G5 G6 H Un Kyrgyzstan G1 G2 G3 G4 G5 G6 Eritrea G1 G2 G3 G4 G5 G6 Afghanistan G1 G2 G3 G4 G5 G6 Cyprus G1 G2 G3 G4 G5 G6 Iran Israil G1 G2 G3 G4 G5 G6 Lebanon G1 G2 G3 G4 G5 G6 Turkey G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un MoroccoG1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Tunisia G1 G2 G3 G4 G5 G6 Nepal G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Pakistan G1 G2 G3 G4 G5 G6 China G1 G2 G3 G4 G5 G6 -Recombination and diversity in Himalayan and Near Himalayan regions Diversity and recombination in PST worldwide populations (Ali et al. 2014; PLOS Pathogens)

0.00 1.00 2.00 3.00 4.00 5.00 6.00 4 5 6 7 Telialproduction Weeks after inoculation China Nepal Pakistan Middle-East Mediterranean France Telial progress curve for 56 PST isolates according to their geographical origin Reduction in PST sexual reproduction ability in worldwide populations (Ali et al. 2010; Fungal Genetics and Biology)

France G1 G2 G3 G4 G5 G6 H Un Azerbaijan G1 G2 G3 G4 G5 G6 H Un France G1 G2 G3 G4 G5 G6 H Un Kyrgyzstan G1 G2 G3 G4 G5 G6 Eritrea G1 G2 G3 G4 G5 G6 Afghanistan G1 G2 G3 G4 G5 G6 Cyprus G1 G2 G3 G4 G5 G6 Iran Israil G1 G2 G3 G4 G5 G6 Lebanon G1 G2 G3 G4 G5 G6 Turkey G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un MoroccoG1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Tunisia G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Nepal G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Pakistan G1 G2 G3 G4 G5 G6 China G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Denmark G1 G2 G3 G4 G5 G6 H Un -Centre of diversity in Himalayan and Near Himalayan -Reduction in sexual reproduction ability during ancestral invasion (Ali et al. 2014; PLOS Pathogens) Ancestral relaIonship of worldwide PST populaIons Reduction in PST sexual reproduction ability in worldwide populations

Ancestral relationship among worldwide populations ChNpPkChNp Pk Ch Np PkCh NpPk Ch Np Pk (Ali et al. 2014; PLOS Pathogens) ABC analyses with DIYABC (Cornuet et al., 2008)

The six genetic groups together Ancestral relationship among worldwide populations (Ali et al. 2014; PLOS Pathogens)

Mexico G1 G2 G3 G4 G5 G6 Nepal G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Pakistan G1 G2 G3 G4 G5 G6 China G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Azerbaijan G1 G2 G3 G4 G5 G6 H Un France G1 G2 G3 G4 G5 G6 H Un Kyrgyzstan G1 G2 G3 G4 G5 G6 Eritrea G1 G2 G3 G4 G5 G6 Afghanistan G1 G2 G3 G4 G5 G6 Cyprus G1 G2 G3 G4 G5 G6 Iran Israil G1 G2 G3 G4 G5 G6 Lebanon G1 G2 G3 G4 G5 G6 Turkey G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 Yemen G1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un MoroccoG1 G2 G3 G4 G5 G6 France G1 G2 G3 G4 G5 G6 H Un Tunisia G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Early 1900s 1979 1996 Denmark G1 G2 G3 G4 G5 G6 H Un 2000 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 Mexico G1 G2 G3 G4 G5 G6 ? -The worldwide spread with population subdivision and loss of sexual reproduction - PST possibly originated in the Himalayan and Near Himalayan region -Re-emergence linked with the recent invasions Overall conclusions on worldwide populations (Ali et al. 2014; PLOS Pathogens)

Khyber Peshawar Nowshera Haripur Abbottabad Tanawal Mansehra Attarshisha Balakot Siran Battal Oghi Skardu Battagram Islamabad KASHMIR PAKISTAN Berberis zone Non-Berberis zone Sampled in 2010 Sampled in 2011 Off-season Berberis zone 200 km Pakistani PST population; a high genetic diversity Temporal maintenance of Pakistani PST populaIons

Development of rapid genotyping DNA extraction Directly from infected lesion (Ali et al. 2011; BMC Research Notes) Pakistani PST population; a high genetic diversity

ns ns ns ns ns ns ns ns ns ns ns ns ns ns 0 0.1 0.2 0.3 0.4 0.5 Heterozygosity Geographical region He Ho Year Location Sample size MLGs Genotypic diversity 2010 Khyber 35 25 0.971 Peshawar 12 9 0.939 Nowshera 19 8 0.673 Haripur 42 20 0.864 Abbottabad 34 31 0.993 Mansehra 28 27 0.997 Battal 46 33 0.967 Battagram 9 9 1 Attarshisha 30 22 0.966 Balakot 24 18 0.975 Skardu 63 54 0.991 2011 Peshawar 33 23 0.973 Nowshera 16 10 0.825 Abbottabad 18 9 0.706 Mansehra 61 54 0.991 Tanawal 22 19 0.983 Siran 51 33 0.939 Oghi 19 16 0.977 Battal 35 30 0.99 Battagram 20 17 0.984 Attarshisha 40 31 0.953 Balakot 27 21 0.977 A high genotypic diversity and non-significant FIS across all locations (Ali et al. 2014; Molecular Ecology) Pakistani PST population; a high genetic diversity

A high pathotypic diversity 53 pathotypes out of 127 isolates tested With varying virulence frequencies across locations Location Number of isolates pathotyped No. of different pathotypes detected No. of different virulences detected Khyber 18 14 16 Peshawar 4 2 13 Nowshera 5 3 15 Haripur 20 13 15 Abbottabad 14 10 15 Mansehra 5 5 15 Battal 24 18 17 Battagram 6 6 15 Attarshisha 16 11 12 Balakot 11 11 16 Skardu 4 4 17 Maximum no. of virulences tested - 26 Maximum no. of pathotypes observed 53 - - Vr2, Vr6, Vr7, Vr8, Vr9 and VrSu in a high frequency - Vr5, Vr15 detected - AvrVicto detected Pakistani PST population; a high genetic diversity

Comparison of eight temporally sampled populations Locations Sample size FST between 2010 and 2011 Number of resampled MLGs and their frequency over two years* Peshawar 45 0.0719 1 (5:3) Nowshera 35 0.0863 1 (13:1) Abbottabad 52 0.2375 1 (1:11) Mansehra 89 0.0522 2 (2:2) (1:6) Battal 81 0.0740 0 Battagram 29 0.0661 0 Attarshisha 70 0.1359 0 Balakot 51 0.0609 1 (1:5) (Ali et al. 2014; Molecular Ecology) Pakistani PST population; a high genetic diversity

- Resistance genes deployment considering the information on worldwide population subdivision and diversity - Worldwide virulence structure to be re-analyzed Overall conclusions and perspectives - Future invasion risks assessment and management - Broader perspective for breeders, considering invasions - Role of sexual reproduction in PST biology in Pakistan and China - What strategy to adapt while considering the Berberis spp.?

Current work: - Wheat sampling in 2013 in all major PST regions -To be SSR genotyped -Berberis sampling from the Berberis zone -Sequence characterization to identify the species (and f.sp.) HEC-Start-up Research Grant Rust-Fight Danish Council for Strategic Research The University of Agriculture, Peshawar, Pakistan Pakistani PST population; a high genetic diversity Current work

Thanks to all those who contributed to the worldwide yellow rust collection French project: EMERFUNDIS, ANR 07-BDIV-003 European Integrated Project: BIOEXPLOIT, FOOD- CT-2005-513959 Higher Education Commission, Pakistan

Thank you Picture provided by Tarnab Agricultural Research Station, Peshawar, Pakistan